溪黄草黄酮对肝纤维化及急性肝损伤治疗作用的实验研究
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摘要
第一部分溪黄草总黄酮膜分离技术研究
研究背景及目的：
溪黄草属于唇形科香茶属植物,是一种多年生草本植物,含黄酮类、酚类等多种化学成分。溪黄草具有清热祛湿、凉血散瘀之功效,对急性肝炎、肠炎、痢疾等有较好效果。近年来,中草药溪黄草在华南各地临床上广为应用,并出现了一些以其为主要原料的保健产品,具有较好的开发应用前景。溪黄草现有制剂工艺过程均采用传统水煎醇沉除杂工艺,工艺复杂,具有一定的不足。膜分离技术应用于中药制剂的生产过程具有效率高、降低污染、节省能源等优点。无机陶瓷膜具有机械强度高、耐腐蚀等特点,对中药提取液的纯化除杂有其独特的优点。本实验拟定了陶瓷膜微滤参数,最有效地为溪黄草黄酮类物质的提取、纯化等提供支持。
研究方法：
本实验将溪黄草水提取液用无机陶瓷膜微滤机以错流方式进行循环微滤,测定各孔径陶瓷膜的滤过情况、各孔径陶瓷膜总黄酮含量及转移率、除固率、膜通量,从而确定最佳陶瓷膜孔径；测定六个温度对有效成分转移率、膜通量、总黄酮含量的影响,从而确定最佳微滤温度；分别考察四个操作压力对有效成分转移率、膜通量的影响,确定最佳操作压力；取溪黄草水提液在确定条件下进行微滤,测定总黄酮转移率,从而确定最佳顶洗条件。通过测量通量恢复率测试每步清洗对膜通量恢复的影响；
研究结果：
1.最佳陶瓷膜孔径：0.1μm、0.2μm、0.5μm、0.8μm各孔径陶瓷膜滤液的收得率为93%、92%、86%、90%；总黄酮累计转移率为82%、87.1%、82%、87.2%；除了0.8μm外,其余三个孔径除固率都在20%左右；0.2μm孔径陶瓷膜微滤过程中通量衰减较小,稳定时膜通量最大。
2.最佳微滤温度：45℃-75℃范围的膜通量增长比较稳定,约为300L·h-1·-2,25℃-45℃范围内微滤温度的总黄酮转移率约为95%。
3.最佳操作压力：在恒定的微滤温度下25℃,0.15MPa压力下总黄酮转移率为96%。
4.最佳顶洗条件的确定：顶洗1.5倍量残存液的顶洗量,顶洗两次,总黄酮累计转移率为99.79%,更高效地获得溪黄草总黄酮。
5.清洗对膜通量恢复的影响：对于膜孔径较小的0.1μm、0.2μm陶瓷膜,除纯水外,酸洗和碱洗对膜通量的恢复有一定作用,0.5%NaOH+0.5%多聚磷酸钠+0.5%EDTA二钠盐洗后膜通量能达到过滤前原始纯水通量的90%, NaClO+硝酸浸泡一晚后通量能恢复到100%。
研究结论
陶瓷膜较适用于溪黄草水提液的纯化精制,利用陶瓷膜微滤技术可有效除去溪黄草水提物中的不溶性或大分子杂质,能对总黄酮进行更高效的分离、浓缩、纯化和精制。陶瓷膜微滤最佳条件为：在25℃,0.15MPa下,用0.2μm孔径陶瓷膜,1.5倍量残存液的顶洗量,顶洗两次的条件进行微滤能高效地获得溪黄草总黄酮,采用0.5%NaOH+0.5%多聚磷酸钠+0.5%EDTA二钠盐及NaClO+硝酸浸泡为陶瓷膜的最佳清洗方法。
第二部分溪黄草黄酮抗肝纤维化作用
研究背景及目的：
肝纤维化(hepatic fibrosis, HF)是慢性肝病发展成肝硬化的必经阶段,是多种致病因子引起的肝脏炎症或损伤后组织修复过程中的代偿反应,以细胞外基质(extracellular matrix, ECM)合成大于降解导致ECM的过度沉积的结果。有研究表明,肝纤维化病变经积极治疗可延缓甚至逆转。目前,各国学者从肝纤维化发生发展的不同环节入手,研制了许多抗纤维化药物,但因种种原因尚未找到十分理想的药物。因此,从我国天然植物资源宝库中找寻到抗纤维化的天然有效成分,具有极其重要的社会意义和经济意义。本实验通过三种常用肝纤维化损伤的动物模型,观察溪黄草黄酮(flavonoids from Rabdosia serra (Maxim.) Hara, FRSH)对急性肝损伤的保护作用,为溪黄草黄酮在临床应用提供科学依据。
研究方法：
1、健康SD大鼠随机分为6组,即假手术对照组/正常对照、模型对照组、水飞蓟素组50mg/kg、溪黄草黄酮低剂量组9mg/kg、溪黄草黄酮中剂量组18mg//kg、溪黄草黄酮高剂量组36mg//kg。
2、采用胆总管结扎、腹腔注射猪血清及皮下注射CCl4复合因素三种方法造成肝纤维化动物模型。从血清检测TBIL、DBIL、ALT、AST、HA、LN、PC-Ⅲ、TNF-α、 ALB/GLB水平；肝组织匀浆后检测SOD、MDA、GSH-Px、Hyp,采用ELISA法检测MMP-1和TIMP-1蛋白的表达,通过HE染色观察肝细胞结构和肝纤维化程度,并通过免疫组织化学法(SABC法)检测肝组织α-肌动蛋白(a-SMA)的表达。
研究结果：
1、溪黄草黄酮抗胆总管结扎所致大鼠肝纤维化的实验研究
与假手术组比较,模型对照组大鼠血清中肝功能指标TBIL、DBIL、ALT、AST水平均显著升高(均P=0.000),与模型对照组比较,溪黄草黄酮低、中、高剂量组大鼠血清中肝功能指标显著降低(P<0.05或0.01)。与假手术组比较,模型对照组大鼠肝纤维化指标PC-Ⅲ、HA、LN、Hyp含量均显著升高(均P=0.000),与模型对照组比较,溪黄草黄酮低、中、高剂量组大鼠肝纤维化指标显著降低(P<0.05或0.01)。与正常对照组比较,模型组肝组织GSH-Px活性显著降低(均P=0.000),而MDA、TNF-α含量提高有显著性差异(均P=0.000),与模型对照组比较,FRSH各剂量组肝组织SOD、GSH-Px活性显著升高(P<0.05),而MDA、TNF-α含量降低有统计学差异(P<0.01)。与正常对照组比较,模型组肝组织TIMP-1蛋白表达显著增加(P=0.000),而模型组肝组织MMP-1无统计学意义(P>0.05),与模型对照组比较,溪黄草黄酮低、中、高剂量组大鼠肝组织TIMP-1蛋白表达显著降低(P<0.01)。
2、溪黄草黄酮抗猪血清致大鼠免疫性肝纤维化的实验研究
与正常对照组比较,模型对照组大鼠血清中肝功能指标ALT、AST水平均显著升高(均P=0.000), ALB/GLB值降低有显著性差异(P=0.000),与模型对照组比较,溪黄草黄酮低、中、高剂量组大鼠血清中肝功能指标显著降低(P<0.05或0.01), ALB/GLB值显著升高(P<0.05或0.01)。与正常对照组比较,模型对照组大鼠肝纤维化指标PC-Ⅲ、HA、LN、Hyp含量均显著升高(均P=0.000),与模型对照组比较,溪黄草黄酮低、中、高剂量组大鼠肝纤维化指标显著降低(P<0.05或0.01)。与正常对照组比较,模型组大鼠肝组织TIMP-1蛋白表达显著增加(P=0.000),而MMP-1无统计学意义(P>0.05),与模型对照组比较,溪黄草黄酮低、中、高剂量组大鼠肝组织TIMP-1蛋白表达显著降低(P<0.01)。
3、溪黄草黄酮抗CCl4复合因素所致大鼠肝纤维化的实验研究
与正常对照组比较,模型对照组大鼠血清中肝功能指标TBIL、DBIL、ALT、AST水平均显著升高(均P=0.000),与模型对照组比较,溪黄草黄酮低、中、高剂量组大鼠血清中肝功能指标显著降低(P<0.05或0.01)。与正常对照组比较,模型对照组大鼠肝纤维化指标PC-Ⅲ、HA、LN、Hyp含量均显著升高(均P=0.000),与模型对照组比较,溪黄草黄酮低、中、高剂量组大鼠肝纤维化指标显著降低(P<0.05或0.01)。与正常对照组比较,肝组织SOD、GSH-Px活性显著降低(均P=0.000),而MDA、TNF-α含量提高有显著性差异(均P=0.000),与模型对照组比较,肝组织SOD、GSH-Px活性显著升高(P<0.01),而MDA、TNF-α含量降低有统计学差异(P<0.01)。与正常对照组比较,肝组织TIMP-1蛋白表达显著增加(P=0.000),而肝组织MMP-1无统计学意义(P>0.05),与模型对照组比较,溪黄草黄酮低、中、高剂量组大鼠肝组织TIMP-1蛋白表达显著降低(P<0.05或0.01)。
4、肝组织HE染色结果
三种肝纤维化动物试验研究中,正常对照组肝小叶结构清晰,小叶内肝细胞基本正常,汇管区结构清晰,汇管区及汇管区周围未见小胆管及纤维组织增生；模型对照组见肝小叶结构模糊,小叶内肝细胞水肿,局灶肝细胞坏死,小胆管大量增生,以汇管区为中心放射状向肝实质内延伸,增生的胆管呈花环样,增生胆管周围伴纤维组织增生及炎细胞浸润；水飞蓟素组见肝小叶结构较清晰,小叶内局灶肝细胞呈点状坏死,肝内小胆管以汇管区为中心轻度增生,增生胆管周围伴轻度纤维组织增生及少量炎细胞浸润；溪黄草黄酮组可见肝小叶结构较清晰,小胆管以汇管区为中心轻度增生,增生胆管周围伴轻度纤维组织增生。
5、肝组织α-SMA蛋白表达免疫组化染色结果
三种肝纤维化动物试验研究中,正常对照组可见α-SMA在肝组织汇管区少量阳性表达；模型对照组可见α-SMA在肝组织内阳性表达的面积大大增加；水飞蓟素组α-SMA在肝组织内阳性表达的面积少于模型对照组；溪黄草黄酮组α-SMA在肝组织内阳性表达的面积明显少于模型对照组,趋于正常。
研究结论：
溪黄草黄酮对胆总管结扎、腹腔注射猪血清及皮下注射CCl4复合因素三种方法造成的肝纤维化动物模型都具有治疗作用,肝功能指标和肝纤维化指标都趋于正常,该作用与溪黄草黄酮可缓解大鼠肝脏的氧化应激状态、调节脂质代谢,增强肝脏抗氧化,减轻脂质过氧化损伤作用有关。
第三部分溪黄草黄酮对急性肝损伤的预防作用
研究背景及目的：
肝损伤是临床常见的危害人类健康的疾病,肝损伤的常见病因是饮酒过多、熬夜过劳、环境污染、脂肪肝等。肝损伤主要分为酒精性肝损伤、免疫性肝损伤及化学性肝损伤。近年来随着药物滥用,药源性肝损伤发病呈逐年增加趋势。肝损伤的防治目前已成为国内外研究的热点与重点课题,本实验拟以中草药溪黄草重要的有效成分黄酮,考察其对多种因素诱导的的急性肝损伤模型的预防作用,进一步研究溪黄草黄酮的保肝、护肝作用,并对其机制进行初步探讨。
研究方法：
1、健康昆明种小鼠随机分为6组,即正常对照组、模型对照组、联苯双酯组100mg/kg、溪黄草黄酮低剂量组13mg/kg、溪黄草黄酮中剂量组26mg/kg、溪黄草黄酮高剂量组52mg/kg。
2、经过CC14、BCG-LPS及酒精处理后造成急性肝损伤模型,取血测定AST、ALT活性；肝组织匀浆后检测相应的MDA、GSH、SOD、NOS指标并进行肝组织病理组织学检查。
研究结果：
1、溪黄草黄酮对CCl4致小鼠急性肝损伤的预防作用
与正常对照组比较,模型对照组小鼠血清中肝功能指标ALT、AST水平均显著升高(均P=0.000),与模型对照组比较,溪黄草黄酮低、中、高剂量组的血清中ALT、AST活性显著降低(P<0.05或0.01)。与正常对照组比较,模型对照组小鼠肝组织MDA含量显著升高,GSH含量和SOD活性显著降低,有统计学意义(均P=0.000),与模型对照组比较,联苯双酯组与溪黄草黄酮低、中、高剂量组小鼠肝组织MDA含量显著降低而GSH含量和SOD活性显著升高(P<0.05或0.01)。
2、溪黄草黄酮对卡介苗联合脂多糖所致小鼠急性肝损伤的预防作用
与正常对照组比较,模型对照组小鼠血清中肝功能指标ALT、AST水平均显著升高(均P=0.000),与模型对照组比较,联苯双酯组与溪黄草黄酮低、中、高剂量组的血清中ALT、AST活性显著降低(P<0.05或0.01)；与正常对照组比较,模型对照组小鼠肝组织MDA含量和NOS活性显著升高,GSH含量和SOD活性显著降低(均P=0.000),与模型对照组比较,联苯双酯组与溪黄草黄酮低、中、高剂量组小鼠肝组织MDA含量和NOS活性显著降低而GSH含量和SOD活性显著升高(P<0.05或0.01)。
3、溪黄草黄酮对酒精致小鼠急性肝损伤的预防作用
与正常对照组比较,模型对照组小鼠血清中肝功能指标ALT、AST均显著升高(均P=0.000),与模型对照组比较,联苯双酯组与溪黄草黄酮低、中、高剂量组的血清中ALT、AST活性显著降低(P<0.05或0.01)；与正常对照组比较,模型组小鼠肝组织MDA含量显著升高,GSH含量和SOD活性显著降低,有统计学意义(均P=0.000),与模型对照组比较,联苯双酯组与溪黄草黄酮低、中、高剂量组小鼠肝组织MDA含量显著降低而GSH含量和SOD活性显著升高(P<0.05或0.01)。
4、病理组织学检查结果
三种急性肝损伤动物模型试验中,正常对照组小鼠肝脏颜色红润,有光泽且富于弹性,光学显微镜下可见肝小叶结构完整,肝细胞胞质丰富,核大而圆,核仁清晰,中央静脉及门管区正常,细胞排列整齐。模型对照组的肝脏发生明显病理改变,主要表现为灰黄色点状坏死灶,表面无光泽,质地稍脆,光镜下,肝小叶中央静脉周围坏死,坏死细胞轮廓不清,肝细胞出现中至重度病变,胞浆疏松、淡染,细胞核多皱缩,细胞间隙增大,且肝细胞多见点状坏死灶及门管区见炎细胞浸润。联苯双酯组小鼠的肝小叶内见点状坏死,坏死处炎细胞浸润,病理肝损伤减轻。溪黄草黄酮组小鼠的肝小叶结构完整,肝索呈放射状排列,部分肝细胞仅见轻度的病理改变,肝细胞轻度水肿,胞浆疏松、淡染,肝脏组织结构与模型组比较,病理损伤减轻明显。
研究结论：
溪黄草黄酮对化学性、免疫性及酒精所导致的急性肝损伤都具有预防性保护作用,该作用可能与溪黄草黄酮能够提高小鼠肝组织的抗氧化应激能力,减少细胞质膜的脂质过氧化水平,从而修复细胞质膜,降低肝损伤有密切关系。
Part1Research on the separation of FRSH with inorganic ceramic membrane
Background:Rabdosia serra (Maxim.) Hara(RSH), a labiatae and perennial herbaceous plant, contains flavonoids, phenols and other chemical compositions. It is widely used for the treatment of acute hepatitis, enteritis and diarrhea in south China and provides main raw material for health care products. So, this plant is worthy of extensive study and development. However, existing preparation adopts the traditional water extract-alcohol sedimentation process; the process is complex and contains certain shortcomings. Application of membrane separation technology to the extraction process of traditional Chinese medicine (TCM) is a desirable choice and it has advantages of high efficiency, low pollution and low consumption of energy. Inorganic ceramic membrane has the characteristics of high mechanical strength and corrosion resistance; it has unique advantages on the extraction of traditional Chinese herb and purification in removing impurities. This study focused on the most optimal parameters for ceramic membrane microfiltration, it provides methodological support for the extraction and purification of total flavonoids from RSH.
Methods:
Water extract of RSH was filtrated through inorganic ceramic membrane filtration system with cross-flow microfiltration. the pore size of ceramic membrane, the total content of flavonoids, transfer rate and removal rate of solid impurities were measured, so as to determine the best condition for ceramic membrane microfiltration; the cleaning effect on membrane flux recovery was determined by measuring the flux recovery rate in each step; effects of six various temperatures on the components transfer rate, membrane flux, flavonoids content were measured, so as to determine the best microfiltration temperature; transfer rate and membrane flux of active ingredients with four operating pressure were investigated respectively to investigate the optimum operating pressure; water solution of RSH was filtrated under the determined condition of microfiltration, the transfer rate of flavones was evaluated, so as to determine the best washing condition.
Results:
1. The best ceramic membrane pore size:The filtrate rate of various pore size of ceramic membrane (0.1μm,0.2μm,0.5μm,0.8μm) was93%,92%,86%,90%and the total cumulative flavonoids transfer rate was82%,87.1%,82%and87.2%respectively, in addition to0.8μm membrane, the removal rate of solid impurities was about20%,0.2μm aperture in the process of the ceramic microfiltration membrane flux attenuation of the membrane with0.2μm pore is small and the membrane flux is the largest in stable condition.
2. The optimum microfiltration temperature:the membrane flux is about300L·h-1·m-2when the temperature set at45℃-75℃25℃, the flavonoids transfer rate is about95%when the temperature was25℃-45℃.
3. The best operating pressure:In a constant microfiltration temperature (25℃), flavonoids transfer rate was96%under0.15MPa.
4. The determination of optimum washing conditions:The cumulative total flavonoids transfer rate was99.79%when washed twice with1.5times the amount of residual liquid.
5. The influence of washing on the recovery of membrane flux:For the membrane with small pore (0.1μm and0.2μm), besides pure water, acid and alkaline cleaning membrane flux can be achieved about90%of the pure water flux, flux can be recovered to100%after soaking with NaClO and nitric acid overnight.
Conclusions:microfiltration can be most efficiently obtained for FESH with a suitable membrane (0.2μm pore), at a suitable temperatyre (25℃), under a suitable pressure (0.15MPa) and washed twice with1.5times residual liquid. Ceramic membrane can effectively remove insoluble or macromolecular impurities in the water extract of RSH and it is suitable for the purification and refine of the total flavonoids in RSH.
Part2Effect of flavonoids from Rabdosia serra (Maxim.) Hara on liver fibrosis
Backgrounds:
Liver fibrosis (hepatic fibrosis, HF) is a necessary stage to develop liver cirrhosis, the main causes of LF including liver inflammation caused by a variety of pathogenic factors and compensatory responses during the process of tissue repair after liver injury. During the course, the synthesis of extracellular matrix (ECM) is greater than the degradation of ECM, this response leads to the excessive deposition of ECM and the formation of LF. Studies have shown that pathological changes of liver fibrosis could be reversed or slowed down after effective treatment. So far, even many anti fibrosis drugs have been developed under the efforts of pharmacologists from all over the world, however, the ideal compounds/drugs have not yet been synthesed or found. In the present study, we aimed to identify effective natural ingredients for the treatment of liver fibrosis from the natural plants in China. Three different kinds of commonly used animal models of hepatic fibrosis were adopted and the protective effects of flavonoids from Rabdosia serra (Maxim.) Hara (FRSH) against acute liver injury were evaluated. Our research provided the experimental basis for its clinical application.
Methods
1. Healthy SD rats were randomly divided into6groups, normal control group, model group, Silibinin group (50mg/kg), FRSH group (9mg/kg), FRSH group (18mg/kg) and FRSH group (36mg/kg).
2. Animal models of liver fibrosis were constructed through ligation of common bile duct, intraperitoneal injections of pig serum or subcutaneous injection of CCl4、the levels of TBIL、DBIL、ALT、AST、HA、LN、PC-Ⅲ、TNF-α、ALB/GLB in the serum were detected, the activities or amount of SOD、MDA、GSH-Px、Hyp in the homogenate of liver tissue were analyzed, ELISA assay was used to identify the expression of MMP-1and TIMP-1protein in liver tissue, the structure of liver cell and the degree of liver fibrosis was observed by HE staining and the protein level of a-SMA was obtained by immunohistochemical method.
Results:
1. Effect of FRSH against liver fibrosis induced by Bile duct ligation (BDL) Compared with the Sham group, the levels of TBIL、DBIL、ALT、AST were increased significantly in the model group(P=0.000), Compared with the model group, levels of TBIL、DBIL、ALT、AST in the rats treated with FRSH were decreased significantly(P<0.05or0.01). Compared with the Sham group, the content of PC-Ⅲ、HA、LN、Hyp were increased significantly in model group(P=0.000), Compared with the model group, the rats treated with FRSH were decreased significantly(P<0.05or0.01).Compared with the Sham group, the activities of SOD and GSH-Px were decreased in model group (P=0.000). Meanwhile, the content of MDA、TNF-α were increased significantly (P=0.000). Compared with the model group, the activities of SOD and GSH-Px were increased and the content of MDA, TNF-α were decrease significantly (P<0.05) in three kind of FRSH group. Compared with the Sham group, the protein expression of TIMP-1were increased (P<0.01), However, there is no marked alteration was observed concerning the content of MMP-1(P>0.05). Compared with the model group, the protein expression of TIMP-1were decreased (P<0.01) after FRSH treatment.
2. Effect of FRSH against liver fibrosis induced by porcine serum Compared with the control group, the levels of ALT、AST were increased significantly in model group(P=0.000), the injury of liver decreased the ratio of ALB/GLB(P=0.000). Compared with the model group, The index of liver function were decreased significantly in three kind of FRSH group(P<0.05or0.01). Compared with the control group, the content of PC-Ⅲ、HA、LN、Hyp were increased significantly in model group (P=0.000), Compared with the model group, the index of liver fibrosis were decrease(P<0.05or0.01). Compared with the control group, the protein expression of TIMP-1were increased (P=0.000), However, there is no marked alteration was observed concerning the content of MMP-1(P>0.05). Compared with the model group, the protein expression of TIMP-1were decreased (P<0.01) after FRSH treatment.
3. Effect of FRSH against liver fibrosis induced by CCl4compounded other factors Compared with the control group, the levels of TBIL、DBIL、ALT、AST were increased significantly in model group(P=0.000), Compared with the model group, the rats treated with FRSH were decreased significantly(P<0.05or0.01), Compared with the control group, the content of PC-Ⅲ、HA、LN、Hyp were increased significantly in model group(P=0.000), Compared with the model group, the rats treated with FRSH were decreased significantly(P<0.05or0.01). Compared with the control group, the activities of SOD and GSH-Px were decreased in model group (P=0.000). Meanwhile, the content of MDA、TNF-α were increased significantly (P=0.000). Compared with the model group, the activities of SOD and GSH-Px were increased and the content of MDA、TNF-α were decrease significantly (P<0.05) in three kind of FRSH group. Compared with the control group, the protein expression of TIMP-1were increased (P<0.01), However, there is no marked alteration was observed concerning the content of MMP-1(P>0.05). Compared with the model group, the protein expression of TIMP-1were decreased (P<0.01) after FRSH treatment.
4. liver HE staining showed that hepatic lobule structure was clear and intact in the normal control group, no cellular proliferation of the small bile ducts and fibrous tissue was observed as well; In model group, There was no discernable normal lobular architecture, obvious edema and focal necrosis were observed in liver cells, small bile duct proliferation with radial extension within the liver parenchyma could be seen under an electron microscope, fibrous tissue hyperplasia and inflammatory cell infiltration were formed around the bile duct, hepatic lobule structure in the silymarin groups was clear, however, lobules in focal liver cells exhibited spotty necrosis, the small bile ducts in the liver portal area were centered with mild hyperplasia, mild hyperplasia of fibrous tissue and a small amount of inflammatory cell infiltration could be observed. Based on these findings, we further investigated the protective effect of FRSH against liver injury in these animal models, we found that treatment with middle dose of FRSH attenuated the pathological characteristics of the liver cells, the hepatic lobule structure was still clear, small duct in portal area was suffered with mild hyperplasia and around with proliferating fibrous tissues.
5. Immunohistochemical staining of a-SMA protein expression in the liver showed that a-SMA proteins in the liver tissue were mainly distributed in portal area. In normal control group, only a few positive expression of a-SMA were visible in liver tissue portal area; while, in model control group, a-SMA positive expression in the liver tissue area were greatly increased; a-SMA positive expression in the liver tissue area of silymarin group was less than that in the model control group; FRSH protected the structure of liver cells and attenuated the expression of a-SMA in the liver tissue.
Conclusions:
FRSH has a therapeutic effect on hepatic fibrosis in the animal model of common bile duct ligation, intraperitoneal injections of pig serum and subcutaneous injection of CCl4; liver functions and liver fibrosis index are remarkably improved in all cases, the protective role of FRSH may be related to its alleviation of oxidative stress and inflammatory response in rats.
Part3The Preventive effects of flavonoids from Rabdosia serra (Maxim.) Hara on acute liver injury
Background:Liver injury is a common disease in clinic, which refers to the hepatic lesion caused by invasion of external factors. According to its etiology, it can be divided into biological liver injury, autoimmune liver injury and chemical liver injury. Common causes of liver damage including drinking too much, critical fatigue or exhaustion, environmental pollution, alcoholic hepatitis and fatty liver disease. In recent years, with the enhanced rate of drug abuse, the incidence of drug-induced liver injury is increasing year by year. Prevention and treatment of liver injury have become a hot research topic and also the core of this thesis. We focused on the total flanovoids, which is an important extracted part from Rabdosia serra (Maxim.) Hara. We intend to examining the protective effects of FRSH against liver injury in various acute liver injury models, further study on the preliminary mechanisms will also be performed.
Methods:
1. Healthy mice were randomly divided into6groups, namely, control group, model group, Bifendate group (100mg/kg)、FRSH group(13mg/kg)、FRSH group(26mg/kg) and FRSH group(52mg/kg).
2. liver injury models were constructed based on treatment with CCl4、BCG-LPS or Alcohol; Blood samples were taken to measure the activities of AST and ALT. The contents of MDA、GS、SOD、NOS in liver were measured by chemical colorimetry method; Pathological examinations were conducted to investigate the severity of liver injury.
Results
1. Effect of FRSH against acute liver injury induced by CCl4Compared with normal control group, levels of ALT and AST in the serum of rats in model control group were significantly increased (P<0.01); compared with model control group, the activities of ALT and AST in blood decreased remarkably and showed a dose effect relationship in each dosage group of FRSH (P<0.05or P<0.01). Compared with normal control group, the content of MDA was increased, while the levels of GSH and SOD in the liver tissue were decreased in model control group; both bifendate and FRSH downregulated the expression of MDA and enhanced the activities of GSH and SOD significantly (P<0.05or P<0.01).
2. Effect of FRSH against acute liver injury induced by combined treatment with bccillus calmette guerin and lipopolysaccharide
Compared with normal control group, levels of ALT and AST in the serum of rats in model control group were significantly increased (P<0.01); compared with model control group, the activities of ALT and AST in blood decreased remarkably and showed a dose effect relationship in each dosage group of FRSH and positive control group (P<0.05or P<0.01). Compared with normal control group, the content of MDA and NOS were increased, while the levels of GSH and SOD in the liver tissue were decreased in model control group; both bifendate and FRSH reduced the levels of both MDA and NOS, on the other hand, the activities of GSH and SOD were increased significantly after treatment with FRSH or bifendate group (P<0.05or P<0.01).
3. Effect of FRSH against acute liver injury induced by alcohol.
Compared with normal control group, levels of ALT and AST in the serum of rats in model control group were significantly increased (P<0.01); compared with model control group, the activities of ALT and AST in blood decreased remarkably and showed a dose effect relationship in each dosage group of FRSH (P<0.05or P<0.01). Compared with normal control group, the content of MDA was increased, while the levels of GSH and SOD in the liver tissue were decreased in model control group; both bifendate and FRSH downregulated the expression of MDA and enhanced the activities of GSH and SOD significantly (P<0.05or P<0.01).
4. Histopathologic examination
Livers in the mice of normal control group mice were ruddy, luster and elastic, the structure of lobules was intact and normal, the cytoplasm of liver cells was homogeneous and the nucleus was round in shape. Cells around central vein were were basically complete, and neatly arranged as well. On the contrary, hepatocyte showed multiple spotty necrosis in the model group, the main pathologic changes including yellowish gray dot necrosis, slightly crispy texture; By light microscopy, the hepatocytes were necrotic with fuzzy boundary around the central vein, the cytoplasm was light color, the nucleus was shrink and the intercellular space was increased, around central vein, inflammatory cell infiltration is also evident. Dotted necrosis in the hepatic lobule and inflammatory cells infiltration was also observed in the positive control group; however, the changes described above were markedly abated. After treatment with FRSH, the lobular architecture was relatively complete and the liver shows only mild necrosis,
Conclusions:FRSH has markedly protective effect on liver injury induced by CCl4or combined treatment with bccillus calmette guerin and lipopolysaccharide or alcohol, this effect may be attributed to the property that FRSH could improve the resistance to oxidative stress, reduce the cytoplasm membrane lipid peroxidation, so as to fix the cytoplasm membrane and reduce liver damage.
研究背景及目的：
溪黄草属于唇形科香茶属植物,是一种多年生草本植物,含黄酮类、酚类等多种化学成分。溪黄草具有清热祛湿、凉血散瘀之功效,对急性肝炎、肠炎、痢疾等有较好效果。近年来,中草药溪黄草在华南各地临床上广为应用,并出现了一些以其为主要原料的保健产品,具有较好的开发应用前景。溪黄草现有制剂工艺过程均采用传统水煎醇沉除杂工艺,工艺复杂,具有一定的不足。膜分离技术应用于中药制剂的生产过程具有效率高、降低污染、节省能源等优点。无机陶瓷膜具有机械强度高、耐腐蚀等特点,对中药提取液的纯化除杂有其独特的优点。本实验拟定了陶瓷膜微滤参数,最有效地为溪黄草黄酮类物质的提取、纯化等提供支持。
研究方法：
本实验将溪黄草水提取液用无机陶瓷膜微滤机以错流方式进行循环微滤,测定各孔径陶瓷膜的滤过情况、各孔径陶瓷膜总黄酮含量及转移率、除固率、膜通量,从而确定最佳陶瓷膜孔径；测定六个温度对有效成分转移率、膜通量、总黄酮含量的影响,从而确定最佳微滤温度；分别考察四个操作压力对有效成分转移率、膜通量的影响,确定最佳操作压力；取溪黄草水提液在确定条件下进行微滤,测定总黄酮转移率,从而确定最佳顶洗条件。通过测量通量恢复率测试每步清洗对膜通量恢复的影响；
研究结果：
1.最佳陶瓷膜孔径：0.1μm、0.2μm、0.5μm、0.8μm各孔径陶瓷膜滤液的收得率为93%、92%、86%、90%；总黄酮累计转移率为82%、87.1%、82%、87.2%；除了0.8μm外,其余三个孔径除固率都在20%左右；0.2μm孔径陶瓷膜微滤过程中通量衰减较小,稳定时膜通量最大。
2.最佳微滤温度：45℃-75℃范围的膜通量增长比较稳定,约为300L·h-1·-2,25℃-45℃范围内微滤温度的总黄酮转移率约为95%。
3.最佳操作压力：在恒定的微滤温度下25℃,0.15MPa压力下总黄酮转移率为96%。
4.最佳顶洗条件的确定：顶洗1.5倍量残存液的顶洗量,顶洗两次,总黄酮累计转移率为99.79%,更高效地获得溪黄草总黄酮。
5.清洗对膜通量恢复的影响：对于膜孔径较小的0.1μm、0.2μm陶瓷膜,除纯水外,酸洗和碱洗对膜通量的恢复有一定作用,0.5%NaOH+0.5%多聚磷酸钠+0.5%EDTA二钠盐洗后膜通量能达到过滤前原始纯水通量的90%, NaClO+硝酸浸泡一晚后通量能恢复到100%。
研究结论
陶瓷膜较适用于溪黄草水提液的纯化精制,利用陶瓷膜微滤技术可有效除去溪黄草水提物中的不溶性或大分子杂质,能对总黄酮进行更高效的分离、浓缩、纯化和精制。陶瓷膜微滤最佳条件为：在25℃,0.15MPa下,用0.2μm孔径陶瓷膜,1.5倍量残存液的顶洗量,顶洗两次的条件进行微滤能高效地获得溪黄草总黄酮,采用0.5%NaOH+0.5%多聚磷酸钠+0.5%EDTA二钠盐及NaClO+硝酸浸泡为陶瓷膜的最佳清洗方法。
第二部分溪黄草黄酮抗肝纤维化作用
研究背景及目的：
肝纤维化(hepatic fibrosis, HF)是慢性肝病发展成肝硬化的必经阶段,是多种致病因子引起的肝脏炎症或损伤后组织修复过程中的代偿反应,以细胞外基质(extracellular matrix, ECM)合成大于降解导致ECM的过度沉积的结果。有研究表明,肝纤维化病变经积极治疗可延缓甚至逆转。目前,各国学者从肝纤维化发生发展的不同环节入手,研制了许多抗纤维化药物,但因种种原因尚未找到十分理想的药物。因此,从我国天然植物资源宝库中找寻到抗纤维化的天然有效成分,具有极其重要的社会意义和经济意义。本实验通过三种常用肝纤维化损伤的动物模型,观察溪黄草黄酮(flavonoids from Rabdosia serra (Maxim.) Hara, FRSH)对急性肝损伤的保护作用,为溪黄草黄酮在临床应用提供科学依据。
研究方法：
1、健康SD大鼠随机分为6组,即假手术对照组/正常对照、模型对照组、水飞蓟素组50mg/kg、溪黄草黄酮低剂量组9mg/kg、溪黄草黄酮中剂量组18mg//kg、溪黄草黄酮高剂量组36mg//kg。
2、采用胆总管结扎、腹腔注射猪血清及皮下注射CCl4复合因素三种方法造成肝纤维化动物模型。从血清检测TBIL、DBIL、ALT、AST、HA、LN、PC-Ⅲ、TNF-α、 ALB/GLB水平；肝组织匀浆后检测SOD、MDA、GSH-Px、Hyp,采用ELISA法检测MMP-1和TIMP-1蛋白的表达,通过HE染色观察肝细胞结构和肝纤维化程度,并通过免疫组织化学法(SABC法)检测肝组织α-肌动蛋白(a-SMA)的表达。
研究结果：
1、溪黄草黄酮抗胆总管结扎所致大鼠肝纤维化的实验研究
与假手术组比较,模型对照组大鼠血清中肝功能指标TBIL、DBIL、ALT、AST水平均显著升高(均P=0.000),与模型对照组比较,溪黄草黄酮低、中、高剂量组大鼠血清中肝功能指标显著降低(P<0.05或0.01)。与假手术组比较,模型对照组大鼠肝纤维化指标PC-Ⅲ、HA、LN、Hyp含量均显著升高(均P=0.000),与模型对照组比较,溪黄草黄酮低、中、高剂量组大鼠肝纤维化指标显著降低(P<0.05或0.01)。与正常对照组比较,模型组肝组织GSH-Px活性显著降低(均P=0.000),而MDA、TNF-α含量提高有显著性差异(均P=0.000),与模型对照组比较,FRSH各剂量组肝组织SOD、GSH-Px活性显著升高(P<0.05),而MDA、TNF-α含量降低有统计学差异(P<0.01)。与正常对照组比较,模型组肝组织TIMP-1蛋白表达显著增加(P=0.000),而模型组肝组织MMP-1无统计学意义(P>0.05),与模型对照组比较,溪黄草黄酮低、中、高剂量组大鼠肝组织TIMP-1蛋白表达显著降低(P<0.01)。
2、溪黄草黄酮抗猪血清致大鼠免疫性肝纤维化的实验研究
与正常对照组比较,模型对照组大鼠血清中肝功能指标ALT、AST水平均显著升高(均P=0.000), ALB/GLB值降低有显著性差异(P=0.000),与模型对照组比较,溪黄草黄酮低、中、高剂量组大鼠血清中肝功能指标显著降低(P<0.05或0.01), ALB/GLB值显著升高(P<0.05或0.01)。与正常对照组比较,模型对照组大鼠肝纤维化指标PC-Ⅲ、HA、LN、Hyp含量均显著升高(均P=0.000),与模型对照组比较,溪黄草黄酮低、中、高剂量组大鼠肝纤维化指标显著降低(P<0.05或0.01)。与正常对照组比较,模型组大鼠肝组织TIMP-1蛋白表达显著增加(P=0.000),而MMP-1无统计学意义(P>0.05),与模型对照组比较,溪黄草黄酮低、中、高剂量组大鼠肝组织TIMP-1蛋白表达显著降低(P<0.01)。
3、溪黄草黄酮抗CCl4复合因素所致大鼠肝纤维化的实验研究
与正常对照组比较,模型对照组大鼠血清中肝功能指标TBIL、DBIL、ALT、AST水平均显著升高(均P=0.000),与模型对照组比较,溪黄草黄酮低、中、高剂量组大鼠血清中肝功能指标显著降低(P<0.05或0.01)。与正常对照组比较,模型对照组大鼠肝纤维化指标PC-Ⅲ、HA、LN、Hyp含量均显著升高(均P=0.000),与模型对照组比较,溪黄草黄酮低、中、高剂量组大鼠肝纤维化指标显著降低(P<0.05或0.01)。与正常对照组比较,肝组织SOD、GSH-Px活性显著降低(均P=0.000),而MDA、TNF-α含量提高有显著性差异(均P=0.000),与模型对照组比较,肝组织SOD、GSH-Px活性显著升高(P<0.01),而MDA、TNF-α含量降低有统计学差异(P<0.01)。与正常对照组比较,肝组织TIMP-1蛋白表达显著增加(P=0.000),而肝组织MMP-1无统计学意义(P>0.05),与模型对照组比较,溪黄草黄酮低、中、高剂量组大鼠肝组织TIMP-1蛋白表达显著降低(P<0.05或0.01)。
4、肝组织HE染色结果
三种肝纤维化动物试验研究中,正常对照组肝小叶结构清晰,小叶内肝细胞基本正常,汇管区结构清晰,汇管区及汇管区周围未见小胆管及纤维组织增生；模型对照组见肝小叶结构模糊,小叶内肝细胞水肿,局灶肝细胞坏死,小胆管大量增生,以汇管区为中心放射状向肝实质内延伸,增生的胆管呈花环样,增生胆管周围伴纤维组织增生及炎细胞浸润；水飞蓟素组见肝小叶结构较清晰,小叶内局灶肝细胞呈点状坏死,肝内小胆管以汇管区为中心轻度增生,增生胆管周围伴轻度纤维组织增生及少量炎细胞浸润；溪黄草黄酮组可见肝小叶结构较清晰,小胆管以汇管区为中心轻度增生,增生胆管周围伴轻度纤维组织增生。
5、肝组织α-SMA蛋白表达免疫组化染色结果
三种肝纤维化动物试验研究中,正常对照组可见α-SMA在肝组织汇管区少量阳性表达；模型对照组可见α-SMA在肝组织内阳性表达的面积大大增加；水飞蓟素组α-SMA在肝组织内阳性表达的面积少于模型对照组；溪黄草黄酮组α-SMA在肝组织内阳性表达的面积明显少于模型对照组,趋于正常。
研究结论：
溪黄草黄酮对胆总管结扎、腹腔注射猪血清及皮下注射CCl4复合因素三种方法造成的肝纤维化动物模型都具有治疗作用,肝功能指标和肝纤维化指标都趋于正常,该作用与溪黄草黄酮可缓解大鼠肝脏的氧化应激状态、调节脂质代谢,增强肝脏抗氧化,减轻脂质过氧化损伤作用有关。
第三部分溪黄草黄酮对急性肝损伤的预防作用
研究背景及目的：
肝损伤是临床常见的危害人类健康的疾病,肝损伤的常见病因是饮酒过多、熬夜过劳、环境污染、脂肪肝等。肝损伤主要分为酒精性肝损伤、免疫性肝损伤及化学性肝损伤。近年来随着药物滥用,药源性肝损伤发病呈逐年增加趋势。肝损伤的防治目前已成为国内外研究的热点与重点课题,本实验拟以中草药溪黄草重要的有效成分黄酮,考察其对多种因素诱导的的急性肝损伤模型的预防作用,进一步研究溪黄草黄酮的保肝、护肝作用,并对其机制进行初步探讨。
研究方法：
1、健康昆明种小鼠随机分为6组,即正常对照组、模型对照组、联苯双酯组100mg/kg、溪黄草黄酮低剂量组13mg/kg、溪黄草黄酮中剂量组26mg/kg、溪黄草黄酮高剂量组52mg/kg。
2、经过CC14、BCG-LPS及酒精处理后造成急性肝损伤模型,取血测定AST、ALT活性；肝组织匀浆后检测相应的MDA、GSH、SOD、NOS指标并进行肝组织病理组织学检查。
研究结果：
1、溪黄草黄酮对CCl4致小鼠急性肝损伤的预防作用
与正常对照组比较,模型对照组小鼠血清中肝功能指标ALT、AST水平均显著升高(均P=0.000),与模型对照组比较,溪黄草黄酮低、中、高剂量组的血清中ALT、AST活性显著降低(P<0.05或0.01)。与正常对照组比较,模型对照组小鼠肝组织MDA含量显著升高,GSH含量和SOD活性显著降低,有统计学意义(均P=0.000),与模型对照组比较,联苯双酯组与溪黄草黄酮低、中、高剂量组小鼠肝组织MDA含量显著降低而GSH含量和SOD活性显著升高(P<0.05或0.01)。
2、溪黄草黄酮对卡介苗联合脂多糖所致小鼠急性肝损伤的预防作用
与正常对照组比较,模型对照组小鼠血清中肝功能指标ALT、AST水平均显著升高(均P=0.000),与模型对照组比较,联苯双酯组与溪黄草黄酮低、中、高剂量组的血清中ALT、AST活性显著降低(P<0.05或0.01)；与正常对照组比较,模型对照组小鼠肝组织MDA含量和NOS活性显著升高,GSH含量和SOD活性显著降低(均P=0.000),与模型对照组比较,联苯双酯组与溪黄草黄酮低、中、高剂量组小鼠肝组织MDA含量和NOS活性显著降低而GSH含量和SOD活性显著升高(P<0.05或0.01)。
3、溪黄草黄酮对酒精致小鼠急性肝损伤的预防作用
与正常对照组比较,模型对照组小鼠血清中肝功能指标ALT、AST均显著升高(均P=0.000),与模型对照组比较,联苯双酯组与溪黄草黄酮低、中、高剂量组的血清中ALT、AST活性显著降低(P<0.05或0.01)；与正常对照组比较,模型组小鼠肝组织MDA含量显著升高,GSH含量和SOD活性显著降低,有统计学意义(均P=0.000),与模型对照组比较,联苯双酯组与溪黄草黄酮低、中、高剂量组小鼠肝组织MDA含量显著降低而GSH含量和SOD活性显著升高(P<0.05或0.01)。
4、病理组织学检查结果
三种急性肝损伤动物模型试验中,正常对照组小鼠肝脏颜色红润,有光泽且富于弹性,光学显微镜下可见肝小叶结构完整,肝细胞胞质丰富,核大而圆,核仁清晰,中央静脉及门管区正常,细胞排列整齐。模型对照组的肝脏发生明显病理改变,主要表现为灰黄色点状坏死灶,表面无光泽,质地稍脆,光镜下,肝小叶中央静脉周围坏死,坏死细胞轮廓不清,肝细胞出现中至重度病变,胞浆疏松、淡染,细胞核多皱缩,细胞间隙增大,且肝细胞多见点状坏死灶及门管区见炎细胞浸润。联苯双酯组小鼠的肝小叶内见点状坏死,坏死处炎细胞浸润,病理肝损伤减轻。溪黄草黄酮组小鼠的肝小叶结构完整,肝索呈放射状排列,部分肝细胞仅见轻度的病理改变,肝细胞轻度水肿,胞浆疏松、淡染,肝脏组织结构与模型组比较,病理损伤减轻明显。
研究结论：
溪黄草黄酮对化学性、免疫性及酒精所导致的急性肝损伤都具有预防性保护作用,该作用可能与溪黄草黄酮能够提高小鼠肝组织的抗氧化应激能力,减少细胞质膜的脂质过氧化水平,从而修复细胞质膜,降低肝损伤有密切关系。
Part1Research on the separation of FRSH with inorganic ceramic membrane
Background:Rabdosia serra (Maxim.) Hara(RSH), a labiatae and perennial herbaceous plant, contains flavonoids, phenols and other chemical compositions. It is widely used for the treatment of acute hepatitis, enteritis and diarrhea in south China and provides main raw material for health care products. So, this plant is worthy of extensive study and development. However, existing preparation adopts the traditional water extract-alcohol sedimentation process; the process is complex and contains certain shortcomings. Application of membrane separation technology to the extraction process of traditional Chinese medicine (TCM) is a desirable choice and it has advantages of high efficiency, low pollution and low consumption of energy. Inorganic ceramic membrane has the characteristics of high mechanical strength and corrosion resistance; it has unique advantages on the extraction of traditional Chinese herb and purification in removing impurities. This study focused on the most optimal parameters for ceramic membrane microfiltration, it provides methodological support for the extraction and purification of total flavonoids from RSH.
Methods:
Water extract of RSH was filtrated through inorganic ceramic membrane filtration system with cross-flow microfiltration. the pore size of ceramic membrane, the total content of flavonoids, transfer rate and removal rate of solid impurities were measured, so as to determine the best condition for ceramic membrane microfiltration; the cleaning effect on membrane flux recovery was determined by measuring the flux recovery rate in each step; effects of six various temperatures on the components transfer rate, membrane flux, flavonoids content were measured, so as to determine the best microfiltration temperature; transfer rate and membrane flux of active ingredients with four operating pressure were investigated respectively to investigate the optimum operating pressure; water solution of RSH was filtrated under the determined condition of microfiltration, the transfer rate of flavones was evaluated, so as to determine the best washing condition.
Results:
1. The best ceramic membrane pore size:The filtrate rate of various pore size of ceramic membrane (0.1μm,0.2μm,0.5μm,0.8μm) was93%,92%,86%,90%and the total cumulative flavonoids transfer rate was82%,87.1%,82%and87.2%respectively, in addition to0.8μm membrane, the removal rate of solid impurities was about20%,0.2μm aperture in the process of the ceramic microfiltration membrane flux attenuation of the membrane with0.2μm pore is small and the membrane flux is the largest in stable condition.
2. The optimum microfiltration temperature:the membrane flux is about300L·h-1·m-2when the temperature set at45℃-75℃25℃, the flavonoids transfer rate is about95%when the temperature was25℃-45℃.
3. The best operating pressure:In a constant microfiltration temperature (25℃), flavonoids transfer rate was96%under0.15MPa.
4. The determination of optimum washing conditions:The cumulative total flavonoids transfer rate was99.79%when washed twice with1.5times the amount of residual liquid.
5. The influence of washing on the recovery of membrane flux:For the membrane with small pore (0.1μm and0.2μm), besides pure water, acid and alkaline cleaning membrane flux can be achieved about90%of the pure water flux, flux can be recovered to100%after soaking with NaClO and nitric acid overnight.
Conclusions:microfiltration can be most efficiently obtained for FESH with a suitable membrane (0.2μm pore), at a suitable temperatyre (25℃), under a suitable pressure (0.15MPa) and washed twice with1.5times residual liquid. Ceramic membrane can effectively remove insoluble or macromolecular impurities in the water extract of RSH and it is suitable for the purification and refine of the total flavonoids in RSH.
Part2Effect of flavonoids from Rabdosia serra (Maxim.) Hara on liver fibrosis
Backgrounds:
Liver fibrosis (hepatic fibrosis, HF) is a necessary stage to develop liver cirrhosis, the main causes of LF including liver inflammation caused by a variety of pathogenic factors and compensatory responses during the process of tissue repair after liver injury. During the course, the synthesis of extracellular matrix (ECM) is greater than the degradation of ECM, this response leads to the excessive deposition of ECM and the formation of LF. Studies have shown that pathological changes of liver fibrosis could be reversed or slowed down after effective treatment. So far, even many anti fibrosis drugs have been developed under the efforts of pharmacologists from all over the world, however, the ideal compounds/drugs have not yet been synthesed or found. In the present study, we aimed to identify effective natural ingredients for the treatment of liver fibrosis from the natural plants in China. Three different kinds of commonly used animal models of hepatic fibrosis were adopted and the protective effects of flavonoids from Rabdosia serra (Maxim.) Hara (FRSH) against acute liver injury were evaluated. Our research provided the experimental basis for its clinical application.
Methods
1. Healthy SD rats were randomly divided into6groups, normal control group, model group, Silibinin group (50mg/kg), FRSH group (9mg/kg), FRSH group (18mg/kg) and FRSH group (36mg/kg).
2. Animal models of liver fibrosis were constructed through ligation of common bile duct, intraperitoneal injections of pig serum or subcutaneous injection of CCl4、the levels of TBIL、DBIL、ALT、AST、HA、LN、PC-Ⅲ、TNF-α、ALB/GLB in the serum were detected, the activities or amount of SOD、MDA、GSH-Px、Hyp in the homogenate of liver tissue were analyzed, ELISA assay was used to identify the expression of MMP-1and TIMP-1protein in liver tissue, the structure of liver cell and the degree of liver fibrosis was observed by HE staining and the protein level of a-SMA was obtained by immunohistochemical method.
Results:
1. Effect of FRSH against liver fibrosis induced by Bile duct ligation (BDL) Compared with the Sham group, the levels of TBIL、DBIL、ALT、AST were increased significantly in the model group(P=0.000), Compared with the model group, levels of TBIL、DBIL、ALT、AST in the rats treated with FRSH were decreased significantly(P<0.05or0.01). Compared with the Sham group, the content of PC-Ⅲ、HA、LN、Hyp were increased significantly in model group(P=0.000), Compared with the model group, the rats treated with FRSH were decreased significantly(P<0.05or0.01).Compared with the Sham group, the activities of SOD and GSH-Px were decreased in model group (P=0.000). Meanwhile, the content of MDA、TNF-α were increased significantly (P=0.000). Compared with the model group, the activities of SOD and GSH-Px were increased and the content of MDA, TNF-α were decrease significantly (P<0.05) in three kind of FRSH group. Compared with the Sham group, the protein expression of TIMP-1were increased (P<0.01), However, there is no marked alteration was observed concerning the content of MMP-1(P>0.05). Compared with the model group, the protein expression of TIMP-1were decreased (P<0.01) after FRSH treatment.
2. Effect of FRSH against liver fibrosis induced by porcine serum Compared with the control group, the levels of ALT、AST were increased significantly in model group(P=0.000), the injury of liver decreased the ratio of ALB/GLB(P=0.000). Compared with the model group, The index of liver function were decreased significantly in three kind of FRSH group(P<0.05or0.01). Compared with the control group, the content of PC-Ⅲ、HA、LN、Hyp were increased significantly in model group (P=0.000), Compared with the model group, the index of liver fibrosis were decrease(P<0.05or0.01). Compared with the control group, the protein expression of TIMP-1were increased (P=0.000), However, there is no marked alteration was observed concerning the content of MMP-1(P>0.05). Compared with the model group, the protein expression of TIMP-1were decreased (P<0.01) after FRSH treatment.
3. Effect of FRSH against liver fibrosis induced by CCl4compounded other factors Compared with the control group, the levels of TBIL、DBIL、ALT、AST were increased significantly in model group(P=0.000), Compared with the model group, the rats treated with FRSH were decreased significantly(P<0.05or0.01), Compared with the control group, the content of PC-Ⅲ、HA、LN、Hyp were increased significantly in model group(P=0.000), Compared with the model group, the rats treated with FRSH were decreased significantly(P<0.05or0.01). Compared with the control group, the activities of SOD and GSH-Px were decreased in model group (P=0.000). Meanwhile, the content of MDA、TNF-α were increased significantly (P=0.000). Compared with the model group, the activities of SOD and GSH-Px were increased and the content of MDA、TNF-α were decrease significantly (P<0.05) in three kind of FRSH group. Compared with the control group, the protein expression of TIMP-1were increased (P<0.01), However, there is no marked alteration was observed concerning the content of MMP-1(P>0.05). Compared with the model group, the protein expression of TIMP-1were decreased (P<0.01) after FRSH treatment.
4. liver HE staining showed that hepatic lobule structure was clear and intact in the normal control group, no cellular proliferation of the small bile ducts and fibrous tissue was observed as well; In model group, There was no discernable normal lobular architecture, obvious edema and focal necrosis were observed in liver cells, small bile duct proliferation with radial extension within the liver parenchyma could be seen under an electron microscope, fibrous tissue hyperplasia and inflammatory cell infiltration were formed around the bile duct, hepatic lobule structure in the silymarin groups was clear, however, lobules in focal liver cells exhibited spotty necrosis, the small bile ducts in the liver portal area were centered with mild hyperplasia, mild hyperplasia of fibrous tissue and a small amount of inflammatory cell infiltration could be observed. Based on these findings, we further investigated the protective effect of FRSH against liver injury in these animal models, we found that treatment with middle dose of FRSH attenuated the pathological characteristics of the liver cells, the hepatic lobule structure was still clear, small duct in portal area was suffered with mild hyperplasia and around with proliferating fibrous tissues.
5. Immunohistochemical staining of a-SMA protein expression in the liver showed that a-SMA proteins in the liver tissue were mainly distributed in portal area. In normal control group, only a few positive expression of a-SMA were visible in liver tissue portal area; while, in model control group, a-SMA positive expression in the liver tissue area were greatly increased; a-SMA positive expression in the liver tissue area of silymarin group was less than that in the model control group; FRSH protected the structure of liver cells and attenuated the expression of a-SMA in the liver tissue.
Conclusions:
FRSH has a therapeutic effect on hepatic fibrosis in the animal model of common bile duct ligation, intraperitoneal injections of pig serum and subcutaneous injection of CCl4; liver functions and liver fibrosis index are remarkably improved in all cases, the protective role of FRSH may be related to its alleviation of oxidative stress and inflammatory response in rats.
Part3The Preventive effects of flavonoids from Rabdosia serra (Maxim.) Hara on acute liver injury
Background:Liver injury is a common disease in clinic, which refers to the hepatic lesion caused by invasion of external factors. According to its etiology, it can be divided into biological liver injury, autoimmune liver injury and chemical liver injury. Common causes of liver damage including drinking too much, critical fatigue or exhaustion, environmental pollution, alcoholic hepatitis and fatty liver disease. In recent years, with the enhanced rate of drug abuse, the incidence of drug-induced liver injury is increasing year by year. Prevention and treatment of liver injury have become a hot research topic and also the core of this thesis. We focused on the total flanovoids, which is an important extracted part from Rabdosia serra (Maxim.) Hara. We intend to examining the protective effects of FRSH against liver injury in various acute liver injury models, further study on the preliminary mechanisms will also be performed.
Methods:
1. Healthy mice were randomly divided into6groups, namely, control group, model group, Bifendate group (100mg/kg)、FRSH group(13mg/kg)、FRSH group(26mg/kg) and FRSH group(52mg/kg).
2. liver injury models were constructed based on treatment with CCl4、BCG-LPS or Alcohol; Blood samples were taken to measure the activities of AST and ALT. The contents of MDA、GS、SOD、NOS in liver were measured by chemical colorimetry method; Pathological examinations were conducted to investigate the severity of liver injury.
Results
1. Effect of FRSH against acute liver injury induced by CCl4Compared with normal control group, levels of ALT and AST in the serum of rats in model control group were significantly increased (P<0.01); compared with model control group, the activities of ALT and AST in blood decreased remarkably and showed a dose effect relationship in each dosage group of FRSH (P<0.05or P<0.01). Compared with normal control group, the content of MDA was increased, while the levels of GSH and SOD in the liver tissue were decreased in model control group; both bifendate and FRSH downregulated the expression of MDA and enhanced the activities of GSH and SOD significantly (P<0.05or P<0.01).
2. Effect of FRSH against acute liver injury induced by combined treatment with bccillus calmette guerin and lipopolysaccharide
Compared with normal control group, levels of ALT and AST in the serum of rats in model control group were significantly increased (P<0.01); compared with model control group, the activities of ALT and AST in blood decreased remarkably and showed a dose effect relationship in each dosage group of FRSH and positive control group (P<0.05or P<0.01). Compared with normal control group, the content of MDA and NOS were increased, while the levels of GSH and SOD in the liver tissue were decreased in model control group; both bifendate and FRSH reduced the levels of both MDA and NOS, on the other hand, the activities of GSH and SOD were increased significantly after treatment with FRSH or bifendate group (P<0.05or P<0.01).
3. Effect of FRSH against acute liver injury induced by alcohol.
Compared with normal control group, levels of ALT and AST in the serum of rats in model control group were significantly increased (P<0.01); compared with model control group, the activities of ALT and AST in blood decreased remarkably and showed a dose effect relationship in each dosage group of FRSH (P<0.05or P<0.01). Compared with normal control group, the content of MDA was increased, while the levels of GSH and SOD in the liver tissue were decreased in model control group; both bifendate and FRSH downregulated the expression of MDA and enhanced the activities of GSH and SOD significantly (P<0.05or P<0.01).
4. Histopathologic examination
Livers in the mice of normal control group mice were ruddy, luster and elastic, the structure of lobules was intact and normal, the cytoplasm of liver cells was homogeneous and the nucleus was round in shape. Cells around central vein were were basically complete, and neatly arranged as well. On the contrary, hepatocyte showed multiple spotty necrosis in the model group, the main pathologic changes including yellowish gray dot necrosis, slightly crispy texture; By light microscopy, the hepatocytes were necrotic with fuzzy boundary around the central vein, the cytoplasm was light color, the nucleus was shrink and the intercellular space was increased, around central vein, inflammatory cell infiltration is also evident. Dotted necrosis in the hepatic lobule and inflammatory cells infiltration was also observed in the positive control group; however, the changes described above were markedly abated. After treatment with FRSH, the lobular architecture was relatively complete and the liver shows only mild necrosis,
Conclusions:FRSH has markedly protective effect on liver injury induced by CCl4or combined treatment with bccillus calmette guerin and lipopolysaccharide or alcohol, this effect may be attributed to the property that FRSH could improve the resistance to oxidative stress, reduce the cytoplasm membrane lipid peroxidation, so as to fix the cytoplasm membrane and reduce liver damage.
引文
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