丹参多糖抗小鼠免疫性肝损伤及其免疫调节机制的研究
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摘要
研究背景：
我国肝病的主要发病原因是感染肝炎病毒。流行病学资料表明,我国每年有50万～100万新发病例,而且在全球约3.5亿以上慢性乙型肝炎病毒携带者中,中国人占80%。在HBV感染者中,有10%～15%的患者可发展为慢性HBV感染,从慢性肝炎发展为纤维化一般只需2～6年时,其中25%～40%的病例最终发展为肝硬化乃至肝癌。研究肝炎的发生机理及寻找有效的治疗措施是国内外学者共同研究的热门课题,大量研究表明,免疫功能的异常改变和氧自由基的大量产生是引起肝组织损伤的重要原因。
西医用于肝炎的治疗多见于抗病毒药、免疫调节药及护肝降酶类药物,目前临床上常用的品种有干扰素、阿糖腺苷、干扰素诱导剂聚肌胞、无环鸟苷、左旋咪唑、转移因子、辅酶Q、胸腺素、右旋儿茶素、甘草甜素、齐墩果酸、葫芦素B、葫芦素E、联苯双酯、维生素类等。临床治疗结果表明,这些药物多具有良好的抑制病毒复制,恢复正常肝功能,保护肝细胞等作用,并且疗效迅速,不足之处为远期疗效不稳定,易反复,HBsAg转阴率低,有些药品价格昂贵和表现一定的副作用。
中医对肝炎的认识常见于胁痛、黄疸、积聚及虚劳等病症中。其病因为湿热疫毒或先天胎毒,病机特点为湿热羁留、肝胆不疏,脾胃受损。蔡春江等提出慢性乙肝属伏邪致病,伏邪为“浊”、“毒”之邪,病位在肝,邪伏部位为血分,浊、毒之邪与慢性乙肝关系密切,既是病理产物,又是致病之因。刘兴家等认为乙型肝炎病毒的侵入是致病的主因,而湿热与肝郁是一种诱因。陈良金等认为慢性乙肝的病理基础可概括为郁、湿、热、毒、痰、瘀、虚；病位主要在肝,涉及到脾、肾二脏。裴建宏从中医病因理论着眼,认为慢乙肝的病因当属疫气范畴,其病理属性为湿热疫气侵袭,胶固难解,损及肝脾,伤及气血阴阳,并在病情发展过程中出现正邪盛衰演变的复杂病理格局,病势迁延难愈。陈华东等认为病毒性肝炎急性期以湿热疫毒为主,尤其热重于湿；慢性期则因失治、误治、久治不愈而致毒邪滞留,以湿为主。以具有解毒活血祛湿、滋补肝肾、健脾益气之效的药物予以治疗。中医中药具有不良反应较少,可长期服药的特点。近年来,中药治疗HBV感染取得了良好的疗效。
多聚糖(polysaccharide),简称多糖,常由一百个以上甚至几千个单糖基通过糖苷键连接而成的,其性质已大不同于单糖,如甜味已经基本消失,广泛存在于动物细胞膜和植物、微生物的细胞壁中,是构成生命的四大基本物质之一,与生命功能的维持密切相关。到目前为止,已有几百种多糖从自然界被分离和提纯,多糖不仅是生命有机体的重要组成成分,还具有广泛的生物活性,如免疫调节、抗炎、抗病毒、抗肿瘤、抗凝血、抗氧化、抗辐射、抗衰老、降血糖、降血脂、保肝、抗寄生虫等。多糖作为非细胞毒性物质,对正常细胞几乎无毒副作用。
目前已对数十种多糖进行了含量测定,乌头多糖、东当归多糖、石斛多糖、桔梗多糖、人参多糖、细叶蓟多糖、柴胡多糖、猪苓多糖、红松果多糖、菟丝子多糖、胎盘脂多糖、黔产党参多糖、文蛤多糖、石仙桃多糖、肉苁蓉多糖、胖大海多糖、黄精多糖、耳叶牛皮消多糖、石菖蒲多糖、海藻多糖、黄花倒水莲多糖、商陆多糖、金线莲多糖、灵芝多糖、白花蛇舌草多糖、米糖多糖、芦荟多糖、山药多糖、大枣多糖等进行了较系统的含量研究。应用于临床取得较好疗效的有黄芪多糖、猪苓多糖、茯苓多糖、香菇多糖等。文献已有报道许多多糖对肝脏具有保护作用,并且它们的保肝作用多与免疫因素联系密切例如：虫草多糖对小鼠免疫性肝损伤的保护作用,天麻多糖GEP-2对BCG+LPS致小鼠免疫性肝损伤的保护作用,大蒜多糖C对免疫性肝损伤小鼠外周血淋巴细胞亚群的影响,紫菜多糖对肝损伤小鼠的保护作用,琐琐葡萄多糖对大鼠体外免疫性肝损伤保护作用的研究。
丹参为唇形科植物丹参Salvia miltiorrhiza Bge.的干燥根及根茎,性味苦,微寒,归心、肝经。始载于《神农本草经》,列为草部上品,名为郄蝉草。为多年生直立草本,高40-80cm,全株密被黄白色柔毛及腺毛；根肉质,圆柱形,外皮朱红色。丹参具有活血祛瘀、通经止痛、清热安神的功效。其“善治血分,去滞生新,调经顺脉”之功效尤佳。故有“一味丹参,功同四物”之誉,广泛应用于临床。
经现代研究证明,丹参有效化学成分为脂溶性菲醌色素丹参酮类(如：丹参酮ⅡA等)和一些水溶性的酚性化合物(如丹酚酸类、原儿茶醛等),水溶性和脂溶性成分均有药理活性。由于丹参及其复方制剂对许多疾病如冠心病、中风、肝炎、肿瘤、糖尿病等有良好的疗效,国内外对丹参化学成分、药理作用、临床应用、制剂、质量控制等方面进行了系统的研究,取得了显著的进展。
本课题组在进行药物成分分离的实验中提取到了丹参中的多糖成分,经文献检索发现丹参中作为重要有效部位的多糖成分研究较少。现有文献报道的丹参多糖提取方法为水提醇沉法,其提取方法的缺点是操作复杂、多糖提取率较低,而且关于丹参多糖药效和药理方面的研究更少。本课题组进一步将丹参多糖的提取工艺进行优化,找到一种多糖提取率高、成本低廉、操作简单的提取方法。鉴于大多数多糖的疗效及丹参的临床应用,我们将对丹参多糖对免疫性肝损伤的影响及免疫调节机制进行初步研究,从而为其进一步研究开发奠定理论和实验基础。
研究目的：
通过对丹参多糖提取工艺、丹参多糖抗免疫性肝损伤及其免疫调节机制的实验研究,提供一种从丹参中提取多糖的方法并阐明其抗免疫性肝损伤及免疫调的作用机制,为丹参多糖的进一步开发利用奠定实验基础。
研究方法：
1.丹参多糖的提取
1.1粉碎：将丹参粉碎至粒径为70～90μm的微粉。
1.2脱脂：将丹参微粉用10倍体积的浓度大于或等于85%乙醇回流2小时,过滤,取滤渣挥干溶剂后备用。
1.3酶处理：将脱脂处理后的丹参微粉加到水中,再加入酶,然后用碱将pH值调节至5.5,在70℃下保温1小时；所述的酶可以是纤维素酶、木瓜蛋白酶、果胶酶中的一种,用量为丹参重量1.5%,也可以纤维素酶、木瓜蛋白酶、果胶酶中的两种以上,每种酶的用量为丹参重量1%,最好是纤维素酶和木瓜蛋白酶,每种酶的用量均为丹参重量的1.5%。
1.4超声提取：在频率为59kHz的超声波下处理,过滤,收集滤液,滤液浓缩至1：2(每2ml药液相当于1克药材)。
1.5醇沉：加入70ml的乙醇使其在滤液中的浓度至85%,滤去乙醇即得丹参多糖粗品,再经洗涤得丹参多糖,75℃真空干燥。
2.丹参多糖抗卡介苗(BCG)加脂多糖(LPS)诱导的小鼠免疫性肝损伤的实验研究
2.1实验分组
昆明小鼠,SPF级,体质量18～22g,60只(南方医科大学实验动物中心提供)。采用随机分组的方法分为正常组、模型组、丹参多糖高(360mg/kg)、中(180mg/kg)、低(90mg/kg)剂量组、联苯双酯组(200mg/kg),每组10只,雌雄各半。除正常组小鼠外,其余小鼠在造模的同时灌胃给药,灌胃剂量为0.5ml/20g,正常组小鼠给同等剂量生理盐水。
2.2造模方法
采用BCG加LPS诱导小鼠免疫性肝损伤。小鼠尾静脉注射BCG浆液0.2 ml/只(含菌量5×107单位),致敏后12 d,尾静脉注射LPS 0.2 ml/只(7.5μg)以诱导肝损伤,正常组小鼠尾静脉注射同等剂量生理盐水。
2.3指标检测
2.3.1摘眼球采血后,离心获取血清,按试剂盒说明操作检测血清ALT、AST、NO活性。
2.3.2小鼠处死后,立即取出所需肝脏,称取0.25g肝组织用冷生理盐水洗去浮血,配成10%肝组织匀浆,匀浆液经500转离心20min去胞核碎片后,再经3000转离心15min,取上清液,ELISA方法检测TNF-a、IL-1β含量。
2.3.3肝组织固定、切片和肝组织病变程度分级方法
处死动物,取出肝脏,用冷生理盐水洗去浮血,拭干。于肝门处及右肝叶取材4块,每块大小约为1.5cm×1.5cm×0.4cm,立即用10%中性福尔马林固定。70%、80%、90%、95%、100%乙醇脱水,二甲苯透明,石蜡包埋,切片机作4μm切片,常规脱蜡,HE染色,中性树胶封片。用显微镜观察,以及PM-20型显微摄像系统照相。
光镜下检查,按肝脏坏死程度分级:“-”,未见明显病理改变；“+”,部分肝组织肿胀伴有散在的点状坏死,汇管区有少许炎性细胞浸润；“++”,肝细胞肿胀有点状坏死及小灶性坏死,可见散在的肉芽肿形成,汇管区有大量炎性细胞浸润；“+++”,肝细胞肿胀,有大片状坏死,有肉芽肿形成,汇管区及周围有大量炎性细胞浸润。
3.丹参多糖对免疫功能调节的机制研究
3.1 BALB/c小鼠,SPF级,中山大学实验动物中心提供。分离BALB/c鼠脾细胞,不同浓度(1、10、50、100、200mg/l)丹参多糖对淋巴细胞(浓度1×106cell/ml)作用48h,MTT法检测细胞增殖。
3.2分离B、T淋巴细胞,不同浓度(1、10、50、100、200mg/l)丹参多糖分别对B、T淋巴细胞(浓度1×106cell/ml)作用48h并与分裂素ConA(T细胞)、LPS(B细胞)(最终浓度5ug/ml)作比较,MTT法检测细胞增殖。
3.3不同浓度(1、10、50、100、200mg/l)丹参多糖作用于T淋巴细胞72h,流式技术分析CD4+、CD8+的变化。
3.4 200mg/l浓度丹参多糖作用于T细胞不同时间(12、24、48、72h),ELISA分析IL-2、IL-4浓度。
3.5 200mg/l浓度丹参多糖作用于T细胞不同时间(24、48、72h),荧光定量PCR分析IL-2、IL-4水平变化。
4.统计处理
实验结果用均数(x)±标准差(s)表示,组间比较用one-way ANOVA。方差齐性时,采用LSD方法进行组间多重比较；方差不齐时,采用Welch法,组间多重比较采用DunnettT3方法进行。两组等级资料的比较采用两个独立样本的非参数检验Mann-WhitneyU法,多组等级资料的比较采用多个独立样本的非参数检验KrusKal-WallisH法,用统计软件SPSS13.0进行分析,以P<0.05定为差异有统计学意义。
结果：
1.采用粉碎、脱脂、酶处理、超声提取、醇沉的方法对丹参中的多糖成分进行提取,采用正交设计法进一步优化超声法的工艺条件,最终经统计学分析确定最优工艺条件为：以加水为12倍药材重量,粒度为过20目筛,超声提取时间20min为最佳提取条件。
2.在丹参多糖抗免疫性肝损伤的实验中,血清ALT、AST水平丹参多糖高、中、低剂量组与模型组相比有显著差异(P=0.000,P=0.003,P=0.044)。阳性药对照组与模型组相比有显著差异(P=0.000)；血清NO和肝组织匀浆IL-1β、TNF-α的水平丹参多糖高剂量组与模型组相比有显著差异(P=0.000),丹参多糖中剂量组与模型组相比TNF-α、IL-1β有显著差异(P=0.048),但是NO差异不显著(P=0.057)。丹参多糖低剂量组与模型组相比TNF-α(P=0.187)、NO(P=0.097)、IL-1β(P=0.145)差异不显著。联苯双酯滴丸组与模型组相比NO有显著差异(P=0.037),但是TNF-a(P=0.064)、IL-1β(P=0.882)差异不显著；脏器系数丹参多糖高剂量组与模型组相比有显著差异(P=0.000,P=0.005,P=0.018)。丹参多糖中剂量组与模型组相比肝脏系数(P=0.043)、脾脏系数(P=0.016)有显著差异。丹参多糖低剂量组与模型组相比无显著差异(P=0.178,P=0.175,P=0.329)。阳性药物对照组与模型组相比除脾脏系数差异不显著外(P=0.059),其两项均有显著差异(P=0.000,P=0.042)；与正常组相比较,模型组肝脏病理变化显著(P=0.000)。丹参多糖高剂量组(P=0.032)、阳性药物对照组(P=0.016)与模型组相比肝脏病理变化显著。丹参多糖中剂量组(P=0.240)、丹参多糖低剂量组(P=0.547)与模型组相比肝脏病理变化不显著。
3.在丹参多糖免疫调节机制的研究中,在10～200mg/l剂量范围内,丹参多糖不同剂量组均能显著促进脾细胞的增殖(P=0.000),与对照组比有显著性差异(P=0.020,P=0.000),丹参多糖200mg/l组显示出更高的活性；实验结果显示在100～200mg/l剂量范围内,丹参多糖组能显著促进T淋巴细胞的增殖,与对照组比较有显著性差异(P=0.042,P=0.000),同时丝裂原ConA与对照组比较有显著性差异(P=0.000)。丹参多糖浓度在50～200mg/l范围内,丹参多糖组能显著促进B淋巴细胞的增殖,与对照组比较有显著性差异(P=0.033,P=0.000),同时丝裂原LPS与对照组比较有显著性差异(P=0.000)；用200mg/l浓度的丹参多糖对小鼠淋巴细胞干预12h、24h、48h、72h后,IL-2水平升高,各时间段之间比较具有显著差别(P=0.000)。IL-4水平也有显著增强,各时间段之间比较具有显著差别(P=0.005),但是升高趋势较IL-2水平平缓；荧光定量PCR检测结果显示,经浓度为200mg/l的丹参多糖干预24h、48h、72h后IL-2 mRNA和IL-4 mRNA表达存在差异(P=0.016,P=0.169)；与空白对照相比,干预72h后IL-2 mRNA和IL-4mRNA表达均有所有提高,差异有统计学意义(P=0.022,P=0.036)；干预24h、48h后的IL-2mRNA和IL-4 mRNA表达差异不显著(P=0.753,P=0.400,P=0.177,P=0.208)。说明丹参多糖在干预72h后可有效增强IL-2 mRNA和IL-4 mRNA的表达；丹参多糖在1～200 mg/l剂量范围内,随着剂量的升高对CD4+、CD8+比例的影响逐渐增强,显示出明显的剂量依赖性,同时与对照组比较较高浓度组差异有统计学意义(P=0.028,P=0.007,P=0.008,P=0.027,P=0.000)。
结论：
1.确定最优丹参多糖提取工艺条件为：以加水为12倍药材重量,粒度为过20目筛,超声提取时间20min为最佳提取条件,本提取方法具有低耗能、提取率高、操作简单等优点。
2.结果显示丹参多糖有抗免疫性肝损伤的作用,这种作用的实现不仅表现在直接的保肝降酶方面,还对肝损伤后小鼠的免疫功能起到调节作用,通过免疫功能的调控进一步发挥保肝降酶作用。
3.体外实验说明丹参多糖在一定浓度范围内对淋巴细胞有较强的增殖作用,并且还有促进相关细胞因子表达及分泌的作用,与体内试验的结果相一致,进一步说明丹参多糖在免疫调节方面有很好的疗效。
Background:
The main causes of liver disease in China are infected with hepatitis virus. Epidemiological data show that our country have about 50 million to 1 million new cases every year, also in the world for more than about 350 million chronic hepatitis B virus carriers, accounts for 80% in China. In HBV infection,10% to 15% of patients may develop into chronic HBV infection, from chronic hepatitis develop into liver fibrosis only need 2 to 6 years in general.25%～40% of the patients eventually develop into cirrhosis of the liver and even hepatocellular carcinoma. The of study occurrence of hepatitis mechanism and to find effective treatment measures are the most popular in domestic and foreign scholars, a large number of studies have shown that abnormal changes in immune function and oxygen free radicals production are the cause of a large number of important reasons for the liver tissue damage.
Doctors prevalently use anti-viral drugs, immunomodulatory drugs and liver protection drugs to treat hepatitis in western medicine. These drugs commonly contain varieties interferon, vidarabine, interferon-inducing agent polyinosinic, acyclovir, levamisole, transfer factor, coenzyme Q, thymosin, cianidanol, glycyrrhizin, oleanolic acid, cucurbitacin B, cucurbitacin E, bifendate, vitamins and so on. Clinical results show that many of these drugs have good control of virus replication and restore normal liver function, protect liver cells and so on with efficacy of rapid. Disadvantage is unstable long-term efficacy, easily repeated, low rate of HBsAg darkening, expensive and the certain side effects.
Knowledge of traditional Chinese medicine for hepatitis common is Xie Tong, jaundice, accumulation and consumption diseases. The cause is hot and humid disease or congenital erupted fetal disease, pathogenesis characteristic is hot and humid detention, liver and gallbladder blockage, the spleen and stomach damage. Caichun jiang et al proposed an Fuxie pathogenesis of chronic hepatitis B, Fuxie is "muddy" and "poisonous",sickness locate in the liver, disease sub-site in the blood, "muddy" and "poisonous" are closely related to chronic hepatitis B both the pathological product and the result of illness. Liu xing jia presume that the intrusion of hepatitis B virus is the main cause of disease, while the hot and humid with liver depression is a kind of inducement. Chen Jin presume that the pathological basis of chronic hepatitis B can be summarized as depression, wet, heat, poison, phlegm, stasis, weak; Disease localization is mainly in the liver, involving the spleen and kidney. Pei Jianhong presumes the cause of chronic hepatitis B epidemic course ought to YiQi from the theory of traditional Chinese medicine. The pathological properties is hot and humid YiQi attack, damage liver and spleen, cause injuries to yin and yang, qi and blood, and the progression of the disease relapse during the evolution of the intricately illness.Chen Huadong et al presume that the acute phase of viral hepatitis is base on wet and heat; chronic phase due to incurable disease, mistreatment and long term therapy. They usually use HuoXueQuSi drug, ZhiBuGanShen drug, JianPiYiQi drug to treat this disease.Chinese medicine has fewer side effects and long-term medication characteristics. In recent years, treatment of Chinese medicine in HBV infection achieved good effect.
Polysaccharide referred to as polysaccharides, often composed by more than one hundred and even thousands of simple sugars through glycosidic, its nature has been very different from simple sugars, such as sweet has basically disappeared, widely found in animal and plant cell membrane, microorganisms of the cell wall, one of the four basic material of constitute life, and is closely related to the maintenance of life functions. So far, hundreds of kinds of polysaccharides have been isolated and purified from nature, polysaccharide is not only an important component of living organisms, but also has a wide range of biological activity, such as immune regulation, anti-inflammatory, anti-virus, anti-tumor, anti-coagulation, anti-oxidation, anti-radiation, anti-aging, lowering blood sugar and blood fat, liver protection, anti-parasites. Polysaccharide as non-cell-toxic substances has almost innocuity effects on normal cells.
Many polysaccharide has been carried out assaying, aconitane polysaccharide, east angelica polysaccharide, Dendrobium polysaccharide, Campanulaceae polysaccharide, ginseng polysaccharides, thistle polysaccharide, Bupleurum polysaccharide, grifola polysaccharide, red cone polysaccharide, Dodder polysaccharide, placenta polysaccharide, Codonopsis polysaccharide, hard-shelled clam polysaccharide, Chinese Pholidota Pseudobulb or Herb polysaccharide, Desertliving Cistanche Herb polysaccharide, Sterculia lychnophora polysaccharide, Siberian Solomonseal Rhizome polysaccharide, Cynanchum auriculatum Royle ex Wight polysaccharide, Tatarinow Sweetflag Rhizome polysaccharides, seaweed polysaccharides, False-yellowflower Milkwort Root polysaccharide, Indian Pokwees Root Polysaccharide, Roxburgh Anoectochilus Herb polysaccharide, Ganoderma lucidum polysaccharides, Spreading Hedyotis Herb polysaccharide, Mi Tang polysaccharides,aloe polysaccharide,Common Yam Rhizome polysaccharide,jujube polysaccharide and so on. Astragalin polysaccharide, grifola polysaccharide, pachyman polysaccharide, Lentinan polysaccharide has been applied to achieve better clinical efficacy. Documents has been reported a number of polysaccharides have a protective effect on the liver, and their role of protecting liver is more closely with immune factors, for example:Cordyceps polysaccharide on immunological liver injury in rats, Tall Gastrodia Rhizome polysaccharide GEP-2 against BCG+ LPS-induced immune liver injury in rats, garlic polysaccharide C on immunological liver injury in mice peripheral blood lymphocyte subsets, Sweet Porphyra polysaccharides on hepatic injury of mice, Haloxylon grape polysaccharides' protective effect on immunological liver injury in rats in vitro.
Salvia miltiorrhiza belongs to the plant family oregano's dried root and rhizome, sexual bitter, slightly cold, ascription heart and liver. It was first indexed in the Shen Nong's Materia Medica as a promoting blood flow-removing blood stasis and tonic herb of the nontoxic superior class. It is a erect perennial herb, high 40-80cm, whole-plant density is yellow-white hair and soft glandular hairs; roots fleshy, cylindrical, red skin. Salvia miltiorrhiza has effects of promoting blood flow-removing blood stasis, inducing menstruation to stop pain, clearing heat tranquilization. It has better efficacy of curing postamenorrhea edema, freshing new, activating blood circulation to dissipate blood stasis. And therefore, Salvia miltiorrhiza is considered equivalently with the SiWu tang, and is widely used in clinical.
Modern studies have shown that the effective chemical composition of fat-soluble pigment phenanthraquinone tanshinone classes (such as tanshinone II A, etc.), and some water-soluble phenolic compounds (such as Salvia miltiorrhizanolic acid, protocatechualdehyde, etc.), both water-soluble and fat-soluble constituents have pharmacologically active. Because Salvia miltiorrhiza and its compound preparation have a good effect on many diseases such as coronary heart disease, stroke, hepatitis, cancer, diabetes. At domestic and abroad, Salvia miltiorrhiza's chemical composition, pharmacological effects, clinical application, formulation, quality control and other aspects have made remarkable progress.
Our research group extracts the polysaccharide components from Salvia miltiorrhiza when carrying out drug ingredients separate experiments. We found that polysaccharide as important and effective parts of Salvia miltiorrhiza have been studied less. Current reported method of Salvia miltiorrhiza polysaccharides extraction is water extraction and alcohol precipitation, the shortcomings of its extraction method is complicated to operate, low rates of polysaccharide extraction, and the efficacy and pharmacological research of Salvia miltiorrhiza polysaccharides have been studied less. Our research group's purpose is to find a method to extract Salvia miltiorrhiza polysaccharide with a high rate, low cost,easy-to-extraction. Because of most the efficacy of polysaccharides and Salvia miltiorrhiza's clinical application, we will study Salvia miltiorrhiza polysaccharides on immunological liver injury in rats and the immune regulatory mechanisms as a preliminary research in order to establish its further research and development of theoretical and experimental basis.
Objectives:
Through the experimental research of extraction technology, immunological liver injury and its immune regulation mechanism to provide a polysaccharide extracted method from Salvia miltiorrhiza, and to clarify its anti-immune liver injury and the role of the immune adjustment mechanism of Salvia miltiorrhiza polysaccharides to establish the experimental basis for further development and utilization.
Methods:
1.Extraction of Salvia miltiorrhiza polysaccharides
1.1 Crush:The Salvia miltiorrhiza is crushed to 70～90μm particle size of the powder.
1.2 Degreasing:The Salvia miltiorrhiza powder with 10 times the volume of the concentration that is greater than or equal to 85% ethanol, reflux 2 hours, filter, take residues to dry and to reserve.
1.3 Enzyme treatment:The treatments of Salvia miltiorrhiza degreasing powder added to the water, then add enzymes, and then adjust pH to 5.5,at 70℃for 1 hour; The enzymes could be one of cellulase, papain, pectinase, and the dosage is the weight of 1.5% Salvia miltiorrhiza.The enzymes could be two or more of cellulase, papain, pectinase, and each enzyme dosage is the weight of 1% Salvia miltiorrhiza. The best method is cellulase and papain, and each enzyme dosage is the weight of 1.5% Salvia miltiorrhiza.
1.4 Ultrasonic extraction:Under the 59 kHz of ultrasound, filtration, collecting the filtrate, the filtrate concentrated to 1:2 (equivalent to 1 gram per 2ml physic liquor).
1.5 Alcohol precipitation:Add 70ml ethanol to the filtrate for the density to 85%, filter ethanol for gaining crude Salvia miltiorrhiza polysaccharide, and then wash and gain Salvia miltiorrhiza polysaccharides,75℃vacuum drying.
2. This study was designed to evaluate the hepatoprotective effects of Salvia miltiorrhiza polysaccharides in immunological liver injury induced by Bacille-Calmette-Guerin (BCG) and lipopolysaccharide (LPS) in mice.
2.1 Experiment grouping
Kunming mice, SPF grade,18～22g,60 (purchased from the Laboratory Animal Center of Southern Medical University). A randomized method was divided into normal group, model group, Salvia miltiorrhiza polysaccharide high-dose (360mg/kg), Salvia miltiorrhiza polysaccharide middle-dose(180mg/kg), Salvia miltiorrhiza polysaccharide low-dose (90mg/kg) dose group, Bifendate group (200mg/kg) (n=10 in each group), evenly divided male and female. In addition to normal mice, the remaining mice were gavage and the dose was 0.5ml/20g. Normal mice were given the same dose of saline.
2.2 Method
0.2mL BCG culture (approximately 5×107 viable units per mouse) was injected via the tail vein into mice, with the exception of the control group, who received saline alone. The control group was given an equal volume of saline. Twelve days later, the mice were given 7.5μg of LPS in 0.2ml saline (control received saline alone) via lateral tail vein injection.
2.3 Index Detection
2.3.1 The mice were weighed and the blood was collected from mice orbit, the blood was centrifugated to obtain serum, according to kit instructions to study serum ALT, AST,NO activity.
2.3.2 After the mice were killed and immediately took out the necessary liver,0.25g liver tissue was obtained and the blood were washed away with cold saline, dubbed into 10% liver tissue homogenate, homogenate centrifugated for 20min at 500 rotation speed, then at 3000 rotation speed for 15min, getted the supernatant, ELISA to detected TNF-a, IL-1βlevels.
2.3.3 Liver tissue fixation, sectioning and liver tissue pathological classification
Animals were killed, taken out the liver, washed floating blood with cold saline for drying. The right hepatic leaves were drawn into four. Each block size was about 1.5cm×1.5cm×0.4cm, and immediately fixed with 10% neutral formalin.70%, 80%,90%,95%,100% ethanol dehydration, xylene transparent, embedded in paraffin, slicing machine for 4μm slices, conventional dewaxing, HE staining, neutral gum mounting. As well as taked pictures with PM-20 type microscope camera system, observed with a microscope.
Using light microscope to examine, the degree of liver damage was categorized according to the degree of liver necrosis rating:"-",no significant pathological changes in liver tissue;"+",part of the scattered point-like necrosis, a little periportal infiltration of inflammatory cells;"++",liver cell necrosis and swelling and scattered granuloma formation can be seen, periportal inflammatory cell infiltration;"+++",a large number of liver cell swelling,necrosis, and granuloma formation of periportal areas is surrounded by a large number of inflammatory cell infiltration.
3.Salvia miltiorrhiza polysaccharides on immunomodulatory mechanism
3.1 BALB/c mice, SPF grade, purchased from Experimental Animal Center of Sun Yat-sen. Separated BALB/c mouse spleen cells, at different concentrations (1,10,50,100,200 mg/1) of Salvia miltiorrhiza polysaccharides on lymphocytes (concentration of 1×106 cell/ml) for 48h, MTT assayed cell proliferation.
3.2 Separated B, T lymphocytes, at different concentrations(1,10,50,100,200 mg/1) of Salvia miltiorrhiza polysaccharides respectively on B, T lymphocytes (concentration of 1×106cell/ml) for 48h and with the mitogen ConA (T cell),LPS (B cells) (final concentration 5mg/l) for comparison, MTT assayed cell proliferation.
3.3 Used different concentrations(1，10，50，100，200, mg/l) of Salvia-miltiorrhiza polysaccharides on T lymphocytes for 72h, streaming technology assayed CD4+ and CD8+.
3.4 200mg/l concentration of Salvia miltiorrhiza polysaccharides acted on T cells at different time(12,24,48,72 h), ELISA analyzed IL-2 and IL-4 concentration.
3.5 200mg/l concentration of Salvia miltiorrhiza polysaccharides acted on T cells at different time (24,48,72 h), fluorescence quantitative PCR analyzed IL-2, IL-4.
4. Statistical analysis
All experiments quantitative data were expressed as mean±S.D and assessed by the one-way analysis of variance (ANOVA).If the variances between groups were homogenous (Levene's test), groups were subjected to the multiple comparison Least Significant Differences (LSD) test. In case of no homogeneity variances, differences were evaluated by Welch and the groups were subjected to the multiple comparisons Dunnett's T3 test. The Mann-Whitney rank-sum test was used for the degree of histopathological liver injury. The statistical significance between groups was indicated using superscript signs in the figures and tables. Significance was defined as P<0.05.
Results:
1.With the method of grinding, degreasing, enzyme treatment, ultrasonic extraction, alcohol precipitation, the polysaccharide was extracted from Salvia miltiorrhiza. Using orthogonal design method to optimize the ultrasound process conditions and ultimately by the statistical analysis to determine the optimal process conditions:The water is 12 times of the herbs weight, particle size is 20 mesh sieve, ultrasonic extraction time is 20min for the best extraction conditions.
2.In the experiment of Salvia miltiorrhiza polysaccharide against immunological liver injury in mice. The serum levels of ALT, AST in Salvia miltiorrhiza polysaccharides high-dose, middle-dose, low-dose group compared with the model group were significantly different(P=0.000,P=0.003,P=0.044). Control group and model group were significantly different(P=0.000);The levels of serum NO and liver tissue homogenate IL-1β, TNF-a in Salvia miltiorrhiza polysaccharide high-dose group and model group were significantly different(P=0.000), and Salvia miltiorrhiza polysaccharides middle dose group compared with model group, TNF-a, IL-1βwere significantly different(P=0.048), but NO was not significantly different(P=0.057). Salvia miltiorrhiza polysaccharide low dose group compared with model group TNF-a(P=0.187), NO(P=0.097)and IL-1β(P=0.145)were not significantly different. Bifendate Pills group compared with model group NO was significantly different (P=0.037), but TNF-a(P=0.064)and IL-1β(P=0.882)were not significantly different; organ coefficient of Salvia miltiorrhiza polysaccharide high dose group and model group were significant difference(P=0.000,P=0.005,P=0.018). Salvia miltiorrhiza polysaccharide middle dose group compared with model group, liver index(P=0.043), spleen index(P=0.016)were significantly different. Salvia miltiorrhiza polysaccharide low dose group and model group showed no significant difference(P=0.178,P=0.175, P=0.329). Except spleen index(P=0.059), control group compared with model group were significantly different(P=0.000,P=0.042); Compared with the normal group, model group pathological changes in liver was significantly. Compared with model group, Salvia miltiorrhiza polysaccharide high dose group(P=0.032) and control group(P=0.016) pathological changes in liver were significantly. Compared with model group, Salvia miltiorrhiza polysaccharide middle dose group(P=0.240) and Salvia miltiorrhiza polysaccharide low dose group(P=0.547) showed no significant pathological changes in liver.
3.In the experiment of Salvia miltiorrhiza polysaccharide on immunomodulatory mechanism, in the 10～200mg/l range of different doses of Salvia miltiorrhiza polysaccharides can significantly promote the proliferation of spleen cells, compared with the control group were significant difference(P=0.020,P=0.000), Salvia miltiorrhiza polysaccharides 200mg/l group showed higher activity; Experimental results showed that 100～200mg/l dose range Salvia miltiorrhiza polysaccharide group could significantly promote the proliferation of T lymphocytes, compared with the control group were significant difference (P=0.042,P=0.000), while mitogen ConA compared with the control group was significant difference(P=0.000). In the concentration of 50-200mg/l, Salvia miltiorrhiza polysaccharide group could significantly promote the proliferation of B lymphocytes,compared with-the-control group there was significant difference(P=0.033,P=0.000), while mitogen LPS and the control group were significant difference (P=0.000); 200mg/l concentrations of Salvia miltiorrhiza polysaccharides on mouse lymphocytes for 12h,24h,48h,72h, IL-2 levels increased,each time showed significant difference(P=0.000). IL-4 levels significantly increased, making comparisons between different time periods with a significant difference(P=0.005), but mildly compared with the levels of IL-2; Fluorescence quantitative PCR results showed that, after 200mg/l concentration of Salvia miltiorrhiza polysaccharides interfereing for 24h,48h,72h,IL-2 mRNA and IL-4 mRNA expression were differences; Compared with the control, IL-2 mRNA and IL-4mRNA expression increased after 72h, and the difference was statistical significance(P=0.022,P=0.036); IL-2 mRNA and IL-4 mRNA expression were no significant difference after 24h,48h(P=0.753,P=0.400,P=0.177,P=0.208). It was accounted that Salvia miltiorrhiza polysaccharides effectively increased IL-2 mRNA and IL-4 mRNA expression after interfereing for 72h; With the higher dose in 1～200mg/l range of Salvia miltiorrhiza polysaccharides, the ratio of CD4+, CD8+ gradual enhanced, showing a clear dose-dependent, while higher concentrations group compared with control group showed statistically significant differences(P=0.028,P=0.007,P=0.008,P=0.027,P=0.000).
Conclusion:
1.To determine the optimal extraction conditions of Salvia miltiorrhiza polysaccharides:The water was 12 times of the herbs weight, particle size was 20 mesh sieves, ultrasonic extraction time was 20 min for the best extraction conditions, and the extraction method had low energy consumption, high extraction rate, advantages of simple operation.
2.The results showed that Salvia miltiorrhiza polysaccharides had the role of anti-immune liver injury. This role displayed not only in direct hepatoprotection, but also in regulatory role of immune function in liver injury, through the immune function further played the role of hepatoprotection.
3.Salvia miltiorrhiza polysaccharides in vitro showed a stronger proliferation of lymphocytes in certain concentration,and also promoted expression and secretion of cytokines. Consistent with the results in vivo experiments to further clarify the Salvia miltiorrhiza polysaccharides had immune regulation function.
我国肝病的主要发病原因是感染肝炎病毒。流行病学资料表明,我国每年有50万～100万新发病例,而且在全球约3.5亿以上慢性乙型肝炎病毒携带者中,中国人占80%。在HBV感染者中,有10%～15%的患者可发展为慢性HBV感染,从慢性肝炎发展为纤维化一般只需2～6年时,其中25%～40%的病例最终发展为肝硬化乃至肝癌。研究肝炎的发生机理及寻找有效的治疗措施是国内外学者共同研究的热门课题,大量研究表明,免疫功能的异常改变和氧自由基的大量产生是引起肝组织损伤的重要原因。
西医用于肝炎的治疗多见于抗病毒药、免疫调节药及护肝降酶类药物,目前临床上常用的品种有干扰素、阿糖腺苷、干扰素诱导剂聚肌胞、无环鸟苷、左旋咪唑、转移因子、辅酶Q、胸腺素、右旋儿茶素、甘草甜素、齐墩果酸、葫芦素B、葫芦素E、联苯双酯、维生素类等。临床治疗结果表明,这些药物多具有良好的抑制病毒复制,恢复正常肝功能,保护肝细胞等作用,并且疗效迅速,不足之处为远期疗效不稳定,易反复,HBsAg转阴率低,有些药品价格昂贵和表现一定的副作用。
中医对肝炎的认识常见于胁痛、黄疸、积聚及虚劳等病症中。其病因为湿热疫毒或先天胎毒,病机特点为湿热羁留、肝胆不疏,脾胃受损。蔡春江等提出慢性乙肝属伏邪致病,伏邪为“浊”、“毒”之邪,病位在肝,邪伏部位为血分,浊、毒之邪与慢性乙肝关系密切,既是病理产物,又是致病之因。刘兴家等认为乙型肝炎病毒的侵入是致病的主因,而湿热与肝郁是一种诱因。陈良金等认为慢性乙肝的病理基础可概括为郁、湿、热、毒、痰、瘀、虚；病位主要在肝,涉及到脾、肾二脏。裴建宏从中医病因理论着眼,认为慢乙肝的病因当属疫气范畴,其病理属性为湿热疫气侵袭,胶固难解,损及肝脾,伤及气血阴阳,并在病情发展过程中出现正邪盛衰演变的复杂病理格局,病势迁延难愈。陈华东等认为病毒性肝炎急性期以湿热疫毒为主,尤其热重于湿；慢性期则因失治、误治、久治不愈而致毒邪滞留,以湿为主。以具有解毒活血祛湿、滋补肝肾、健脾益气之效的药物予以治疗。中医中药具有不良反应较少,可长期服药的特点。近年来,中药治疗HBV感染取得了良好的疗效。
多聚糖(polysaccharide),简称多糖,常由一百个以上甚至几千个单糖基通过糖苷键连接而成的,其性质已大不同于单糖,如甜味已经基本消失,广泛存在于动物细胞膜和植物、微生物的细胞壁中,是构成生命的四大基本物质之一,与生命功能的维持密切相关。到目前为止,已有几百种多糖从自然界被分离和提纯,多糖不仅是生命有机体的重要组成成分,还具有广泛的生物活性,如免疫调节、抗炎、抗病毒、抗肿瘤、抗凝血、抗氧化、抗辐射、抗衰老、降血糖、降血脂、保肝、抗寄生虫等。多糖作为非细胞毒性物质,对正常细胞几乎无毒副作用。
目前已对数十种多糖进行了含量测定,乌头多糖、东当归多糖、石斛多糖、桔梗多糖、人参多糖、细叶蓟多糖、柴胡多糖、猪苓多糖、红松果多糖、菟丝子多糖、胎盘脂多糖、黔产党参多糖、文蛤多糖、石仙桃多糖、肉苁蓉多糖、胖大海多糖、黄精多糖、耳叶牛皮消多糖、石菖蒲多糖、海藻多糖、黄花倒水莲多糖、商陆多糖、金线莲多糖、灵芝多糖、白花蛇舌草多糖、米糖多糖、芦荟多糖、山药多糖、大枣多糖等进行了较系统的含量研究。应用于临床取得较好疗效的有黄芪多糖、猪苓多糖、茯苓多糖、香菇多糖等。文献已有报道许多多糖对肝脏具有保护作用,并且它们的保肝作用多与免疫因素联系密切例如：虫草多糖对小鼠免疫性肝损伤的保护作用,天麻多糖GEP-2对BCG+LPS致小鼠免疫性肝损伤的保护作用,大蒜多糖C对免疫性肝损伤小鼠外周血淋巴细胞亚群的影响,紫菜多糖对肝损伤小鼠的保护作用,琐琐葡萄多糖对大鼠体外免疫性肝损伤保护作用的研究。
丹参为唇形科植物丹参Salvia miltiorrhiza Bge.的干燥根及根茎,性味苦,微寒,归心、肝经。始载于《神农本草经》,列为草部上品,名为郄蝉草。为多年生直立草本,高40-80cm,全株密被黄白色柔毛及腺毛；根肉质,圆柱形,外皮朱红色。丹参具有活血祛瘀、通经止痛、清热安神的功效。其“善治血分,去滞生新,调经顺脉”之功效尤佳。故有“一味丹参,功同四物”之誉,广泛应用于临床。
经现代研究证明,丹参有效化学成分为脂溶性菲醌色素丹参酮类(如：丹参酮ⅡA等)和一些水溶性的酚性化合物(如丹酚酸类、原儿茶醛等),水溶性和脂溶性成分均有药理活性。由于丹参及其复方制剂对许多疾病如冠心病、中风、肝炎、肿瘤、糖尿病等有良好的疗效,国内外对丹参化学成分、药理作用、临床应用、制剂、质量控制等方面进行了系统的研究,取得了显著的进展。
本课题组在进行药物成分分离的实验中提取到了丹参中的多糖成分,经文献检索发现丹参中作为重要有效部位的多糖成分研究较少。现有文献报道的丹参多糖提取方法为水提醇沉法,其提取方法的缺点是操作复杂、多糖提取率较低,而且关于丹参多糖药效和药理方面的研究更少。本课题组进一步将丹参多糖的提取工艺进行优化,找到一种多糖提取率高、成本低廉、操作简单的提取方法。鉴于大多数多糖的疗效及丹参的临床应用,我们将对丹参多糖对免疫性肝损伤的影响及免疫调节机制进行初步研究,从而为其进一步研究开发奠定理论和实验基础。
研究目的：
通过对丹参多糖提取工艺、丹参多糖抗免疫性肝损伤及其免疫调节机制的实验研究,提供一种从丹参中提取多糖的方法并阐明其抗免疫性肝损伤及免疫调的作用机制,为丹参多糖的进一步开发利用奠定实验基础。
研究方法：
1.丹参多糖的提取
1.1粉碎：将丹参粉碎至粒径为70～90μm的微粉。
1.2脱脂：将丹参微粉用10倍体积的浓度大于或等于85%乙醇回流2小时,过滤,取滤渣挥干溶剂后备用。
1.3酶处理：将脱脂处理后的丹参微粉加到水中,再加入酶,然后用碱将pH值调节至5.5,在70℃下保温1小时；所述的酶可以是纤维素酶、木瓜蛋白酶、果胶酶中的一种,用量为丹参重量1.5%,也可以纤维素酶、木瓜蛋白酶、果胶酶中的两种以上,每种酶的用量为丹参重量1%,最好是纤维素酶和木瓜蛋白酶,每种酶的用量均为丹参重量的1.5%。
1.4超声提取：在频率为59kHz的超声波下处理,过滤,收集滤液,滤液浓缩至1：2(每2ml药液相当于1克药材)。
1.5醇沉：加入70ml的乙醇使其在滤液中的浓度至85%,滤去乙醇即得丹参多糖粗品,再经洗涤得丹参多糖,75℃真空干燥。
2.丹参多糖抗卡介苗(BCG)加脂多糖(LPS)诱导的小鼠免疫性肝损伤的实验研究
2.1实验分组
昆明小鼠,SPF级,体质量18～22g,60只(南方医科大学实验动物中心提供)。采用随机分组的方法分为正常组、模型组、丹参多糖高(360mg/kg)、中(180mg/kg)、低(90mg/kg)剂量组、联苯双酯组(200mg/kg),每组10只,雌雄各半。除正常组小鼠外,其余小鼠在造模的同时灌胃给药,灌胃剂量为0.5ml/20g,正常组小鼠给同等剂量生理盐水。
2.2造模方法
采用BCG加LPS诱导小鼠免疫性肝损伤。小鼠尾静脉注射BCG浆液0.2 ml/只(含菌量5×107单位),致敏后12 d,尾静脉注射LPS 0.2 ml/只(7.5μg)以诱导肝损伤,正常组小鼠尾静脉注射同等剂量生理盐水。
2.3指标检测
2.3.1摘眼球采血后,离心获取血清,按试剂盒说明操作检测血清ALT、AST、NO活性。
2.3.2小鼠处死后,立即取出所需肝脏,称取0.25g肝组织用冷生理盐水洗去浮血,配成10%肝组织匀浆,匀浆液经500转离心20min去胞核碎片后,再经3000转离心15min,取上清液,ELISA方法检测TNF-a、IL-1β含量。
2.3.3肝组织固定、切片和肝组织病变程度分级方法
处死动物,取出肝脏,用冷生理盐水洗去浮血,拭干。于肝门处及右肝叶取材4块,每块大小约为1.5cm×1.5cm×0.4cm,立即用10%中性福尔马林固定。70%、80%、90%、95%、100%乙醇脱水,二甲苯透明,石蜡包埋,切片机作4μm切片,常规脱蜡,HE染色,中性树胶封片。用显微镜观察,以及PM-20型显微摄像系统照相。
光镜下检查,按肝脏坏死程度分级:“-”,未见明显病理改变；“+”,部分肝组织肿胀伴有散在的点状坏死,汇管区有少许炎性细胞浸润；“++”,肝细胞肿胀有点状坏死及小灶性坏死,可见散在的肉芽肿形成,汇管区有大量炎性细胞浸润；“+++”,肝细胞肿胀,有大片状坏死,有肉芽肿形成,汇管区及周围有大量炎性细胞浸润。
3.丹参多糖对免疫功能调节的机制研究
3.1 BALB/c小鼠,SPF级,中山大学实验动物中心提供。分离BALB/c鼠脾细胞,不同浓度(1、10、50、100、200mg/l)丹参多糖对淋巴细胞(浓度1×106cell/ml)作用48h,MTT法检测细胞增殖。
3.2分离B、T淋巴细胞,不同浓度(1、10、50、100、200mg/l)丹参多糖分别对B、T淋巴细胞(浓度1×106cell/ml)作用48h并与分裂素ConA(T细胞)、LPS(B细胞)(最终浓度5ug/ml)作比较,MTT法检测细胞增殖。
3.3不同浓度(1、10、50、100、200mg/l)丹参多糖作用于T淋巴细胞72h,流式技术分析CD4+、CD8+的变化。
3.4 200mg/l浓度丹参多糖作用于T细胞不同时间(12、24、48、72h),ELISA分析IL-2、IL-4浓度。
3.5 200mg/l浓度丹参多糖作用于T细胞不同时间(24、48、72h),荧光定量PCR分析IL-2、IL-4水平变化。
4.统计处理
实验结果用均数(x)±标准差(s)表示,组间比较用one-way ANOVA。方差齐性时,采用LSD方法进行组间多重比较；方差不齐时,采用Welch法,组间多重比较采用DunnettT3方法进行。两组等级资料的比较采用两个独立样本的非参数检验Mann-WhitneyU法,多组等级资料的比较采用多个独立样本的非参数检验KrusKal-WallisH法,用统计软件SPSS13.0进行分析,以P<0.05定为差异有统计学意义。
结果：
1.采用粉碎、脱脂、酶处理、超声提取、醇沉的方法对丹参中的多糖成分进行提取,采用正交设计法进一步优化超声法的工艺条件,最终经统计学分析确定最优工艺条件为：以加水为12倍药材重量,粒度为过20目筛,超声提取时间20min为最佳提取条件。
2.在丹参多糖抗免疫性肝损伤的实验中,血清ALT、AST水平丹参多糖高、中、低剂量组与模型组相比有显著差异(P=0.000,P=0.003,P=0.044)。阳性药对照组与模型组相比有显著差异(P=0.000)；血清NO和肝组织匀浆IL-1β、TNF-α的水平丹参多糖高剂量组与模型组相比有显著差异(P=0.000),丹参多糖中剂量组与模型组相比TNF-α、IL-1β有显著差异(P=0.048),但是NO差异不显著(P=0.057)。丹参多糖低剂量组与模型组相比TNF-α(P=0.187)、NO(P=0.097)、IL-1β(P=0.145)差异不显著。联苯双酯滴丸组与模型组相比NO有显著差异(P=0.037),但是TNF-a(P=0.064)、IL-1β(P=0.882)差异不显著；脏器系数丹参多糖高剂量组与模型组相比有显著差异(P=0.000,P=0.005,P=0.018)。丹参多糖中剂量组与模型组相比肝脏系数(P=0.043)、脾脏系数(P=0.016)有显著差异。丹参多糖低剂量组与模型组相比无显著差异(P=0.178,P=0.175,P=0.329)。阳性药物对照组与模型组相比除脾脏系数差异不显著外(P=0.059),其两项均有显著差异(P=0.000,P=0.042)；与正常组相比较,模型组肝脏病理变化显著(P=0.000)。丹参多糖高剂量组(P=0.032)、阳性药物对照组(P=0.016)与模型组相比肝脏病理变化显著。丹参多糖中剂量组(P=0.240)、丹参多糖低剂量组(P=0.547)与模型组相比肝脏病理变化不显著。
3.在丹参多糖免疫调节机制的研究中,在10～200mg/l剂量范围内,丹参多糖不同剂量组均能显著促进脾细胞的增殖(P=0.000),与对照组比有显著性差异(P=0.020,P=0.000),丹参多糖200mg/l组显示出更高的活性；实验结果显示在100～200mg/l剂量范围内,丹参多糖组能显著促进T淋巴细胞的增殖,与对照组比较有显著性差异(P=0.042,P=0.000),同时丝裂原ConA与对照组比较有显著性差异(P=0.000)。丹参多糖浓度在50～200mg/l范围内,丹参多糖组能显著促进B淋巴细胞的增殖,与对照组比较有显著性差异(P=0.033,P=0.000),同时丝裂原LPS与对照组比较有显著性差异(P=0.000)；用200mg/l浓度的丹参多糖对小鼠淋巴细胞干预12h、24h、48h、72h后,IL-2水平升高,各时间段之间比较具有显著差别(P=0.000)。IL-4水平也有显著增强,各时间段之间比较具有显著差别(P=0.005),但是升高趋势较IL-2水平平缓；荧光定量PCR检测结果显示,经浓度为200mg/l的丹参多糖干预24h、48h、72h后IL-2 mRNA和IL-4 mRNA表达存在差异(P=0.016,P=0.169)；与空白对照相比,干预72h后IL-2 mRNA和IL-4mRNA表达均有所有提高,差异有统计学意义(P=0.022,P=0.036)；干预24h、48h后的IL-2mRNA和IL-4 mRNA表达差异不显著(P=0.753,P=0.400,P=0.177,P=0.208)。说明丹参多糖在干预72h后可有效增强IL-2 mRNA和IL-4 mRNA的表达；丹参多糖在1～200 mg/l剂量范围内,随着剂量的升高对CD4+、CD8+比例的影响逐渐增强,显示出明显的剂量依赖性,同时与对照组比较较高浓度组差异有统计学意义(P=0.028,P=0.007,P=0.008,P=0.027,P=0.000)。
结论：
1.确定最优丹参多糖提取工艺条件为：以加水为12倍药材重量,粒度为过20目筛,超声提取时间20min为最佳提取条件,本提取方法具有低耗能、提取率高、操作简单等优点。
2.结果显示丹参多糖有抗免疫性肝损伤的作用,这种作用的实现不仅表现在直接的保肝降酶方面,还对肝损伤后小鼠的免疫功能起到调节作用,通过免疫功能的调控进一步发挥保肝降酶作用。
3.体外实验说明丹参多糖在一定浓度范围内对淋巴细胞有较强的增殖作用,并且还有促进相关细胞因子表达及分泌的作用,与体内试验的结果相一致,进一步说明丹参多糖在免疫调节方面有很好的疗效。
Background:
The main causes of liver disease in China are infected with hepatitis virus. Epidemiological data show that our country have about 50 million to 1 million new cases every year, also in the world for more than about 350 million chronic hepatitis B virus carriers, accounts for 80% in China. In HBV infection,10% to 15% of patients may develop into chronic HBV infection, from chronic hepatitis develop into liver fibrosis only need 2 to 6 years in general.25%～40% of the patients eventually develop into cirrhosis of the liver and even hepatocellular carcinoma. The of study occurrence of hepatitis mechanism and to find effective treatment measures are the most popular in domestic and foreign scholars, a large number of studies have shown that abnormal changes in immune function and oxygen free radicals production are the cause of a large number of important reasons for the liver tissue damage.
Doctors prevalently use anti-viral drugs, immunomodulatory drugs and liver protection drugs to treat hepatitis in western medicine. These drugs commonly contain varieties interferon, vidarabine, interferon-inducing agent polyinosinic, acyclovir, levamisole, transfer factor, coenzyme Q, thymosin, cianidanol, glycyrrhizin, oleanolic acid, cucurbitacin B, cucurbitacin E, bifendate, vitamins and so on. Clinical results show that many of these drugs have good control of virus replication and restore normal liver function, protect liver cells and so on with efficacy of rapid. Disadvantage is unstable long-term efficacy, easily repeated, low rate of HBsAg darkening, expensive and the certain side effects.
Knowledge of traditional Chinese medicine for hepatitis common is Xie Tong, jaundice, accumulation and consumption diseases. The cause is hot and humid disease or congenital erupted fetal disease, pathogenesis characteristic is hot and humid detention, liver and gallbladder blockage, the spleen and stomach damage. Caichun jiang et al proposed an Fuxie pathogenesis of chronic hepatitis B, Fuxie is "muddy" and "poisonous",sickness locate in the liver, disease sub-site in the blood, "muddy" and "poisonous" are closely related to chronic hepatitis B both the pathological product and the result of illness. Liu xing jia presume that the intrusion of hepatitis B virus is the main cause of disease, while the hot and humid with liver depression is a kind of inducement. Chen Jin presume that the pathological basis of chronic hepatitis B can be summarized as depression, wet, heat, poison, phlegm, stasis, weak; Disease localization is mainly in the liver, involving the spleen and kidney. Pei Jianhong presumes the cause of chronic hepatitis B epidemic course ought to YiQi from the theory of traditional Chinese medicine. The pathological properties is hot and humid YiQi attack, damage liver and spleen, cause injuries to yin and yang, qi and blood, and the progression of the disease relapse during the evolution of the intricately illness.Chen Huadong et al presume that the acute phase of viral hepatitis is base on wet and heat; chronic phase due to incurable disease, mistreatment and long term therapy. They usually use HuoXueQuSi drug, ZhiBuGanShen drug, JianPiYiQi drug to treat this disease.Chinese medicine has fewer side effects and long-term medication characteristics. In recent years, treatment of Chinese medicine in HBV infection achieved good effect.
Polysaccharide referred to as polysaccharides, often composed by more than one hundred and even thousands of simple sugars through glycosidic, its nature has been very different from simple sugars, such as sweet has basically disappeared, widely found in animal and plant cell membrane, microorganisms of the cell wall, one of the four basic material of constitute life, and is closely related to the maintenance of life functions. So far, hundreds of kinds of polysaccharides have been isolated and purified from nature, polysaccharide is not only an important component of living organisms, but also has a wide range of biological activity, such as immune regulation, anti-inflammatory, anti-virus, anti-tumor, anti-coagulation, anti-oxidation, anti-radiation, anti-aging, lowering blood sugar and blood fat, liver protection, anti-parasites. Polysaccharide as non-cell-toxic substances has almost innocuity effects on normal cells.
Many polysaccharide has been carried out assaying, aconitane polysaccharide, east angelica polysaccharide, Dendrobium polysaccharide, Campanulaceae polysaccharide, ginseng polysaccharides, thistle polysaccharide, Bupleurum polysaccharide, grifola polysaccharide, red cone polysaccharide, Dodder polysaccharide, placenta polysaccharide, Codonopsis polysaccharide, hard-shelled clam polysaccharide, Chinese Pholidota Pseudobulb or Herb polysaccharide, Desertliving Cistanche Herb polysaccharide, Sterculia lychnophora polysaccharide, Siberian Solomonseal Rhizome polysaccharide, Cynanchum auriculatum Royle ex Wight polysaccharide, Tatarinow Sweetflag Rhizome polysaccharides, seaweed polysaccharides, False-yellowflower Milkwort Root polysaccharide, Indian Pokwees Root Polysaccharide, Roxburgh Anoectochilus Herb polysaccharide, Ganoderma lucidum polysaccharides, Spreading Hedyotis Herb polysaccharide, Mi Tang polysaccharides,aloe polysaccharide,Common Yam Rhizome polysaccharide,jujube polysaccharide and so on. Astragalin polysaccharide, grifola polysaccharide, pachyman polysaccharide, Lentinan polysaccharide has been applied to achieve better clinical efficacy. Documents has been reported a number of polysaccharides have a protective effect on the liver, and their role of protecting liver is more closely with immune factors, for example:Cordyceps polysaccharide on immunological liver injury in rats, Tall Gastrodia Rhizome polysaccharide GEP-2 against BCG+ LPS-induced immune liver injury in rats, garlic polysaccharide C on immunological liver injury in mice peripheral blood lymphocyte subsets, Sweet Porphyra polysaccharides on hepatic injury of mice, Haloxylon grape polysaccharides' protective effect on immunological liver injury in rats in vitro.
Salvia miltiorrhiza belongs to the plant family oregano's dried root and rhizome, sexual bitter, slightly cold, ascription heart and liver. It was first indexed in the Shen Nong's Materia Medica as a promoting blood flow-removing blood stasis and tonic herb of the nontoxic superior class. It is a erect perennial herb, high 40-80cm, whole-plant density is yellow-white hair and soft glandular hairs; roots fleshy, cylindrical, red skin. Salvia miltiorrhiza has effects of promoting blood flow-removing blood stasis, inducing menstruation to stop pain, clearing heat tranquilization. It has better efficacy of curing postamenorrhea edema, freshing new, activating blood circulation to dissipate blood stasis. And therefore, Salvia miltiorrhiza is considered equivalently with the SiWu tang, and is widely used in clinical.
Modern studies have shown that the effective chemical composition of fat-soluble pigment phenanthraquinone tanshinone classes (such as tanshinone II A, etc.), and some water-soluble phenolic compounds (such as Salvia miltiorrhizanolic acid, protocatechualdehyde, etc.), both water-soluble and fat-soluble constituents have pharmacologically active. Because Salvia miltiorrhiza and its compound preparation have a good effect on many diseases such as coronary heart disease, stroke, hepatitis, cancer, diabetes. At domestic and abroad, Salvia miltiorrhiza's chemical composition, pharmacological effects, clinical application, formulation, quality control and other aspects have made remarkable progress.
Our research group extracts the polysaccharide components from Salvia miltiorrhiza when carrying out drug ingredients separate experiments. We found that polysaccharide as important and effective parts of Salvia miltiorrhiza have been studied less. Current reported method of Salvia miltiorrhiza polysaccharides extraction is water extraction and alcohol precipitation, the shortcomings of its extraction method is complicated to operate, low rates of polysaccharide extraction, and the efficacy and pharmacological research of Salvia miltiorrhiza polysaccharides have been studied less. Our research group's purpose is to find a method to extract Salvia miltiorrhiza polysaccharide with a high rate, low cost,easy-to-extraction. Because of most the efficacy of polysaccharides and Salvia miltiorrhiza's clinical application, we will study Salvia miltiorrhiza polysaccharides on immunological liver injury in rats and the immune regulatory mechanisms as a preliminary research in order to establish its further research and development of theoretical and experimental basis.
Objectives:
Through the experimental research of extraction technology, immunological liver injury and its immune regulation mechanism to provide a polysaccharide extracted method from Salvia miltiorrhiza, and to clarify its anti-immune liver injury and the role of the immune adjustment mechanism of Salvia miltiorrhiza polysaccharides to establish the experimental basis for further development and utilization.
Methods:
1.Extraction of Salvia miltiorrhiza polysaccharides
1.1 Crush:The Salvia miltiorrhiza is crushed to 70～90μm particle size of the powder.
1.2 Degreasing:The Salvia miltiorrhiza powder with 10 times the volume of the concentration that is greater than or equal to 85% ethanol, reflux 2 hours, filter, take residues to dry and to reserve.
1.3 Enzyme treatment:The treatments of Salvia miltiorrhiza degreasing powder added to the water, then add enzymes, and then adjust pH to 5.5,at 70℃for 1 hour; The enzymes could be one of cellulase, papain, pectinase, and the dosage is the weight of 1.5% Salvia miltiorrhiza.The enzymes could be two or more of cellulase, papain, pectinase, and each enzyme dosage is the weight of 1% Salvia miltiorrhiza. The best method is cellulase and papain, and each enzyme dosage is the weight of 1.5% Salvia miltiorrhiza.
1.4 Ultrasonic extraction:Under the 59 kHz of ultrasound, filtration, collecting the filtrate, the filtrate concentrated to 1:2 (equivalent to 1 gram per 2ml physic liquor).
1.5 Alcohol precipitation:Add 70ml ethanol to the filtrate for the density to 85%, filter ethanol for gaining crude Salvia miltiorrhiza polysaccharide, and then wash and gain Salvia miltiorrhiza polysaccharides,75℃vacuum drying.
2. This study was designed to evaluate the hepatoprotective effects of Salvia miltiorrhiza polysaccharides in immunological liver injury induced by Bacille-Calmette-Guerin (BCG) and lipopolysaccharide (LPS) in mice.
2.1 Experiment grouping
Kunming mice, SPF grade,18～22g,60 (purchased from the Laboratory Animal Center of Southern Medical University). A randomized method was divided into normal group, model group, Salvia miltiorrhiza polysaccharide high-dose (360mg/kg), Salvia miltiorrhiza polysaccharide middle-dose(180mg/kg), Salvia miltiorrhiza polysaccharide low-dose (90mg/kg) dose group, Bifendate group (200mg/kg) (n=10 in each group), evenly divided male and female. In addition to normal mice, the remaining mice were gavage and the dose was 0.5ml/20g. Normal mice were given the same dose of saline.
2.2 Method
0.2mL BCG culture (approximately 5×107 viable units per mouse) was injected via the tail vein into mice, with the exception of the control group, who received saline alone. The control group was given an equal volume of saline. Twelve days later, the mice were given 7.5μg of LPS in 0.2ml saline (control received saline alone) via lateral tail vein injection.
2.3 Index Detection
2.3.1 The mice were weighed and the blood was collected from mice orbit, the blood was centrifugated to obtain serum, according to kit instructions to study serum ALT, AST,NO activity.
2.3.2 After the mice were killed and immediately took out the necessary liver,0.25g liver tissue was obtained and the blood were washed away with cold saline, dubbed into 10% liver tissue homogenate, homogenate centrifugated for 20min at 500 rotation speed, then at 3000 rotation speed for 15min, getted the supernatant, ELISA to detected TNF-a, IL-1βlevels.
2.3.3 Liver tissue fixation, sectioning and liver tissue pathological classification
Animals were killed, taken out the liver, washed floating blood with cold saline for drying. The right hepatic leaves were drawn into four. Each block size was about 1.5cm×1.5cm×0.4cm, and immediately fixed with 10% neutral formalin.70%, 80%,90%,95%,100% ethanol dehydration, xylene transparent, embedded in paraffin, slicing machine for 4μm slices, conventional dewaxing, HE staining, neutral gum mounting. As well as taked pictures with PM-20 type microscope camera system, observed with a microscope.
Using light microscope to examine, the degree of liver damage was categorized according to the degree of liver necrosis rating:"-",no significant pathological changes in liver tissue;"+",part of the scattered point-like necrosis, a little periportal infiltration of inflammatory cells;"++",liver cell necrosis and swelling and scattered granuloma formation can be seen, periportal inflammatory cell infiltration;"+++",a large number of liver cell swelling,necrosis, and granuloma formation of periportal areas is surrounded by a large number of inflammatory cell infiltration.
3.Salvia miltiorrhiza polysaccharides on immunomodulatory mechanism
3.1 BALB/c mice, SPF grade, purchased from Experimental Animal Center of Sun Yat-sen. Separated BALB/c mouse spleen cells, at different concentrations (1,10,50,100,200 mg/1) of Salvia miltiorrhiza polysaccharides on lymphocytes (concentration of 1×106 cell/ml) for 48h, MTT assayed cell proliferation.
3.2 Separated B, T lymphocytes, at different concentrations(1,10,50,100,200 mg/1) of Salvia miltiorrhiza polysaccharides respectively on B, T lymphocytes (concentration of 1×106cell/ml) for 48h and with the mitogen ConA (T cell),LPS (B cells) (final concentration 5mg/l) for comparison, MTT assayed cell proliferation.
3.3 Used different concentrations(1，10，50，100，200, mg/l) of Salvia-miltiorrhiza polysaccharides on T lymphocytes for 72h, streaming technology assayed CD4+ and CD8+.
3.4 200mg/l concentration of Salvia miltiorrhiza polysaccharides acted on T cells at different time(12,24,48,72 h), ELISA analyzed IL-2 and IL-4 concentration.
3.5 200mg/l concentration of Salvia miltiorrhiza polysaccharides acted on T cells at different time (24,48,72 h), fluorescence quantitative PCR analyzed IL-2, IL-4.
4. Statistical analysis
All experiments quantitative data were expressed as mean±S.D and assessed by the one-way analysis of variance (ANOVA).If the variances between groups were homogenous (Levene's test), groups were subjected to the multiple comparison Least Significant Differences (LSD) test. In case of no homogeneity variances, differences were evaluated by Welch and the groups were subjected to the multiple comparisons Dunnett's T3 test. The Mann-Whitney rank-sum test was used for the degree of histopathological liver injury. The statistical significance between groups was indicated using superscript signs in the figures and tables. Significance was defined as P<0.05.
Results:
1.With the method of grinding, degreasing, enzyme treatment, ultrasonic extraction, alcohol precipitation, the polysaccharide was extracted from Salvia miltiorrhiza. Using orthogonal design method to optimize the ultrasound process conditions and ultimately by the statistical analysis to determine the optimal process conditions:The water is 12 times of the herbs weight, particle size is 20 mesh sieve, ultrasonic extraction time is 20min for the best extraction conditions.
2.In the experiment of Salvia miltiorrhiza polysaccharide against immunological liver injury in mice. The serum levels of ALT, AST in Salvia miltiorrhiza polysaccharides high-dose, middle-dose, low-dose group compared with the model group were significantly different(P=0.000,P=0.003,P=0.044). Control group and model group were significantly different(P=0.000);The levels of serum NO and liver tissue homogenate IL-1β, TNF-a in Salvia miltiorrhiza polysaccharide high-dose group and model group were significantly different(P=0.000), and Salvia miltiorrhiza polysaccharides middle dose group compared with model group, TNF-a, IL-1βwere significantly different(P=0.048), but NO was not significantly different(P=0.057). Salvia miltiorrhiza polysaccharide low dose group compared with model group TNF-a(P=0.187), NO(P=0.097)and IL-1β(P=0.145)were not significantly different. Bifendate Pills group compared with model group NO was significantly different (P=0.037), but TNF-a(P=0.064)and IL-1β(P=0.882)were not significantly different; organ coefficient of Salvia miltiorrhiza polysaccharide high dose group and model group were significant difference(P=0.000,P=0.005,P=0.018). Salvia miltiorrhiza polysaccharide middle dose group compared with model group, liver index(P=0.043), spleen index(P=0.016)were significantly different. Salvia miltiorrhiza polysaccharide low dose group and model group showed no significant difference(P=0.178,P=0.175, P=0.329). Except spleen index(P=0.059), control group compared with model group were significantly different(P=0.000,P=0.042); Compared with the normal group, model group pathological changes in liver was significantly. Compared with model group, Salvia miltiorrhiza polysaccharide high dose group(P=0.032) and control group(P=0.016) pathological changes in liver were significantly. Compared with model group, Salvia miltiorrhiza polysaccharide middle dose group(P=0.240) and Salvia miltiorrhiza polysaccharide low dose group(P=0.547) showed no significant pathological changes in liver.
3.In the experiment of Salvia miltiorrhiza polysaccharide on immunomodulatory mechanism, in the 10～200mg/l range of different doses of Salvia miltiorrhiza polysaccharides can significantly promote the proliferation of spleen cells, compared with the control group were significant difference(P=0.020,P=0.000), Salvia miltiorrhiza polysaccharides 200mg/l group showed higher activity; Experimental results showed that 100～200mg/l dose range Salvia miltiorrhiza polysaccharide group could significantly promote the proliferation of T lymphocytes, compared with the control group were significant difference (P=0.042,P=0.000), while mitogen ConA compared with the control group was significant difference(P=0.000). In the concentration of 50-200mg/l, Salvia miltiorrhiza polysaccharide group could significantly promote the proliferation of B lymphocytes,compared with-the-control group there was significant difference(P=0.033,P=0.000), while mitogen LPS and the control group were significant difference (P=0.000); 200mg/l concentrations of Salvia miltiorrhiza polysaccharides on mouse lymphocytes for 12h,24h,48h,72h, IL-2 levels increased,each time showed significant difference(P=0.000). IL-4 levels significantly increased, making comparisons between different time periods with a significant difference(P=0.005), but mildly compared with the levels of IL-2; Fluorescence quantitative PCR results showed that, after 200mg/l concentration of Salvia miltiorrhiza polysaccharides interfereing for 24h,48h,72h,IL-2 mRNA and IL-4 mRNA expression were differences; Compared with the control, IL-2 mRNA and IL-4mRNA expression increased after 72h, and the difference was statistical significance(P=0.022,P=0.036); IL-2 mRNA and IL-4 mRNA expression were no significant difference after 24h,48h(P=0.753,P=0.400,P=0.177,P=0.208). It was accounted that Salvia miltiorrhiza polysaccharides effectively increased IL-2 mRNA and IL-4 mRNA expression after interfereing for 72h; With the higher dose in 1～200mg/l range of Salvia miltiorrhiza polysaccharides, the ratio of CD4+, CD8+ gradual enhanced, showing a clear dose-dependent, while higher concentrations group compared with control group showed statistically significant differences(P=0.028,P=0.007,P=0.008,P=0.027,P=0.000).
Conclusion:
1.To determine the optimal extraction conditions of Salvia miltiorrhiza polysaccharides:The water was 12 times of the herbs weight, particle size was 20 mesh sieves, ultrasonic extraction time was 20 min for the best extraction conditions, and the extraction method had low energy consumption, high extraction rate, advantages of simple operation.
2.The results showed that Salvia miltiorrhiza polysaccharides had the role of anti-immune liver injury. This role displayed not only in direct hepatoprotection, but also in regulatory role of immune function in liver injury, through the immune function further played the role of hepatoprotection.
3.Salvia miltiorrhiza polysaccharides in vitro showed a stronger proliferation of lymphocytes in certain concentration,and also promoted expression and secretion of cytokines. Consistent with the results in vivo experiments to further clarify the Salvia miltiorrhiza polysaccharides had immune regulation function.
引文
[1]冯铃铃,周吉源.丹参的研究现状与应用前景[J].中国野生植物资源,2004；23(2)：4-7.
[2]罗献瑞主编.《实用中草药彩色图集第三册》,广东科技出版社,1994.
[3]Luo H W,Wu B J,Wu M Y,Yong Z G,Jin Y.Isolation and structure of danshenxinkun D[J].Acta pharmaceutica Sinica,1985;20(7):542-544.
[4]Feng B S,Li S R.Studies on the chemical components of Dan-shen (Salvia miltiorrhiza Bunge)[J].Acta pharmaceutica:Sinica,1980;15(8):489-494.
[5]Lee A R,Wu M Y,Chang W L,et al.Isolation and bioactivity of new tanshinones[J].Nat Prod,1987;50:157.
[6]Yang B J,Qian M K,Qin G W,Chen Z X,Studies on the active principles of Dan—Shen V Isolation and structures of przewaquinone A and prezewaquinone B[J].Acta pharmaceutica Sinica,1981;16(11):837-841.
[7]Michavila A,Tone M C,De L,Rodriguez B.Phytochemistrty,1986;25:1935.
[8]Li M,Zhang J S,Chen M Q.A novel dimeric diterpene from salvia prionitis[J].Nat Prod,2001;64:971.
[9]Onitsuka M,Fujiu M,Shinma N,Maruyama H B.New platelet aggregation inhibitors from Tan；/7Shen:radix of Salvia miltiorrhiza Bunge[J].Chemical & pharmaceutical bulletin,1983;31(5):1670-1675.
[10]陈发奎.常用中草药有效成分含量测定[M].北京.人民卫生出版社,1997；139.
[11]Chang H M,Cheng K P,Choang T F,et al.J Org Chem,1990;55,3537.
[12]Luo H,Wu B J,Wu M Y,et al.Pigments from salvia miltiorrhiza[J]. Phytochemitry,1985;24:815.
[13]Luo H W,Hu X J,Wang N,Ji J.Platelet aggregation inhibitors from Salvia miltiorrhiza Bunge[J].Acta pharmaceutica Sinica,1988;23(11):830-834.
[14]Chang H M,Choang T F,Chui K Y,et al.Novel constituents of the Chinese drug Danshen(Salvia miltiorrhiza Bunge):isolation of 6,7,8,9-tetrahydro-1,6,6-trimethylfuro[3,2-c]naphth[2,1-e]oxepine-10,12-dione and its derivatives,secondary metabolites produced by photooxidation reactions[J].J Chem Res,1990;4:114.
[15]Sairafianpour M,Christensen J,Staerk D,Budnik B A,Kharazmi A,Bagherzadeh K,Jaroszewski J W.Leishmanicidal,antiplasmodial and cytotoxic activity of novel diterpenoid 1,2-quinones from Perovskia abrotanoides:new source of tanshinones[J].J Nat Prod,2001;64(11):1398-1403.
[16]Yang B J,Huang X L,Hu Z B,Chen Z X.Studies on the chemical principles of Salvia trijuga Diels(Lahiatae)[J].Acta pharmaceutica Sinica,1982; 17(7): 517-520.
[17]Ulubelen A,Topcu G,Johansson C B.Norditerpenoids and diterpenoids from salvia multicaulis with antituberculous activity[J].Nat Prod,1997;60:1275.
[18]Lin H C,Ding H Y,Chang W L.Two new faty diterpenoids from salvia miltiorrhiza[J].Nat Prod,2001;64:648.
[19]Fang C N,Chang P L,Hsu T P.The antibacterial components of Dan-shen[J]. Acta Chimica Sinica,1976;34:197-209.
[20]Yang B J,Huang X L,Zhou Q R.The structures of four minor diterpenequinones przewaquinones C,D,E and F from the root of Salvia przewalskii Maxim val mandarinorum(Diels)Stib[J].Acta pharmaceutica Sinica,1984; 19(4):274-281.
[21]李家实.中药鉴定学.上海科学技术出版社,1996；169.
[22]Luo H W,Ji J,Wu M Y,et al.Tanshinlactone,a novel seco-abietanoid from salvia miltiorrhiza[J].Chem Pharm Bull,1986;34:3166.
[23]Kong D Y,Liu X J.Structure of dihydroisotanshinone I of danshen[J].Acta pharmaceutica Sinica,1984; 19(10):755-759.
[24]Chen Y Z,Xu S Y,Wang S S,et al.Studies on the constituents of Danshen[J]. Journal of Jinan University(Natural Science),1993;14(3):56-60.
[25]王潮临.丹参对外周血中性粒细胞和单核细胞的调整作用[J].广西医科大学学报,1997；14(3)：9-11.
[26]石乃玉,董华明,黄海金.丹参酮的药理及临床应用[J].中国医师杂志,2001；3(2)：150.
[27]罗厚蔚,韦苞洋.丹参酮类及有关化合物抑菌作用的构效关系[J].中国药科大学学报,2002；33(1)：6-12.
[28]朱雄翔,汤朝武,陈璧.烧伤早期及丹参对中性粒细胞化学发光的影响[J].第四军医大学学报,1999；20(10)：60-62.
[29]Ding M,Ye T X,Zhao G R,et al.A queous extract of Salvia miltiorrhiza attenuats increased endothelial permeability induced by tumor necrosis factor-a[J].Int Immunopharmacol,2005;5(11):1641-1651.
[30]Soung do Y,Rhee S H,Kim J S,et al.Peroxynitrite scavenging activity of lithospermate B from Salvia miltiorrhiza[J].J Pharm Pharmacol,2003;55 (10):1427-1432.
[31]吴呆,何招兵,吴汉斌.丹参酮的药理作用研究进展[J].现代中西医结合杂志,2005；14(10)：1382-1385.
[32]袁淑兰,王修杰.丹参酮的抗肿瘤作用及其机理研究[J].癌症,2003；22(12)：1363-1366.
[33]Yuan S L,Wang X J,Wei Y Q.Anticancer effect of tanshinone and its mechanisms[J].Chin J Cancer,2003;22(12):1363-1366.
[34]Liu J, Yang C F, Wasser S,Shen H M, Tan C E, Ong C N. Protection of salvia miltiorrhiza against aflatoxin-B1-induced hepatocarcinogenesis in Fischer 344 rats dual mechanisms involved[J].Life Sci,2001;69(3):309-326.
[35]袁淑兰等.丹参酮体外诱导分化作用及其机制的研究[J].癌症,1996；15(6)：408.
[36]黄韧敏等.丹参酮诱导HL-60细胞分化及调亡的流式细胞术分析[J].中国肿瘤临床,1997；24(7)：500.
[37]Chen X G, Li Y, Yan C H, Li L N, Han R.Cancer chemopreventive activities of S-3-1, a synthetic derivative of danshinone[J].J Asian Nat Prod Res,2001;3 (1):63-75.
[38]常英姿,梁殿权,王孝铭等.丹参素对氧自由基所致大鼠心肌线粒体H+-ATP酶损伤的保护作用[J].中国病理生理杂志,1991；1(5)：449.
[39]刘启功.丹参注射液对心肌梗塞犬血流变学的观察[J].实用中西医结合杂志,1998；11：605.
[40]杨卫东,朱鸿良.丹参的氧自由基清除作用[J].中国药理学通报,1990；6(2)：118.
[41]Chang P N,Mao J C, Huang S H, et al. Analysis of cardioprotective effects using purified Salvia miltiorrh iza extract on isolated rat hearts[J].J Pharmacol Sci,2006;101(3):245-249.
[42]史举彤,杨文浩,陈长清.丹参素对体外循环中血细胞的影响[J].山东医科大学学报,1991；29(3)：237-239.
[43]赵国胜,杨会杰.川芎嗪和丹参对大鼠心脏氧反常保护作用的比较[J].天津医药,1996；24(5)：289.
[44]陶月玉,严玉兰.丹参对急性缺血后再灌注心肌磷脂肌醇系统变化的影响[J].中国病理生理杂志,1997；13(3)：267.
[45]刘军,匡培根,吴卫平等.大鼠脑缺血再灌注区小胶质细胞反应及丹参的影响[J].中华老年心脑血管病杂志,2002；4(2)：127-129.
[46]Imanshahidi M,Hosseinzadeh H.The pharmacological effects of Salvia species on the central nervous system[J].Phytother Res,2006;20(6):427-437.
[47]Lao C J,Lin J G, Kuo J S, et al. Effect of Salvia miltiorrhiza bunge on cerebral infarct in ischem ia-reperfusion injured rats[J].Am J Chin Med,2003;31 (2)：191-200.
[48]陈刚,刘润侠,陈喜玲.丹参对β2-GPI诱导的自身免疫小鼠aCL产生的影响[J].西安医科大学学报,2001；22(5)：429-431.
[49]许峰,陆伯刚,姚智,等.丹参及丹参素对内毒素刺激下肝巨噬分泌细胞因子影响的动态观察[J].中国危重病急教医学,1996；8(5)：262-264.
[50]JIANG Kai-yu, RUAN Cheng-geng, GU Zhen-lun. Effects of tanshonone ⅡA sulfonate on adhesion molecule expression of endothe-lial cells and platelets in vitro[J].Acta Pharmacological Sinica,1998;19(1):157-158.
[51]蔡建辉,刘维永,易定华等.丹参对兔严重钝性心损伤的治疗作用[J].中国危重病急救医学,1997；9(3)：129-131.
[52]苗戎,陈静,高岚月丹参对肺泡巨噬细胞分泌功能的影响[J].天津中医学院学报,1998；17(1)：389-390.
[53]马雪梅,王宝恩,尤红.丹参对肝星状细胞增殖和凋亡的实验研究[J].中国中医药信息杂志,2000；7(7)：25-26.
[54]周展超,郑家润,徐文严.氧氟沙星及丹参酮Ⅱa对白细胞趋化性的影响[J].中国医学科学院学报,1997；19(3)：232-233.
[55]戚心广.丹参、赤芍对实验性肝损伤肝细胞保护作用的机理研究[J].中西医结合杂志,1991；11(2)：102.
[56]秦嵩,孙备.丹参对肝脏缺血-再灌注损伤的防护作用[J].中医药学报,2001；29(6)：37-38.
[57]沈吉云,燕忠生.丹参保护肝纤化药理作用及应用进展[J].中西医结合肝病杂志,1998；8(2)：124.
[58]高玉芝,王灵芝,唐冀雪.丹参酮的性激素样作用[J].中国医学科学院学报,1980；2：189.
[59]严仲瑜.丹参对胃粘膜屏障的作用[J].中华外科杂志,1990；28(5)：298.
[60]董静,罗桂林,苗维纳.丹参对实验性肺纤维化小鼠病理变化和核因子-KB表达的影响[J].中国药理学通报,2003；19(12)：1428.
[61]吴山,盛延良,蒋志学.丹参对肺水肿大鼠白细胞介素-8和内皮素的影响[J].辽宁中医杂志,2003；30(10)：796.
[1]Karlyshev A V,Linton D,Gregson N A,Wren B W A,Novel paralogous gene family involved in phase-variable flagella-mediated motility in Campylobacter jejuni[J].Microbiology,2002;148(2):473-480.
[2]方积年,王顺春.香菇多糖的研究进展[J].中国药学杂志,1997；32(6)：332-337.
[3]Zheng R,Jie S,Hanchuan D,et al.Characterization and immunomodulating activities of polysaccharide from Lentinus edodes [J].Int Immunopharmacol, 2005;5(5):811-820.
[4]Cheng J J, Huang N K, Chang T T,et al.Study for anti-angiogenic activities of polysaccharides isolated from Antrodia cinnamomea in endothelial [J].Cells Life Sci,2005;76(26):3029-3042.
[5]Fisher M,Yang L X.Anticancer effects and mechanisms of polysaccharide-K (PSK):implications of cancer immunotherapy[J].Anticancer Res,2002;22(3): 1737-1754.
[6]Kobayashi H,Yoshida R,Kanada Y,et al.Suppressing effects of daily oral supplementation of beta-glucan extracted from Agaricus blazei Murill on spontaneous and peritoneal disseminated metastasis in mouse model[J].J Cancer Res Clin Oncol,2005;131(8):527-538.
[7]Popov S V, Popova G Y, Nikolaeva S Y, et al. Immunostimulating activity of pectic polysaccharide from Bergenia crassifolia (L.) Fritsch[J].Phytother Res, 2005;19(12):1052-1056.
[8]Song J Y, Akhalaia M, Platonov A, et al.Effects of polysaccharide ginsan from Panax ginseng on liver function[J].Arch Pharm Res,2004;27(5):531-538.
[9]Zhang M,Chen H,Huang J.Effect of lycium barbarumpolysaccharide on human hepatoma QGY7703 cells:inhibition of proliferation and induction of apoptosis[J]. Life Sci,2005;76(18):2115-2124.
[10]Kim G Y,Choi G S,Lee S H,et al.Acidic polysaccharide isolated from Phellinus linteus enhances through the up-regulation of nitric oxide and tumor necrosis factor-alpha from peritoneal macrophages[J].J Ethnopharmacol,2004;95(1): 69-76.
[11]Kodama N,Murata Y,Nanba H,et al.Administration of a polysaccharide from Grifola f rondosa stimulates immune function of normal mice[J].J Med Food, 2004;7(2):141-145.
[12]Lin Z B, Zhang H N.Antitumor and immunoregulatory activities of Ganoderma lucidum and its possible mechanisms [J].Acta Pharmacol Sin,2004;25(11): 1387-1395.
[13]Bao X F,Zhen Y,Ruan L,et al. Purification, characterization, and modification of T lymphocyte-stimulating polysaccharide from spores of Ganoderma lucidum[J].Chem Pharm Bull (Tokyo),2002;50(5):623-629.
[14]Lin Z B, Zhang H N.Anti-tumor and immunoregulatory activities of Ganoderma lucidum and its possible mechanisms[J].Acta Pharmacol Sin,2004; 25(11):1387-1395.
[15]Schepetkin I A,Quinn M T.Botanical polysaccharides. Macrophage immunomodulation and therapeutic potential[J].Int Immunopharmacol 2006; 6:317-333.
[16]李明春,梁东升,许自明等.灵芝多糖对小鼠腹腔巨噬细胞蛋白激酶A活性的影响[J].中草药,2000；31(5)：353-355.
[17]KM G Y,OH Y H,PARK Y M. Acidic polysaeeharide isolated from Phellinus induces nitric oxide mediated tumorieidal activity of macrophages through protein tyrosine kinase and protein kinase C[J].Bioehem Biophys Res Commun,2003;309 (2):399-407.
[18]陈伟,林新华,陈俊等.库拉索芦荟多糖对小鼠腹腔巨噬细胞的体外激活作用[J].中国药学杂志,2005；40(1)：34-37.
[19]田庚元,冯宇澄,林颖.植物多糖的研究进展[J].中国中药杂志,1995；20(7)：441.
[20]刘彦平,李积东.枸杞多糖对小鼠NK细胞和白细胞活性的免疫调节作用[J].青海医学院学报,2001；22(1)：1-2.
[21]刘彦平,毛辉青,李萍等.枸杞多糖对小鼠T淋巴细胞亚群和淋巴细胞转化作用的研究[J].青海医学院学报,2000；21(4)：4-5.
[22]郑应馨,徐恒卫.具有抗肿瘤活性的多糖及其作用机理研究概况[J].中国药师,2003；6(6)：368-369.
[23]周勇,张丽,赵高原等.仙茅多糖对小鼠免疫功能调节作用实验研究[J].上海 免疫学杂志,1996；16(5)：338.
[24]Zhu Y, Pettolino F, Mau S L. Immunoactive polysaccharide-rich fractions from Panax notoginseng[J].Planta Med,2006;72(13):1193-1199.
[25]Yamada H, Hirano M.Partial structure of an anti-ulcer polysaccharide from the roots of Bupleurum falcataum[J].Carbohydr Res,1991;219:173-192.
[26]Xu H,Zhang Y,Zhang J,et al.Isolation and characterization of an anti-complementary polysaccharide D3-S1 from the roots of Bupleurum smithii[J].Int Immunopharmacol,2007;7(2):175-182.
[27]Olafsdottir E S, Omarsdottir S, Paulsen B S, et al. Immunologically active 6-branched (1→3)-beta-glucan from the lichen Thamnolia vermicularis var subuliformis [J].Phytomedicine,2003;10(4):318-324.
[28]Schepetkin I A,Quinn M T.Botanical polysaccharides Macrophage immunomodulation and therapeutic potential [J].Int Immunopharmacol 2006;6: 317-333.
[29]Liu F,Fung M C,Ooi V E C,et al. Induction in the mouse of gene expression of immunomodulating cytokines by Mushroom polysaccharide-protein complexes [J].Life Science,1996;58(21):1795-1803.
[30]Liu F,Ooi V E C,Fung M C,et al.Analysis of immunomodulating cytokine mRNAs in the mouse induced by Mushroom polysaccharides[J].Life Science, 1999;64(12):1005-1011.
[31]Rout D,Mondal S,Chakraborty I,et al. Chemical-analysis of a new (1→3) 2,(1→6)-branched glucan from an edible mushroom, Pleurotus florida[J]. Carbohydr Res,2005;340(16):2533-2539.
[32]Kanekiyo K, Lee J B.Isolation of an antiviral polysaccharide, nostoflan, from a terrestrial cyanobacterium,Nostoc flagelliforme[J].J Nat Prod,2005;68(7): 1037-1041.
[33]Kimura Y,Sumiyoshi M,Suzuki T,et al. Antitumor and antimetastatic activity of a novel water-soluble low molecular weight beta-1,3-D-glucan (branch beta-1,6) isolated from Aureobasidium pullulans 1A1 strain black yeast [J]. Anticancer Res,2006;26(6B):4131-4141.
[34]Chang R.Bioactive polysaccharides from traditional Chinese medicine herbs as anticancer adjuvants[J].Altern Complement Med,2002;8(5):559-565.
[35]Lee K Y,Lee M H,Chang I Y,et al. Macrophage activation by polysaccharide fraction isolated from Salicornia herbacea[J].J Ethnopharmacol,2006; 103(3): 372-378.
[36]Koyanagi S,Tanigawa N,Nakagawa H,et al.Oversulfation of fucoidan enhances its anti-angiogenic and antitumor activities[J].Biochemi Pharmacol,2003;65: 173-179.
[37]Hatanaka k,Song S C,Maruyama A,et al.A new synthetichypoglycaemic polysacchide[J].Biochem Biophys Res Commun,1992;188(1):16.
[38]Kim K H,Lee Y S,Jung I S,et al.Acidic polysaccharide from Panax ginseng, ginsan, induces Thl cell and macrophage cytokines and generates LAK cells in synergy with rIL-2[J].Planta Med,1998;64(2):110-115.
[39]Kito T,Sobue S.Structural features and hypoglycemic activities of two polysaccharides from a hot-water extract of Agrocybe cylindracea[J]. Carbohydr Res,1994;252:81-87.
[40]刘成梅,付贵明,涂宗财等.百合多糖降血糖功能研究[J].食品科学,2002；23(6)：113-114.
[41]Huang Cheng,Chen Qun li,Sun Jiang tao,et al.Protective effect of lycium barbarum ploysaccharde and its compound recipe on pancreatic islet functiong in rats with streptocototocin induced diabetes mellitus[J].Chinese Journal of Clinical Rehabilitation,2006;10(23):173-175.
[42]Itoh T,Kita N,Kurokawa Y,et al.Suppressive effect of a hot water extract of adzuki beans (Vigna angularis) on hyperglycemia after sucrose loading in mice and diabetic rats[J].Biosci Biotechnol Biochem,2004;68 (12):2421-2426.
[43]Zhang J, Huang Y, Hou T. Hypoglycaemic effect of Artemisia sphaerocephala Krasch seed polysaccharide in alloxan-induced diabetic rats[J].Swiss Med Wkly,2006;136(33-34):529-532.
[44]Shirwaikar A,Rajendran K,Barik R,et al. Effect of aqueous bark extract of Garuga pinnata Roxb. in streptozotocin-nicotinamide induced type-II diabetes mellitus[J].J Ethnopharmacol,2006;19,107(2):285-290.
[45]Ito M, Baba M, Sato A, et al.Inhibitory effect of dextran sulfate and heparin on the replication of human immunodeficiency virus(HIV) in vitro[J].Antiviral Res,1987;7:361-367.
[46]Witvrouw M, Schols D, Andrei G, et al. Newpolyacetal polysulfate active against HIV and other enveloped viruses[J].Antiviral Chem Chemother,1992;3: 351-355.
[47]Talyshinsky MM, Souprun YY, Huleihel MM, et al. Anti-viral activity of red microalgal polysaccharides against retroviruses[J].Cancer Cell Int,2002;2(1):8.
[48]Gerencer M,Turecek P L,Kistner O,et al. In vitro and in vivo anti-retroviral activity of the substance purified from the aqueous extract of Chelidonium majus[J].Antiviral Res,2006;72(2):153-156.
[49]Rusnati M, Urbinati C, Caputo A, et al. Pentosan Polysulfate as an inhibitor of Extracellular HIV-1 Tat[J].Biol Chem,2001;276:22420-22425.
[50]Zhu W,Chiu L C,Ooi V E,et al.Antiviral property and mechanisms of a sulphated polysaccharide from the brown alga Sargassum patens against Herpes simplex virus type 1[J].Phytomedicine,2006;13 (9-10):695-701.
[51]Lee I H, Huang R L, Chen C T, et al. Antrodia camphorate polysaccharides exhibit anti-hepatitis B virus effects[J].FEMS Microbiol Lett,2002;209:63-67.
[52]Naesens L, Bonnafous P, Agut H, et al. Antiviral activity of diverse classes of broad-acting agents and natural compounds in HHV-6-infected lymphoblasts[J].J Clin Virol,2006;37 Suppl 1:S69-75.
[53]Ghosh P, Adhikari U, Ghosal PK, et al.In vitro anti-herpetic activity of sulfated polysaccharide fractions from Caulerpa racemosa[J].Phytochemistry,2004; 65(23):3151-3157.
[54]Lee J B,Hayashi K,Maeda M,et al.Antiherpetic activities of sulfated polysaccharides from green algae [J].Planta Med,2004;70 (9):813-817.
[55]Lee J H, Shim J S, Lee J S, et al.Pectin-like acidic polysaccharide from Panax ginseng with selective antiadhesive activity against pathogenic bacteria[J]. Carbohydr Res,2006;341(9):1154-1163.
[56]Pereda-Miranda R,Kaatz G W,Gibbons S,et al.Polyacylated oligosaccharides from medicinal Mexican morning glory species as antibacterials and inhibitors of multidrug resistance in Staphylococcus aureus[J].J Nat Prod,2006;69(3): 406-409.
[57]王雪萍.植物多糖研究进展[J].粮食与油脂,2005；8：8-10.
[58]张宪党,马弛,张群等.植物多糖抗辐射损伤作用研究进展[J].中国辐射卫生,2003；12(2)：122-123.
[59]Chen Y J,Yang Y C.A novel polysaccharide of black soybean promotes myelopoiesis and reconstitutes bone marrow after 5-flurouracil and irradiation-induced myelosuppression[J]. Life Sci,2005;77(4):400-413.
[60]吴华振.植物多糖的药理作用及应用进展[J].实用医技杂志,2005；12(7)：1803-1804.
[61]Li S P,Zhao K J,Ji Z N,et al. A polysaccharide isolated from Cordyceps sinensis, a traditional Chinese medicine, protects PC 12 cells against hydrogen peroxide-induced injury[J].Life Sciences,2003;73:2503-2513.
[62]Yu M S,Lai S W,Lin K F.Characterization of polysaccharides from the flowers of Nerium indicum and their neuroprotective effects[J].Int J Mol Med,2004; 14(5):917-924.
[63]Sun C,Shan C Y,Gao X D,et al.Protection of PC12 cells from hydrogen peroxide-induced injury by EPS2, an exopolysaccharide from a marine filamentous fungus Keissleriella sp. YS4108J[J]:Biotechnol,2005; 115(2)-137-144.
[64]徐德生,冯怡,林晓.麦冬多糖MDG-1的分离纯化和结构分析[J].药学学报,2005；40(7)：636-639.
[65]Rocha H A, Moraes F A, Trindade E S,et al. Structural and hemostatic activities of a sulfated galactofucan from the brown alga Spatoglossum schroederi. An ideal antithrombotic agent[J].J Biol Chem,2005;280(50): 41278-41288.
[66]杨明,王晓娟,孙红等.树舌多糖抗溃疡作用[J].中药药理与临床, 2004；20(6)：11-13.
[67]傅继华,温涛,徐琛.芦荟多糖对动物实验性胃溃疡的影响[J].中草药,2006；37(6)：894-897.
[1]蔡幼清.丹参根中的精制多糖对大鼠实验性肾病的作用[J].国外医学,中医中药分,1995；17(4)：52-53.
[2]田庚元,冯宇澄,林颖.植物多糖的研究进展[J].中国中药杂志,1995；20(7)：441-441.
[1]Kobayashi S,Nishihira J,Watanabe S, et al.Prevention of lethal acute hepatic failure by antimacrophage migration inhibitory factor antibody in mice treated with bacille Calmette-Guerin and lipopolysaccharide[J].Hepatology,1999;29: 752-759.
[2]Yoneyama H, Harada A, Imai T, et al. Pivotal role of TARC, a CC chemokine, in bacteria-induced fulminant hepatic failure in mice[J].Clin Invest,1998; 102: 1933-1941.
[3]S Kobayashi, J Nishihira, S Watanabe, S Todo. Prevention of lethal acute hepatic failure by antimacrophage migration inhibitory factor antibody in mice treated with bacille Calmette-Guerin and lipopolysaccharide[J].Hepatology, 1999;29:1752-1759.
[4]Wang Y Z, Hu S S.A novel nitric oxide biosensor based on electropolymerization poly (toluidine blue) film electrode and its application to nitric oxide released in liver homogenate [J].Biosens Bioelectron,2006; 22:10-17.
[5]张定凤.乙型肝炎的发病机理及临床[M].重庆出版社,1992：1.
[6]Y H Zou, Y Yang, J Li, W P Li, Q Wu. Prevention of hepatic injury by a traditional Chinese formulation, BJ-JN,in mice treated with Bacille-Calmette-Guerin and lipopolysaccharide[J].J Ethnopharmacol,2006; 107:442-448.
[7]G S Wang, G T Liu. Role of nitric oxide in immunological liver damage in mice[J].Biochem Parmacol,1995;49:1277-1281.
[8]J Nagakawa,I Hishinuma,K Hirota, K Miyamoto, T Yamanaka;,K Tsukidate, K Katayama, I Yamatsu. Involvement of tumor necrosis factor-alpha in the pathogenesis of activated macrophage-mediated hepatitis in mice [J]. Gastroenterology,1990;99:758-765.
[9]H Tsuji, A Harada, N Mukaida, Y Nakanuma, H Bluethmann, S Kaneko, K Yamakawa, S I Nakamura, K I Kobayashi, K Matsushima. Tumor necrosis factor receptor p55 is essential for intrahepatic granuloma formation and hepatocellular apoptosis in a murine model of bacterium-induced fulminant hepatitis[J].Infect Immun,1997; 65:1892-1898.
[10]H Tsutsui, K Matsui, N Kawada, Y Hyodo, N Hayashi, H Okamura, K Higashino, K Nakanishi. IL-18 accounts for both TNF-alpha and Fas ligand-mediated hepatotoxic pathways in endotoxin-induced liver injury in mice[J].J Immunol,1997;159:3961-3967.
[11]N Fujioka, N Mukaida, A Harada, M Akiyama, T Kasahara, K Kuno, A Ooi, M Mai, K Matsushima. Preparation of specific antibodies against murine IL-1ra and the establishment of IL-1ra as an endogenous regulator of bacteria-induced fulminant hepatitis in mice[J]. J Leukoc Biol,1995;58:90-98.
[12]P Liu, H Ohnishi, H Moriwaki,Y Muto.Enhanced tumor necrosis factor and interleukin-1 in an experimental model of massive liver cell necrosis/fatal hepatitis in mice[J].Gastroenterol Jpn,1990;25:339-342.
[13]R Guler, M L Olleros, D Vesin, R Parapanov, C Vesin, S Kantengwa, L Rubbia-Brandt, N Mensi, A Angelillo-Scherrer, E Martinez-Soria, F Tacchini-Cottier, I Garcia. Inhibition of inducible nitric oxide synthase protects against liver injury induced by mycobacterial infection and endotoxins[J].J Hepatol,2004;41:773-781.
[14]K Matsui, T Yoshimoto, H Tsutsui, Y Hyodo, N Hayashi, K Hiroishi, N Kawada, H Okamura, K Nakanishi, K. Higashino. Propionibacterium acnes treatment diminishes CD4+ NK1.1+ T cells but induces type I T cells in the liver by induction of IL-12 and IL-18 production from Kupffer cells[J].J Immunol,1997; 159:97-106.
[15]Y Tanaka, A Takahashi, K Watanabe, K Takayama, T Yahata,S Habu,T Nishimura. A pivotal role of IL-12 in Thl-dependent mouse liver injury[J].Int Immunol,1996;8:569-576.
[16]杨天健,魏怀玲,刘耕陶.16种药物对卡介苗加脂多糖引起小鼠免疫性肝损伤的作用[J].中国药理学报,1997；18(2)：185-188.
[17]张安平,沈玉先,魏伟等.丹皮总甙对小鼠免疫性肝损伤的保护作用[J].中国药理学报,1997；13(3)：435-437.
[18]D L Laskin,M Rodriguez del Valle,D E Heck,S M Hwang,S T Ohnishi,S K Durham,N L Goller,J D Laskin.Hepatic nitric oxide production following acute endotoxemia in rats is mediated by increased inducible nitric oxide synthase gene expression[J].Hepatology,1995;22:223-234.
[19]R Guler, M L. Olleros, D Vesin, R Parapanov, C Vesin, S Kantengwa, L Rubbia-Brandt, N Mensi, A Angelillo-Scherrer, E Martinez-Soria, F Tacchini-Cottier, I Garcia. Inhibition of inducible nitric oxide synthase protects against liver injury induced by mycobacterial infection and endotoxins[J].J Hepatol,2004; 41:773-781.
[20]G Sass, K Koerber, R Bang, H Guehring, G Tiegs. Inducible nitric oxide synthase is critical for immune-mediated liver injury in mice[J].J Clin Invest, 2001; 107:439-447.
[21]M I Luster, P P Simeonova, R M Gallucci, J M Matheson, B Yucesoy. Immunotoxicology:role of inflammation in chemical-induced hepatotoxicity[J]. Int J Immunopharmacol,2000;22:1143-1147.
[22]Y Muto, K T Nouri-Aria, A Meager, G J Alexander, A L Eddleston, R Williams. Enhanced tumor necrosis factor and interleukin-1 in fulminant hepatic failure[J]. Lancet,1988;2:72-74.
[23]M de la Mata, A Meager,N Rolando, H M Daniels,K T Nouri-Aria,A K Goka,A L Eddleston,G J Alexander,R Williams.Tumour necrosis factor production in fulminant hepatic failure:relation to aetiology and superimposed microbial infection[J].Clin Exp Immunol,1990;82:479-484.
[24]H Zylberberg, A C Rimaniol, S Pol, A Masson, D De Groote, P Berthelot, J F Bach, C Brechot, F.Zavala.Soluble tumor necrosis factor receptors in chronic hepatitis C:a correlation with histological fibrosis and activity[J].J Hepatol, 1999; 30:185-191.
[25]M Yin,M D Wheeler, H Kono, B U Bradford, R M Gallucci, MI Luster, R G Thurman.Essential role of tumor necrosis factor alpha in alcohol-induced liver injury in mice[J].Gastroenterology,1999;117:942-952.
[26]H A Rudiger, P A Clavien.Tumor necrosis factor alpha, but not Fas, mediates hepatocellular apoptosis in the murine ischemic liver[J].Gastroenterology,2002; 122:202-210.
[27]J Muntane, F J Rodriguez,O Segado, A Quintero,J M Lozano,E Siendones, C A Pedraza,M Delgado,F O'Valle,R Garcia,J L Montero,M De La Mata, G Mino.TNF-alpha dependent production of inducible nitric oxide is involved in PGE(1) protection against acute liver injury[J]. Gut,2000;47:553-562.
[28]E P Nadler, E C Dickinson, D Beer-Stolz, S M Alber, S C Watkins, D W Pratt, H R Ford.Scavenging nitric oxide reduces hepatocellular injury after endotoxin challenge, Am J Physiol[J].Gastrointest Liver Physiol,2001;281:G173-G181.
[29]P P Simeonova, R M Gallucci, T Hulderman, R Wilson, C Kommineni, M Rao, M I Luster.The role of tumor necrosis factor-alpha in liver toxicity, inflammation and fibrosis induced by carbon tetrachloride[J].Toxicol Appl Pharmacol,2001;177:112-120.
[30]N Tulek, S K Saglam, M Saglam, R Turkyilmaz, M Yildiz. Soluble interleukin-2 receptor and interleukin-10 levels in patients with chronic hepatitis B infection[J].Hepatogastroenterology,2000;47:828-831.
[31]C Basak, S K Pathak, A Bhattacharyya, D Mandal, S Pathak, M Kundu. NF-kappaB-and C/EBPbeta-driven interleukin-1beta gene expression and PAK1-mediated caspase-1 activation play essential roles in interleukin-lbeta release from Helicobacter pylori lipopolysaccharide-stimulated macrophages[J]. J Biol Chem,2005; 280:4279-4288.
[32]C A Dinarello.Interleukin-1 and interleukin-1 antagonism[J].Blood,1991;77: 1627-1652.
[33]A K Nussler,T R Billiar. Inflammation, immunoregulation, and inducible nitric oxide synthase[J].J Leukoc Biol,1993;54:171-178.
[34]J N Dhuley, S R. Naik Moderate protective effect of 6-MFA, a microbial metabolite obtained from Aspergillus ochraceus on immunological liver injury in mice[J].Comp Immunol Microbiol Infect Dis,1999;22:15-25.
[1]Heid C A, Stevens J, Livak K J, et al. Real time quantitative PCR. Genome Res[J].1996;6(10):986-994.
[2]Winer J, Jung C K, Shackel I, et al. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro[J].Anal Biochem,1999;270(1): 41-49.
[3]Kaltenboeck B, Wang C. Advances in real-time PCR:application to clinical laboratory diagnostics[J].Adv Clin Chem,2005;40:219-259.
[4]Valasek M A, Repa J J. The power of real-time PCR[J].Adv Physiol Educ, 2005;29(3):151-159.
[5]Espy M J, Uhl J R, Sloan L M, et al.Real-time PCR in clinical microbiology: applications for routine laboratory testing[J].Clin Microbiol Rev,2006; 19(1):165-256.
[6]Wong M L, Medrano J F. Real-time PCR for mRNA quantitation. Biotechniques[J].2005;39(1):75-85.
[7]Stahlberg A, Zoric N, Aman P, et al. Quantitative real-time PCR for cancer detection:the lymphoma case[J].Expert Rev Mol Diagn,2005;5(2):221-230.
[8]Bubner B, Baldwin I T. Use of real-time PCR for determining copy number and zygosity in transgenic plants[J].Plant Cell Rep,2004;23(5):263-271.
[9]Mackay I M. Real-time PCR in the microbiology laboratory[J].Clin Microbiol Infect,2004; 10(3):190-212.
[10]Gomes-Ruiz A C, Nascimento R T, de Paula SO, et al. SYBR green and TaqMan real-time PCR assays are equivalent for the diagnosis of dengue virus type 3 infections[J].J Med Virol,2006;78(6):760-763.
[11]Liu Y, Cai X, Zhang X, et al. Real time PCR using TaqMan and SYBR Green for detection of Enterobacter sakazakii in infant formula[J].J Microbiol Methods,2006;65(1):21-31.
[12]Suzuki N, Yoshida A, Nakano Y. Quantitative analysis of multi-species oral biofilms by TaqMan Real-Time PCR[J].Clin Med Res,2005;3(3):176-185.
[13]Rousselon N, Delgenes J P, Godon J J. A new real time PCR (TaqMan PCR) system for detection of the16S rDNA gene associated with fecal bacteria[J].J Microbiol Methods,2004;59(1):15-22.
[14]Campsall P A, Au N H, Prendiville J S, et al. Detection and genotyping of varicella-zoster virus by TaqMan allelic discrimination real-time PCR[J].J Clin Microbiol,2004;42(4):1409-1413.
[15]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[J].Methods, 2001;25(4):402-408.
[16]Magram J, Connaughton S E, Warrier R R, et al. IL-12 deficient mice are defective in IFN gamma production and type 1 cytokine responses[J]. Immunity,1996; 4(5):471-481.
[17]Kaplan M H, Sun Y L, Hoey T, et al. Impaired IL-12 responses and enhanced development of Th2 cells in Stat42-deficient mice[J].Nature,1996; 382(657): 174-177.
[2]罗献瑞主编.《实用中草药彩色图集第三册》,广东科技出版社,1994.
[3]Luo H W,Wu B J,Wu M Y,Yong Z G,Jin Y.Isolation and structure of danshenxinkun D[J].Acta pharmaceutica Sinica,1985;20(7):542-544.
[4]Feng B S,Li S R.Studies on the chemical components of Dan-shen (Salvia miltiorrhiza Bunge)[J].Acta pharmaceutica:Sinica,1980;15(8):489-494.
[5]Lee A R,Wu M Y,Chang W L,et al.Isolation and bioactivity of new tanshinones[J].Nat Prod,1987;50:157.
[6]Yang B J,Qian M K,Qin G W,Chen Z X,Studies on the active principles of Dan—Shen V Isolation and structures of przewaquinone A and prezewaquinone B[J].Acta pharmaceutica Sinica,1981;16(11):837-841.
[7]Michavila A,Tone M C,De L,Rodriguez B.Phytochemistrty,1986;25:1935.
[8]Li M,Zhang J S,Chen M Q.A novel dimeric diterpene from salvia prionitis[J].Nat Prod,2001;64:971.
[9]Onitsuka M,Fujiu M,Shinma N,Maruyama H B.New platelet aggregation inhibitors from Tan；/7Shen:radix of Salvia miltiorrhiza Bunge[J].Chemical & pharmaceutical bulletin,1983;31(5):1670-1675.
[10]陈发奎.常用中草药有效成分含量测定[M].北京.人民卫生出版社,1997；139.
[11]Chang H M,Cheng K P,Choang T F,et al.J Org Chem,1990;55,3537.
[12]Luo H,Wu B J,Wu M Y,et al.Pigments from salvia miltiorrhiza[J]. Phytochemitry,1985;24:815.
[13]Luo H W,Hu X J,Wang N,Ji J.Platelet aggregation inhibitors from Salvia miltiorrhiza Bunge[J].Acta pharmaceutica Sinica,1988;23(11):830-834.
[14]Chang H M,Choang T F,Chui K Y,et al.Novel constituents of the Chinese drug Danshen(Salvia miltiorrhiza Bunge):isolation of 6,7,8,9-tetrahydro-1,6,6-trimethylfuro[3,2-c]naphth[2,1-e]oxepine-10,12-dione and its derivatives,secondary metabolites produced by photooxidation reactions[J].J Chem Res,1990;4:114.
[15]Sairafianpour M,Christensen J,Staerk D,Budnik B A,Kharazmi A,Bagherzadeh K,Jaroszewski J W.Leishmanicidal,antiplasmodial and cytotoxic activity of novel diterpenoid 1,2-quinones from Perovskia abrotanoides:new source of tanshinones[J].J Nat Prod,2001;64(11):1398-1403.
[16]Yang B J,Huang X L,Hu Z B,Chen Z X.Studies on the chemical principles of Salvia trijuga Diels(Lahiatae)[J].Acta pharmaceutica Sinica,1982; 17(7): 517-520.
[17]Ulubelen A,Topcu G,Johansson C B.Norditerpenoids and diterpenoids from salvia multicaulis with antituberculous activity[J].Nat Prod,1997;60:1275.
[18]Lin H C,Ding H Y,Chang W L.Two new faty diterpenoids from salvia miltiorrhiza[J].Nat Prod,2001;64:648.
[19]Fang C N,Chang P L,Hsu T P.The antibacterial components of Dan-shen[J]. Acta Chimica Sinica,1976;34:197-209.
[20]Yang B J,Huang X L,Zhou Q R.The structures of four minor diterpenequinones przewaquinones C,D,E and F from the root of Salvia przewalskii Maxim val mandarinorum(Diels)Stib[J].Acta pharmaceutica Sinica,1984; 19(4):274-281.
[21]李家实.中药鉴定学.上海科学技术出版社,1996；169.
[22]Luo H W,Ji J,Wu M Y,et al.Tanshinlactone,a novel seco-abietanoid from salvia miltiorrhiza[J].Chem Pharm Bull,1986;34:3166.
[23]Kong D Y,Liu X J.Structure of dihydroisotanshinone I of danshen[J].Acta pharmaceutica Sinica,1984; 19(10):755-759.
[24]Chen Y Z,Xu S Y,Wang S S,et al.Studies on the constituents of Danshen[J]. Journal of Jinan University(Natural Science),1993;14(3):56-60.
[25]王潮临.丹参对外周血中性粒细胞和单核细胞的调整作用[J].广西医科大学学报,1997；14(3)：9-11.
[26]石乃玉,董华明,黄海金.丹参酮的药理及临床应用[J].中国医师杂志,2001；3(2)：150.
[27]罗厚蔚,韦苞洋.丹参酮类及有关化合物抑菌作用的构效关系[J].中国药科大学学报,2002；33(1)：6-12.
[28]朱雄翔,汤朝武,陈璧.烧伤早期及丹参对中性粒细胞化学发光的影响[J].第四军医大学学报,1999；20(10)：60-62.
[29]Ding M,Ye T X,Zhao G R,et al.A queous extract of Salvia miltiorrhiza attenuats increased endothelial permeability induced by tumor necrosis factor-a[J].Int Immunopharmacol,2005;5(11):1641-1651.
[30]Soung do Y,Rhee S H,Kim J S,et al.Peroxynitrite scavenging activity of lithospermate B from Salvia miltiorrhiza[J].J Pharm Pharmacol,2003;55 (10):1427-1432.
[31]吴呆,何招兵,吴汉斌.丹参酮的药理作用研究进展[J].现代中西医结合杂志,2005；14(10)：1382-1385.
[32]袁淑兰,王修杰.丹参酮的抗肿瘤作用及其机理研究[J].癌症,2003；22(12)：1363-1366.
[33]Yuan S L,Wang X J,Wei Y Q.Anticancer effect of tanshinone and its mechanisms[J].Chin J Cancer,2003;22(12):1363-1366.
[34]Liu J, Yang C F, Wasser S,Shen H M, Tan C E, Ong C N. Protection of salvia miltiorrhiza against aflatoxin-B1-induced hepatocarcinogenesis in Fischer 344 rats dual mechanisms involved[J].Life Sci,2001;69(3):309-326.
[35]袁淑兰等.丹参酮体外诱导分化作用及其机制的研究[J].癌症,1996；15(6)：408.
[36]黄韧敏等.丹参酮诱导HL-60细胞分化及调亡的流式细胞术分析[J].中国肿瘤临床,1997；24(7)：500.
[37]Chen X G, Li Y, Yan C H, Li L N, Han R.Cancer chemopreventive activities of S-3-1, a synthetic derivative of danshinone[J].J Asian Nat Prod Res,2001;3 (1):63-75.
[38]常英姿,梁殿权,王孝铭等.丹参素对氧自由基所致大鼠心肌线粒体H+-ATP酶损伤的保护作用[J].中国病理生理杂志,1991；1(5)：449.
[39]刘启功.丹参注射液对心肌梗塞犬血流变学的观察[J].实用中西医结合杂志,1998；11：605.
[40]杨卫东,朱鸿良.丹参的氧自由基清除作用[J].中国药理学通报,1990；6(2)：118.
[41]Chang P N,Mao J C, Huang S H, et al. Analysis of cardioprotective effects using purified Salvia miltiorrh iza extract on isolated rat hearts[J].J Pharmacol Sci,2006;101(3):245-249.
[42]史举彤,杨文浩,陈长清.丹参素对体外循环中血细胞的影响[J].山东医科大学学报,1991；29(3)：237-239.
[43]赵国胜,杨会杰.川芎嗪和丹参对大鼠心脏氧反常保护作用的比较[J].天津医药,1996；24(5)：289.
[44]陶月玉,严玉兰.丹参对急性缺血后再灌注心肌磷脂肌醇系统变化的影响[J].中国病理生理杂志,1997；13(3)：267.
[45]刘军,匡培根,吴卫平等.大鼠脑缺血再灌注区小胶质细胞反应及丹参的影响[J].中华老年心脑血管病杂志,2002；4(2)：127-129.
[46]Imanshahidi M,Hosseinzadeh H.The pharmacological effects of Salvia species on the central nervous system[J].Phytother Res,2006;20(6):427-437.
[47]Lao C J,Lin J G, Kuo J S, et al. Effect of Salvia miltiorrhiza bunge on cerebral infarct in ischem ia-reperfusion injured rats[J].Am J Chin Med,2003;31 (2)：191-200.
[48]陈刚,刘润侠,陈喜玲.丹参对β2-GPI诱导的自身免疫小鼠aCL产生的影响[J].西安医科大学学报,2001；22(5)：429-431.
[49]许峰,陆伯刚,姚智,等.丹参及丹参素对内毒素刺激下肝巨噬分泌细胞因子影响的动态观察[J].中国危重病急教医学,1996；8(5)：262-264.
[50]JIANG Kai-yu, RUAN Cheng-geng, GU Zhen-lun. Effects of tanshonone ⅡA sulfonate on adhesion molecule expression of endothe-lial cells and platelets in vitro[J].Acta Pharmacological Sinica,1998;19(1):157-158.
[51]蔡建辉,刘维永,易定华等.丹参对兔严重钝性心损伤的治疗作用[J].中国危重病急救医学,1997；9(3)：129-131.
[52]苗戎,陈静,高岚月丹参对肺泡巨噬细胞分泌功能的影响[J].天津中医学院学报,1998；17(1)：389-390.
[53]马雪梅,王宝恩,尤红.丹参对肝星状细胞增殖和凋亡的实验研究[J].中国中医药信息杂志,2000；7(7)：25-26.
[54]周展超,郑家润,徐文严.氧氟沙星及丹参酮Ⅱa对白细胞趋化性的影响[J].中国医学科学院学报,1997；19(3)：232-233.
[55]戚心广.丹参、赤芍对实验性肝损伤肝细胞保护作用的机理研究[J].中西医结合杂志,1991；11(2)：102.
[56]秦嵩,孙备.丹参对肝脏缺血-再灌注损伤的防护作用[J].中医药学报,2001；29(6)：37-38.
[57]沈吉云,燕忠生.丹参保护肝纤化药理作用及应用进展[J].中西医结合肝病杂志,1998；8(2)：124.
[58]高玉芝,王灵芝,唐冀雪.丹参酮的性激素样作用[J].中国医学科学院学报,1980；2：189.
[59]严仲瑜.丹参对胃粘膜屏障的作用[J].中华外科杂志,1990；28(5)：298.
[60]董静,罗桂林,苗维纳.丹参对实验性肺纤维化小鼠病理变化和核因子-KB表达的影响[J].中国药理学通报,2003；19(12)：1428.
[61]吴山,盛延良,蒋志学.丹参对肺水肿大鼠白细胞介素-8和内皮素的影响[J].辽宁中医杂志,2003；30(10)：796.
[1]Karlyshev A V,Linton D,Gregson N A,Wren B W A,Novel paralogous gene family involved in phase-variable flagella-mediated motility in Campylobacter jejuni[J].Microbiology,2002;148(2):473-480.
[2]方积年,王顺春.香菇多糖的研究进展[J].中国药学杂志,1997；32(6)：332-337.
[3]Zheng R,Jie S,Hanchuan D,et al.Characterization and immunomodulating activities of polysaccharide from Lentinus edodes [J].Int Immunopharmacol, 2005;5(5):811-820.
[4]Cheng J J, Huang N K, Chang T T,et al.Study for anti-angiogenic activities of polysaccharides isolated from Antrodia cinnamomea in endothelial [J].Cells Life Sci,2005;76(26):3029-3042.
[5]Fisher M,Yang L X.Anticancer effects and mechanisms of polysaccharide-K (PSK):implications of cancer immunotherapy[J].Anticancer Res,2002;22(3): 1737-1754.
[6]Kobayashi H,Yoshida R,Kanada Y,et al.Suppressing effects of daily oral supplementation of beta-glucan extracted from Agaricus blazei Murill on spontaneous and peritoneal disseminated metastasis in mouse model[J].J Cancer Res Clin Oncol,2005;131(8):527-538.
[7]Popov S V, Popova G Y, Nikolaeva S Y, et al. Immunostimulating activity of pectic polysaccharide from Bergenia crassifolia (L.) Fritsch[J].Phytother Res, 2005;19(12):1052-1056.
[8]Song J Y, Akhalaia M, Platonov A, et al.Effects of polysaccharide ginsan from Panax ginseng on liver function[J].Arch Pharm Res,2004;27(5):531-538.
[9]Zhang M,Chen H,Huang J.Effect of lycium barbarumpolysaccharide on human hepatoma QGY7703 cells:inhibition of proliferation and induction of apoptosis[J]. Life Sci,2005;76(18):2115-2124.
[10]Kim G Y,Choi G S,Lee S H,et al.Acidic polysaccharide isolated from Phellinus linteus enhances through the up-regulation of nitric oxide and tumor necrosis factor-alpha from peritoneal macrophages[J].J Ethnopharmacol,2004;95(1): 69-76.
[11]Kodama N,Murata Y,Nanba H,et al.Administration of a polysaccharide from Grifola f rondosa stimulates immune function of normal mice[J].J Med Food, 2004;7(2):141-145.
[12]Lin Z B, Zhang H N.Antitumor and immunoregulatory activities of Ganoderma lucidum and its possible mechanisms [J].Acta Pharmacol Sin,2004;25(11): 1387-1395.
[13]Bao X F,Zhen Y,Ruan L,et al. Purification, characterization, and modification of T lymphocyte-stimulating polysaccharide from spores of Ganoderma lucidum[J].Chem Pharm Bull (Tokyo),2002;50(5):623-629.
[14]Lin Z B, Zhang H N.Anti-tumor and immunoregulatory activities of Ganoderma lucidum and its possible mechanisms[J].Acta Pharmacol Sin,2004; 25(11):1387-1395.
[15]Schepetkin I A,Quinn M T.Botanical polysaccharides. Macrophage immunomodulation and therapeutic potential[J].Int Immunopharmacol 2006; 6:317-333.
[16]李明春,梁东升,许自明等.灵芝多糖对小鼠腹腔巨噬细胞蛋白激酶A活性的影响[J].中草药,2000；31(5)：353-355.
[17]KM G Y,OH Y H,PARK Y M. Acidic polysaeeharide isolated from Phellinus induces nitric oxide mediated tumorieidal activity of macrophages through protein tyrosine kinase and protein kinase C[J].Bioehem Biophys Res Commun,2003;309 (2):399-407.
[18]陈伟,林新华,陈俊等.库拉索芦荟多糖对小鼠腹腔巨噬细胞的体外激活作用[J].中国药学杂志,2005；40(1)：34-37.
[19]田庚元,冯宇澄,林颖.植物多糖的研究进展[J].中国中药杂志,1995；20(7)：441.
[20]刘彦平,李积东.枸杞多糖对小鼠NK细胞和白细胞活性的免疫调节作用[J].青海医学院学报,2001；22(1)：1-2.
[21]刘彦平,毛辉青,李萍等.枸杞多糖对小鼠T淋巴细胞亚群和淋巴细胞转化作用的研究[J].青海医学院学报,2000；21(4)：4-5.
[22]郑应馨,徐恒卫.具有抗肿瘤活性的多糖及其作用机理研究概况[J].中国药师,2003；6(6)：368-369.
[23]周勇,张丽,赵高原等.仙茅多糖对小鼠免疫功能调节作用实验研究[J].上海 免疫学杂志,1996；16(5)：338.
[24]Zhu Y, Pettolino F, Mau S L. Immunoactive polysaccharide-rich fractions from Panax notoginseng[J].Planta Med,2006;72(13):1193-1199.
[25]Yamada H, Hirano M.Partial structure of an anti-ulcer polysaccharide from the roots of Bupleurum falcataum[J].Carbohydr Res,1991;219:173-192.
[26]Xu H,Zhang Y,Zhang J,et al.Isolation and characterization of an anti-complementary polysaccharide D3-S1 from the roots of Bupleurum smithii[J].Int Immunopharmacol,2007;7(2):175-182.
[27]Olafsdottir E S, Omarsdottir S, Paulsen B S, et al. Immunologically active 6-branched (1→3)-beta-glucan from the lichen Thamnolia vermicularis var subuliformis [J].Phytomedicine,2003;10(4):318-324.
[28]Schepetkin I A,Quinn M T.Botanical polysaccharides Macrophage immunomodulation and therapeutic potential [J].Int Immunopharmacol 2006;6: 317-333.
[29]Liu F,Fung M C,Ooi V E C,et al. Induction in the mouse of gene expression of immunomodulating cytokines by Mushroom polysaccharide-protein complexes [J].Life Science,1996;58(21):1795-1803.
[30]Liu F,Ooi V E C,Fung M C,et al.Analysis of immunomodulating cytokine mRNAs in the mouse induced by Mushroom polysaccharides[J].Life Science, 1999;64(12):1005-1011.
[31]Rout D,Mondal S,Chakraborty I,et al. Chemical-analysis of a new (1→3) 2,(1→6)-branched glucan from an edible mushroom, Pleurotus florida[J]. Carbohydr Res,2005;340(16):2533-2539.
[32]Kanekiyo K, Lee J B.Isolation of an antiviral polysaccharide, nostoflan, from a terrestrial cyanobacterium,Nostoc flagelliforme[J].J Nat Prod,2005;68(7): 1037-1041.
[33]Kimura Y,Sumiyoshi M,Suzuki T,et al. Antitumor and antimetastatic activity of a novel water-soluble low molecular weight beta-1,3-D-glucan (branch beta-1,6) isolated from Aureobasidium pullulans 1A1 strain black yeast [J]. Anticancer Res,2006;26(6B):4131-4141.
[34]Chang R.Bioactive polysaccharides from traditional Chinese medicine herbs as anticancer adjuvants[J].Altern Complement Med,2002;8(5):559-565.
[35]Lee K Y,Lee M H,Chang I Y,et al. Macrophage activation by polysaccharide fraction isolated from Salicornia herbacea[J].J Ethnopharmacol,2006; 103(3): 372-378.
[36]Koyanagi S,Tanigawa N,Nakagawa H,et al.Oversulfation of fucoidan enhances its anti-angiogenic and antitumor activities[J].Biochemi Pharmacol,2003;65: 173-179.
[37]Hatanaka k,Song S C,Maruyama A,et al.A new synthetichypoglycaemic polysacchide[J].Biochem Biophys Res Commun,1992;188(1):16.
[38]Kim K H,Lee Y S,Jung I S,et al.Acidic polysaccharide from Panax ginseng, ginsan, induces Thl cell and macrophage cytokines and generates LAK cells in synergy with rIL-2[J].Planta Med,1998;64(2):110-115.
[39]Kito T,Sobue S.Structural features and hypoglycemic activities of two polysaccharides from a hot-water extract of Agrocybe cylindracea[J]. Carbohydr Res,1994;252:81-87.
[40]刘成梅,付贵明,涂宗财等.百合多糖降血糖功能研究[J].食品科学,2002；23(6)：113-114.
[41]Huang Cheng,Chen Qun li,Sun Jiang tao,et al.Protective effect of lycium barbarum ploysaccharde and its compound recipe on pancreatic islet functiong in rats with streptocototocin induced diabetes mellitus[J].Chinese Journal of Clinical Rehabilitation,2006;10(23):173-175.
[42]Itoh T,Kita N,Kurokawa Y,et al.Suppressive effect of a hot water extract of adzuki beans (Vigna angularis) on hyperglycemia after sucrose loading in mice and diabetic rats[J].Biosci Biotechnol Biochem,2004;68 (12):2421-2426.
[43]Zhang J, Huang Y, Hou T. Hypoglycaemic effect of Artemisia sphaerocephala Krasch seed polysaccharide in alloxan-induced diabetic rats[J].Swiss Med Wkly,2006;136(33-34):529-532.
[44]Shirwaikar A,Rajendran K,Barik R,et al. Effect of aqueous bark extract of Garuga pinnata Roxb. in streptozotocin-nicotinamide induced type-II diabetes mellitus[J].J Ethnopharmacol,2006;19,107(2):285-290.
[45]Ito M, Baba M, Sato A, et al.Inhibitory effect of dextran sulfate and heparin on the replication of human immunodeficiency virus(HIV) in vitro[J].Antiviral Res,1987;7:361-367.
[46]Witvrouw M, Schols D, Andrei G, et al. Newpolyacetal polysulfate active against HIV and other enveloped viruses[J].Antiviral Chem Chemother,1992;3: 351-355.
[47]Talyshinsky MM, Souprun YY, Huleihel MM, et al. Anti-viral activity of red microalgal polysaccharides against retroviruses[J].Cancer Cell Int,2002;2(1):8.
[48]Gerencer M,Turecek P L,Kistner O,et al. In vitro and in vivo anti-retroviral activity of the substance purified from the aqueous extract of Chelidonium majus[J].Antiviral Res,2006;72(2):153-156.
[49]Rusnati M, Urbinati C, Caputo A, et al. Pentosan Polysulfate as an inhibitor of Extracellular HIV-1 Tat[J].Biol Chem,2001;276:22420-22425.
[50]Zhu W,Chiu L C,Ooi V E,et al.Antiviral property and mechanisms of a sulphated polysaccharide from the brown alga Sargassum patens against Herpes simplex virus type 1[J].Phytomedicine,2006;13 (9-10):695-701.
[51]Lee I H, Huang R L, Chen C T, et al. Antrodia camphorate polysaccharides exhibit anti-hepatitis B virus effects[J].FEMS Microbiol Lett,2002;209:63-67.
[52]Naesens L, Bonnafous P, Agut H, et al. Antiviral activity of diverse classes of broad-acting agents and natural compounds in HHV-6-infected lymphoblasts[J].J Clin Virol,2006;37 Suppl 1:S69-75.
[53]Ghosh P, Adhikari U, Ghosal PK, et al.In vitro anti-herpetic activity of sulfated polysaccharide fractions from Caulerpa racemosa[J].Phytochemistry,2004; 65(23):3151-3157.
[54]Lee J B,Hayashi K,Maeda M,et al.Antiherpetic activities of sulfated polysaccharides from green algae [J].Planta Med,2004;70 (9):813-817.
[55]Lee J H, Shim J S, Lee J S, et al.Pectin-like acidic polysaccharide from Panax ginseng with selective antiadhesive activity against pathogenic bacteria[J]. Carbohydr Res,2006;341(9):1154-1163.
[56]Pereda-Miranda R,Kaatz G W,Gibbons S,et al.Polyacylated oligosaccharides from medicinal Mexican morning glory species as antibacterials and inhibitors of multidrug resistance in Staphylococcus aureus[J].J Nat Prod,2006;69(3): 406-409.
[57]王雪萍.植物多糖研究进展[J].粮食与油脂,2005；8：8-10.
[58]张宪党,马弛,张群等.植物多糖抗辐射损伤作用研究进展[J].中国辐射卫生,2003；12(2)：122-123.
[59]Chen Y J,Yang Y C.A novel polysaccharide of black soybean promotes myelopoiesis and reconstitutes bone marrow after 5-flurouracil and irradiation-induced myelosuppression[J]. Life Sci,2005;77(4):400-413.
[60]吴华振.植物多糖的药理作用及应用进展[J].实用医技杂志,2005；12(7)：1803-1804.
[61]Li S P,Zhao K J,Ji Z N,et al. A polysaccharide isolated from Cordyceps sinensis, a traditional Chinese medicine, protects PC 12 cells against hydrogen peroxide-induced injury[J].Life Sciences,2003;73:2503-2513.
[62]Yu M S,Lai S W,Lin K F.Characterization of polysaccharides from the flowers of Nerium indicum and their neuroprotective effects[J].Int J Mol Med,2004; 14(5):917-924.
[63]Sun C,Shan C Y,Gao X D,et al.Protection of PC12 cells from hydrogen peroxide-induced injury by EPS2, an exopolysaccharide from a marine filamentous fungus Keissleriella sp. YS4108J[J]:Biotechnol,2005; 115(2)-137-144.
[64]徐德生,冯怡,林晓.麦冬多糖MDG-1的分离纯化和结构分析[J].药学学报,2005；40(7)：636-639.
[65]Rocha H A, Moraes F A, Trindade E S,et al. Structural and hemostatic activities of a sulfated galactofucan from the brown alga Spatoglossum schroederi. An ideal antithrombotic agent[J].J Biol Chem,2005;280(50): 41278-41288.
[66]杨明,王晓娟,孙红等.树舌多糖抗溃疡作用[J].中药药理与临床, 2004；20(6)：11-13.
[67]傅继华,温涛,徐琛.芦荟多糖对动物实验性胃溃疡的影响[J].中草药,2006；37(6)：894-897.
[1]蔡幼清.丹参根中的精制多糖对大鼠实验性肾病的作用[J].国外医学,中医中药分,1995；17(4)：52-53.
[2]田庚元,冯宇澄,林颖.植物多糖的研究进展[J].中国中药杂志,1995；20(7)：441-441.
[1]Kobayashi S,Nishihira J,Watanabe S, et al.Prevention of lethal acute hepatic failure by antimacrophage migration inhibitory factor antibody in mice treated with bacille Calmette-Guerin and lipopolysaccharide[J].Hepatology,1999;29: 752-759.
[2]Yoneyama H, Harada A, Imai T, et al. Pivotal role of TARC, a CC chemokine, in bacteria-induced fulminant hepatic failure in mice[J].Clin Invest,1998; 102: 1933-1941.
[3]S Kobayashi, J Nishihira, S Watanabe, S Todo. Prevention of lethal acute hepatic failure by antimacrophage migration inhibitory factor antibody in mice treated with bacille Calmette-Guerin and lipopolysaccharide[J].Hepatology, 1999;29:1752-1759.
[4]Wang Y Z, Hu S S.A novel nitric oxide biosensor based on electropolymerization poly (toluidine blue) film electrode and its application to nitric oxide released in liver homogenate [J].Biosens Bioelectron,2006; 22:10-17.
[5]张定凤.乙型肝炎的发病机理及临床[M].重庆出版社,1992：1.
[6]Y H Zou, Y Yang, J Li, W P Li, Q Wu. Prevention of hepatic injury by a traditional Chinese formulation, BJ-JN,in mice treated with Bacille-Calmette-Guerin and lipopolysaccharide[J].J Ethnopharmacol,2006; 107:442-448.
[7]G S Wang, G T Liu. Role of nitric oxide in immunological liver damage in mice[J].Biochem Parmacol,1995;49:1277-1281.
[8]J Nagakawa,I Hishinuma,K Hirota, K Miyamoto, T Yamanaka;,K Tsukidate, K Katayama, I Yamatsu. Involvement of tumor necrosis factor-alpha in the pathogenesis of activated macrophage-mediated hepatitis in mice [J]. Gastroenterology,1990;99:758-765.
[9]H Tsuji, A Harada, N Mukaida, Y Nakanuma, H Bluethmann, S Kaneko, K Yamakawa, S I Nakamura, K I Kobayashi, K Matsushima. Tumor necrosis factor receptor p55 is essential for intrahepatic granuloma formation and hepatocellular apoptosis in a murine model of bacterium-induced fulminant hepatitis[J].Infect Immun,1997; 65:1892-1898.
[10]H Tsutsui, K Matsui, N Kawada, Y Hyodo, N Hayashi, H Okamura, K Higashino, K Nakanishi. IL-18 accounts for both TNF-alpha and Fas ligand-mediated hepatotoxic pathways in endotoxin-induced liver injury in mice[J].J Immunol,1997;159:3961-3967.
[11]N Fujioka, N Mukaida, A Harada, M Akiyama, T Kasahara, K Kuno, A Ooi, M Mai, K Matsushima. Preparation of specific antibodies against murine IL-1ra and the establishment of IL-1ra as an endogenous regulator of bacteria-induced fulminant hepatitis in mice[J]. J Leukoc Biol,1995;58:90-98.
[12]P Liu, H Ohnishi, H Moriwaki,Y Muto.Enhanced tumor necrosis factor and interleukin-1 in an experimental model of massive liver cell necrosis/fatal hepatitis in mice[J].Gastroenterol Jpn,1990;25:339-342.
[13]R Guler, M L Olleros, D Vesin, R Parapanov, C Vesin, S Kantengwa, L Rubbia-Brandt, N Mensi, A Angelillo-Scherrer, E Martinez-Soria, F Tacchini-Cottier, I Garcia. Inhibition of inducible nitric oxide synthase protects against liver injury induced by mycobacterial infection and endotoxins[J].J Hepatol,2004;41:773-781.
[14]K Matsui, T Yoshimoto, H Tsutsui, Y Hyodo, N Hayashi, K Hiroishi, N Kawada, H Okamura, K Nakanishi, K. Higashino. Propionibacterium acnes treatment diminishes CD4+ NK1.1+ T cells but induces type I T cells in the liver by induction of IL-12 and IL-18 production from Kupffer cells[J].J Immunol,1997; 159:97-106.
[15]Y Tanaka, A Takahashi, K Watanabe, K Takayama, T Yahata,S Habu,T Nishimura. A pivotal role of IL-12 in Thl-dependent mouse liver injury[J].Int Immunol,1996;8:569-576.
[16]杨天健,魏怀玲,刘耕陶.16种药物对卡介苗加脂多糖引起小鼠免疫性肝损伤的作用[J].中国药理学报,1997；18(2)：185-188.
[17]张安平,沈玉先,魏伟等.丹皮总甙对小鼠免疫性肝损伤的保护作用[J].中国药理学报,1997；13(3)：435-437.
[18]D L Laskin,M Rodriguez del Valle,D E Heck,S M Hwang,S T Ohnishi,S K Durham,N L Goller,J D Laskin.Hepatic nitric oxide production following acute endotoxemia in rats is mediated by increased inducible nitric oxide synthase gene expression[J].Hepatology,1995;22:223-234.
[19]R Guler, M L. Olleros, D Vesin, R Parapanov, C Vesin, S Kantengwa, L Rubbia-Brandt, N Mensi, A Angelillo-Scherrer, E Martinez-Soria, F Tacchini-Cottier, I Garcia. Inhibition of inducible nitric oxide synthase protects against liver injury induced by mycobacterial infection and endotoxins[J].J Hepatol,2004; 41:773-781.
[20]G Sass, K Koerber, R Bang, H Guehring, G Tiegs. Inducible nitric oxide synthase is critical for immune-mediated liver injury in mice[J].J Clin Invest, 2001; 107:439-447.
[21]M I Luster, P P Simeonova, R M Gallucci, J M Matheson, B Yucesoy. Immunotoxicology:role of inflammation in chemical-induced hepatotoxicity[J]. Int J Immunopharmacol,2000;22:1143-1147.
[22]Y Muto, K T Nouri-Aria, A Meager, G J Alexander, A L Eddleston, R Williams. Enhanced tumor necrosis factor and interleukin-1 in fulminant hepatic failure[J]. Lancet,1988;2:72-74.
[23]M de la Mata, A Meager,N Rolando, H M Daniels,K T Nouri-Aria,A K Goka,A L Eddleston,G J Alexander,R Williams.Tumour necrosis factor production in fulminant hepatic failure:relation to aetiology and superimposed microbial infection[J].Clin Exp Immunol,1990;82:479-484.
[24]H Zylberberg, A C Rimaniol, S Pol, A Masson, D De Groote, P Berthelot, J F Bach, C Brechot, F.Zavala.Soluble tumor necrosis factor receptors in chronic hepatitis C:a correlation with histological fibrosis and activity[J].J Hepatol, 1999; 30:185-191.
[25]M Yin,M D Wheeler, H Kono, B U Bradford, R M Gallucci, MI Luster, R G Thurman.Essential role of tumor necrosis factor alpha in alcohol-induced liver injury in mice[J].Gastroenterology,1999;117:942-952.
[26]H A Rudiger, P A Clavien.Tumor necrosis factor alpha, but not Fas, mediates hepatocellular apoptosis in the murine ischemic liver[J].Gastroenterology,2002; 122:202-210.
[27]J Muntane, F J Rodriguez,O Segado, A Quintero,J M Lozano,E Siendones, C A Pedraza,M Delgado,F O'Valle,R Garcia,J L Montero,M De La Mata, G Mino.TNF-alpha dependent production of inducible nitric oxide is involved in PGE(1) protection against acute liver injury[J]. Gut,2000;47:553-562.
[28]E P Nadler, E C Dickinson, D Beer-Stolz, S M Alber, S C Watkins, D W Pratt, H R Ford.Scavenging nitric oxide reduces hepatocellular injury after endotoxin challenge, Am J Physiol[J].Gastrointest Liver Physiol,2001;281:G173-G181.
[29]P P Simeonova, R M Gallucci, T Hulderman, R Wilson, C Kommineni, M Rao, M I Luster.The role of tumor necrosis factor-alpha in liver toxicity, inflammation and fibrosis induced by carbon tetrachloride[J].Toxicol Appl Pharmacol,2001;177:112-120.
[30]N Tulek, S K Saglam, M Saglam, R Turkyilmaz, M Yildiz. Soluble interleukin-2 receptor and interleukin-10 levels in patients with chronic hepatitis B infection[J].Hepatogastroenterology,2000;47:828-831.
[31]C Basak, S K Pathak, A Bhattacharyya, D Mandal, S Pathak, M Kundu. NF-kappaB-and C/EBPbeta-driven interleukin-1beta gene expression and PAK1-mediated caspase-1 activation play essential roles in interleukin-lbeta release from Helicobacter pylori lipopolysaccharide-stimulated macrophages[J]. J Biol Chem,2005; 280:4279-4288.
[32]C A Dinarello.Interleukin-1 and interleukin-1 antagonism[J].Blood,1991;77: 1627-1652.
[33]A K Nussler,T R Billiar. Inflammation, immunoregulation, and inducible nitric oxide synthase[J].J Leukoc Biol,1993;54:171-178.
[34]J N Dhuley, S R. Naik Moderate protective effect of 6-MFA, a microbial metabolite obtained from Aspergillus ochraceus on immunological liver injury in mice[J].Comp Immunol Microbiol Infect Dis,1999;22:15-25.
[1]Heid C A, Stevens J, Livak K J, et al. Real time quantitative PCR. Genome Res[J].1996;6(10):986-994.
[2]Winer J, Jung C K, Shackel I, et al. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro[J].Anal Biochem,1999;270(1): 41-49.
[3]Kaltenboeck B, Wang C. Advances in real-time PCR:application to clinical laboratory diagnostics[J].Adv Clin Chem,2005;40:219-259.
[4]Valasek M A, Repa J J. The power of real-time PCR[J].Adv Physiol Educ, 2005;29(3):151-159.
[5]Espy M J, Uhl J R, Sloan L M, et al.Real-time PCR in clinical microbiology: applications for routine laboratory testing[J].Clin Microbiol Rev,2006; 19(1):165-256.
[6]Wong M L, Medrano J F. Real-time PCR for mRNA quantitation. Biotechniques[J].2005;39(1):75-85.
[7]Stahlberg A, Zoric N, Aman P, et al. Quantitative real-time PCR for cancer detection:the lymphoma case[J].Expert Rev Mol Diagn,2005;5(2):221-230.
[8]Bubner B, Baldwin I T. Use of real-time PCR for determining copy number and zygosity in transgenic plants[J].Plant Cell Rep,2004;23(5):263-271.
[9]Mackay I M. Real-time PCR in the microbiology laboratory[J].Clin Microbiol Infect,2004; 10(3):190-212.
[10]Gomes-Ruiz A C, Nascimento R T, de Paula SO, et al. SYBR green and TaqMan real-time PCR assays are equivalent for the diagnosis of dengue virus type 3 infections[J].J Med Virol,2006;78(6):760-763.
[11]Liu Y, Cai X, Zhang X, et al. Real time PCR using TaqMan and SYBR Green for detection of Enterobacter sakazakii in infant formula[J].J Microbiol Methods,2006;65(1):21-31.
[12]Suzuki N, Yoshida A, Nakano Y. Quantitative analysis of multi-species oral biofilms by TaqMan Real-Time PCR[J].Clin Med Res,2005;3(3):176-185.
[13]Rousselon N, Delgenes J P, Godon J J. A new real time PCR (TaqMan PCR) system for detection of the16S rDNA gene associated with fecal bacteria[J].J Microbiol Methods,2004;59(1):15-22.
[14]Campsall P A, Au N H, Prendiville J S, et al. Detection and genotyping of varicella-zoster virus by TaqMan allelic discrimination real-time PCR[J].J Clin Microbiol,2004;42(4):1409-1413.
[15]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[J].Methods, 2001;25(4):402-408.
[16]Magram J, Connaughton S E, Warrier R R, et al. IL-12 deficient mice are defective in IFN gamma production and type 1 cytokine responses[J]. Immunity,1996; 4(5):471-481.
[17]Kaplan M H, Sun Y L, Hoey T, et al. Impaired IL-12 responses and enhanced development of Th2 cells in Stat42-deficient mice[J].Nature,1996; 382(657): 174-177.