开口箭多糖免疫调节作用的实验研究
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摘要
多糖(polysaccharide)是生物体内普遍存在的一类高分子化合物,具有许多重要的生理功能。多糖的药用研究始于1943年,60多年来,人们从动物、植物、微生物中提取分离了大量活性多糖,发现多糖具有促进机体免疫功能、抗肿瘤、抗病毒、抗菌、抗衰老、抗骨髓抑制和抗辐射等一系列作用,在临床上已用于治疗肝炎、艾滋病、癌症和许多其它疾病。免疫活性多糖因其来源广泛、效果确切、毒副作用小等受到了普遍重视。
开口箭(Tupistra chinensis Baker)又名牛尾七、竹根七等,主要分布于我国西南地区,民间主要用于治疗咽喉肿痛、风湿痹痛、跌打损伤、胃痛、毒蛇狂犬咬伤、蚊虫叮咬等。开口箭属植物主要活性成分为皂苷,从该属植物根茎中已分离提纯出多种皂苷成份。研究发现,开口箭具有良好的抗炎作用,有良好的开发利用价值。皂苷是其抗炎的重要物质基础之一,抑制炎症介质PGE2、NO的产生是其作用机理；开口箭皂苷体外对HL-60、Caski、251肿瘤细胞具有明显的抑制作用,体内外对S180肉瘤细胞也有很强的抑制作用；同时开口箭皂苷还能够抑制血小板的聚集与活化。也有研究报道,开口箭水提物能明显抑制炎症早期的水肿和渗出,具有抗炎镇痛的功效。我们在活性筛选时发现,开口箭水提物具有一定的免疫活性,从而引起了我们的研究兴趣。本文对分离得到的开口箭多糖组分TCBPⅡ(Tupistra chinensis Baker polysaccharidesⅡ, TCBPⅡ)的免疫调节作用及其机制进行了初步研究。
目的：
1.提取分离开口箭粗多糖并筛选出活性成分。
2.研究开口箭多糖(TCBPⅡ)体外对正常小鼠脾细胞代谢MTT活力及产生肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)、白细胞介素-2(IL-2)的影响；体外对正常小鼠腹腔渗出细胞代谢MTT活力及产生TNF-α的影响；体内对正常小鼠迟发型超敏反应(DTH)、血清溶血素水平的影响。
3.研究开口箭多糖(TCBPⅡ)体内对环磷酰胺诱导免疫抑制小鼠的DTH、单核巨噬细胞吞噬功能、血清溶血素水平的影响。
4.研究开口箭多糖(TCBPⅡ)对荷S180实体瘤小鼠抑瘤作用、血清TNF-α、IFN-γ含量及腹腔单核巨噬细胞吞噬功能的影响。
方法：
1.采用热水提取、乙醇沉淀法从开口箭植物的根茎中提取粗制多糖,再用Sevage法去蛋白。经DEAE-Sepharose fast flow阴离子交换柱对粗制多糖进行分离纯化,得到不同组分开口箭多糖。通过测定不同组分多糖对正常小鼠脾脏淋巴细胞增殖作用筛选出活性成分,命名TCBPⅡ
2.取正常小鼠脾细胞,加入开口箭多糖(TCBPⅡ),在37℃下培养48h,收集培养上清,用ELISA方法检测其中TNF-α、IFN-γ、IL-2的含量,或者用MTT比色法检测细胞代谢MTT活力。
3.取正常小鼠腹腔渗出细胞,加入开口箭多糖(TCBPⅡ),在37℃下培养10h,收集培养上清,用ELISA方法检测其中TNF-α的含量,或者用MTT比色法检测细胞代谢MTT活力。
4.正常小鼠50只,随机分为5组,生理盐水对照组、阳性药物(灵芝多糖100mg/kg)组、TCBPⅡ低、中、高剂量(50,100,200mg/kg)组,给药组小鼠分别连续腹腔注射(ip)干预药物7d。于给药后第1日用1%二硝基氟苯(dinitro-fluorobenzene, DNFB)腹部致敏,末次给药后将1%DNFB涂抹于5组实验小鼠右侧耳廓,24h后处死动物,用打孔器取相同面积两侧耳片称重,比较给药组与对照组耳廓肿胀程度。
5.正常小鼠50只,随机分为5组,生理盐水对照组、阳性药物(灵芝多糖100mg/kg)组、TCBPⅡ低、中、高剂量(50,100,200mg/kg)组,给药组小鼠分别连续ip干预药物7d。给药后第2天上午,各鼠腹腔注射20%(V/V)的生理盐水兔红细胞0.2 ml进行免疫,同日下午继续给药,免疫后d6(免疫当天为d0),动物摘眼球取血,分离和收集血清用于血清溶血素水平测定。
6.皮下注射环磷酰胺(CTX)复制免疫功能低下的动物模型。小鼠50只,随机分为5组,正常对照组、CTX模型组、TCBPⅡ低、中、高剂量(50,100,200mg/kg)+CTX组,采用DNFB诱导迟发型变态反应(delayed-type hypersensitivity, DTH)模型,观察小鼠细胞免疫功能；碳廓清法检测单核巨噬细胞的吞噬功能；兔红细胞免疫法检测体液免疫功能。
7.建立荷S180实体瘤小鼠动物模型,生理盐水对照组：每天腹腔注射(ip)无菌生理盐水0.1ml/10g体重,其他组给药体积与此相同；阳性对照组：每天ip灵芝多糖100mg/kg; TCBPⅡ低、中、高剂量组：每天分别ip TCBPⅡ50、100、200mg/kg;连续给药7d后次日上午,分离血清用于TNF-α和IFN-γ的含量测定。取血后,分离瘤体,称重,并计算抑瘤率。
8.取荷S180实体瘤小鼠50只,随机分为生理盐水对照组、阳性药物(灵芝多糖100mg/kg)组、TCBPⅡ低、中、高剂量(50,100,200mg/kg)组,每组10只,腹腔注射给药,每天1次,连续7天。停药24h后,每只小鼠腹腔注射10%鸡红细胞悬液1ml,30min后,脱颈椎处死动物,检测腹腔巨噬细胞的吞噬活力。
9.统计分析采用SPSS 13.0统计软件,两样本均数比较,用独立样本t检验(Independent-Sample T Test)。多个样本均数比较,经方差齐性检验后进行单因素方差分析(One-Way ANOVA),方差齐时,采用LSD方法进行组间两两比较；方差不齐时,作Welch检验,再用DunnettT3方法进行组间两两比较,显著性水准取a=0.05,以P<0.05时,组间差异判断为具有统计学意义。
结果：
1.洗脱得三个多糖组分,分别命名为TCBPⅠ、TCBPⅡ、TCBPⅢ；TCBPⅠ含量最多,TCBPⅡ次之,TCBPⅢ最少。TCBPⅠ低、中、高浓度(50、100、200μg/ml)均无促进脾细胞代谢MTT活力作用(P=0.597,P=0.398,P=0.073),TCBPⅢ低、中浓度(50、100μg/ml)无促进脾细胞代谢MTT活力(P=0.497,P=0.398),高浓度(200μg/ml)可显著促进脾细胞代谢MTT活力(P=0.034)。因此,TCBPⅡ活性最高,用于后续研究。
2. TCBPⅡ低、中、高浓度(50,100,200μg/ml)均显著促进正常小鼠脾细胞代谢MTT活力(P=0.048,P=0.001,P=0.000),促进正常小鼠脾细胞分泌细胞因子TNF-α(P=0.016, P=0.000, P=0.000),且在一定的浓度范围内具有剂量依赖趋势,但对IFN-γ、IL-2的分泌无促进作用；TCBPⅡ低、中、高浓度(50,100,200μg/ml)显著增强正常小鼠腹腔渗出细胞代谢MTT活力(P=0.004,P=0.000,P=0.000),促进腹腔渗出细胞分泌TNF-α(P=0.044, P=0.001, P=0.000); TCBPⅡ低、中、高剂量(50,100,200mg/kg)对正常小鼠DTH反应耳廓肿胀度和兔红细胞免疫所致血清溶血素水平均无影响。
3. TCBPⅡ中、高剂量(100,200mg/kg)可显著提高环磷酰胺诱导免疫抑制小鼠DTH反应耳廓肿胀度(P=0.014,P=0.006),提高免疫抑制小鼠细胞免疫功能；TCBPⅡ中、高剂量(100,200mg/kg)可显著提高环磷酰胺诱导免疫抑制小鼠体内碳粒廓清能力(P=0.017,P=0.012),提高免疫抑制小鼠腹腔巨噬细胞吞噬功能；TCBPⅡ低、中、高剂量(50,100,200mg/kg)均可显著提高兔红细胞所致环磷酰胺诱导免疫抑制小鼠血清溶血素水平(P=0.011,P=0.001,P=0.000),提高免疫抑制小鼠体液免疫功能。
4. TCBPⅡ低、中、高剂量(50,100,200mg/kg)对小鼠S180实体瘤生长均有显著的抑制作用(P=0.000,P=0.000,P=0.000),抑瘤率分别为26.7%,32.5%,40.7%；同时,TCBPⅡ中、高剂量(100,200mg/kg)可显著提高荷S180实体瘤小鼠血清中TNF-α的水平(P=0.039,P=0.001),但对IFN-γ的水平无影响。TCBPⅡ中、高剂量(100,200mg/kg)可显著促进荷S180实体瘤小鼠腹腔巨噬细胞吞噬鸡红细胞(CRBC)的吞噬率(P=0.005,P=0.000)和吞噬指数(P=0.011,P=0.003)。
结论：
1.开口箭多糖(TCBPⅡ)是筛选的含量较多且活性最强部位。
2. TCBPⅡ体外可以显著增强正常小鼠免疫细胞代谢活力,促进正常小鼠免疫细胞分泌细胞因子TNF-α,但对IFN-γ、IL-2的分泌无促进作用。TCBPⅡ整体水平上无促进正常小鼠DTH和血清溶血素抗体生成作用。
3.采用环磷酰胺成功建立免疫功能低下的小鼠实验模型。
4. TCBPⅡ能显著增强环磷酰胺诱导的免疫抑制小鼠的DTH,增加校正碳粒廓清指数(α),提高吞噬活性,促进血清溶血素抗体生成,提示TCBPⅡ能够显著促进环磷酰胺诱导的免疫功能抑制小鼠的非特异性和特异性(体液免疫和细胞免疫)免疫功能。
5.成功建立了荷S180实体瘤小鼠动物模型。
6. TCBPⅡ能显著抑制荷S180小鼠实体瘤的生长,提高荷S180实体瘤小鼠血清中TNF-α的水平,增强其腹腔巨噬细胞吞噬鸡红细胞的吞噬率和吞噬指数,提示TCBPⅡ能够显著改善荷S180实体瘤小鼠的免疫功能。
7.初步证明开口箭多糖是一种良好的免疫调节剂,其免疫调节机制可能与活化或增强巨噬细胞的功能、促进TNF-α的产生或分泌有关。
Polysaccharides, the macromolecules widely existing in variable organisms ranging from mammals, higher plants, bacteria, viruses, fungi and many other lower organisms, exhibit unusual biological properties. While many kinds of polysaccharides have been isolated and purified from all kinds of organisms, it has been thought that polysaccharides might be good material for the development of new therapeutic agents. The polysaccharides can be used as vaccines against infection of pathogenic bacteria and are also useful against diseases caused by aberrant and abnormal cell-cell interaction, such as cancer metastasis and inflammation. Polysaccharides immunomodulators were first discovered 60 years ago. Although very few has been rigorously studied, certain polysaccharides immunomodulators have been identified that have profound effects in the regulation of immune responses during the progression of infections diseases, and studies have begun to define structural aspects of these molecules that govern their function and interaction with cells of the host immune system.
Tupistra chinensis Baker, also called niuweiqi, zhugenqi and so on in Chinese, is endemic in south-western regions of the People's Republic of China. As a Chinese folk medicine, this species has usually been used for treatment of rheumatic diseases, snake-bite and so on. Many steroidal saponins were isolated from rhizomes of Tupistra chinensis Baker, which was the main active component. It was found that Tupistra chinensis Baker had good anti-inflammatory activity and favorable exploitation value. And saponin is a very important substance for treatment of inflammation. The mechanism involves decreasing the quantity of mediators of inflammation like prostaglandin E2 and nitrogen monoxidum. Saponins of Tupistra chinensis Baker had significant cytotoxicity on HL-60, Caski and U251 tumor cells, strong inhibitory effect on S180 sarcoma cells in vivo and in vitro, and inhibitory effect on the platelets'aggregation and activation induced by TNBS. Also, it was reported that water extraction of Tupistra chinensis Baker could inhibit the early inflammatory exudation and edema, and had anti-inflammatory and analgesic effect. In the screening for immune active substances, we found that Tupistra chinensis Baker polysaccharides had immunological activity, which attracted our interest to study further. This paper reports the immunomodulatory effects of Tupistra chinensis Baker polysaccharidesⅡ.
Objectives:
1. To isolate the crude polysaccharides of Tupistra chinensis Baker and identify the active fraction.
2. To study the effect of Tupistra chinensis Baker polysaccharides (TCBPⅡ) on the production of tumor necrosis factor a (TNF-α), interferon y (INF-y) and interleukin 2 (IL-2) by murine spleen cells, and the effect on spleen cells metabolic activity with methylthiazolyl tetrazolium(MTT) colorimetry assay. To investigate the effect of Tupistra chinensis Baker polysaccharides (TCBPⅡ) on the production of tumor necrosis factor a (TNF-a) by murine peritoneal exudate cells and the effect on peritoneal exudate cells metabolic activity with methylthiazolyl tetrazolium (MTT) colorimetry assay. To study the effect of Tupistra chinensis Baker polysaccharides (TCBPⅡ) on the swelling degree in DTH and the level of serum hemolysin induced by RRBC immunization in normal mice in vivo.
3. To study the effect of Tupistra chinensis Baker polysaccharides (TCBPⅡ) on the swelling degree in DTH, the phagocytic function of macrophages and the level of serum hemolysin induced by RRBC immunization in immunosuppressed mice induced by CTX in vivo.
4. To study the effect of Tupistra chinensis Baker polysaccharides (TCBPⅡ) on the serum amount of tumor necrosis factorα(TNF-α) and interferonγ(INF-γ), the tumor growth inhibition rate, and the phagocytic function of macrophages in S180 tumor-bearing mice.
Methods:
1. Hot water extraction and ethanol precipitation were used to isolate the crude polysaccharides from the rhizomes of Tupistra chinensis, then Sevage method was adopted for deproteination. After that, further separation and purification was carried out by anion-exchange chromatography with DEAE-Sepharose fast flow.
2. Splenocytes from normal mice were incubated at 37℃in the presence of Tupistra chinensis Baker polysaccharides (TCBPⅡ) for 48h. ELISA was employed to determine the amount of TNF-α, IFN-γ, and IL-2 in the culture supernatants. And MTT colorimetry was used for the assay of cell metabolic activity.
3. Peritoneal exudate cells from normal mice were incubated at 37℃in the presence of Tupistra chinensis Baker polysaccharides (TCBPⅡ) for 10h. ELISA was employed to determine the amount of TNF-αin the culture supernatants. And MTT colorimetry was used for the assay of cell metabolic activity.
4.50 normal mice were randomly divided into 5 groups, including normal saline group, positive control group and TCBPⅡ(50,100, and 200mg/kg) groups, and the treatment was given once daily for 7 consecutive days. Mouse model for DTH was established by priming the mice with 1% dinitrofluorobenzene(DNFB) applied to the shaved abdomen skin and attacking on the right ear 7 days later. Twenty four hours after attack, the weight of double ears was weighed to evaluate the influence of TCBPⅡon the swelling of the ear tissues.
5.50 normal mice were randomly divided into 5 groups, including normal saline group, positive control group and TCBPⅡ(50,100, and 200mg/kg) groups, and the treatment was given once daily for 7 consecutive days. The next day after treatment, each mouse was then administered intraperitoneally with 20% RRBCs in a total volume of 0.2 ml.The sixth day after the immunization, the serum samples were collected for haemolysin assay.
6. The immunosuppressed mice were induced by cyclophosphamide (CTX). Cellular immunity was assessed with the mouse delayed type hypersensitivity (DTH) model induced by DNFB, Phagocytosis of mononuclear macrophage was determined by the method of carbon particle clearance test, and humoral immunity was evaluated with the production of specific antibody (haemolysin) against rabbit red blood cells.
7. Mouse models bearing S180 solid tumor were established. The control group was then administered intraperitoneally with sterile physiological saline solution at 0.1ml/10g body weight. The administered volume of other groups was the same as that of control. The positive control group was administered with Ganoderma lucidum polysaccharides(GLP), at 100 mg/kg intraperitoneally and the low, median and high dose groups of Tupistra chinensis Baker polysaccharides (TCBPⅡ)were administered at 50,100, and 200mg/kg intraperitoneally. After 7 days, the serum samples were collected. Then TNF-αand IFN-γlevels in the samples were measured by using enzyme-linked immunosorbent assay (ELISA). Then the implanted tumors and spleens were removed and weighed, and the tumor growth inhibition rate.
8.50 normal mice were randomly divided into 5 groups, including normal saline group, Ganoderma lucidum polysaccharides(GLP100mg/kg) and TCBPⅡ (50,100, and 200mg/kg) groups, and the treatment was given once daily for 7 consecutive days. Phagocytosis of macrophages in S180 tumor-bearing mice was evaluated with percentage of phagocytized chicken red blood cells(CRBC).
Results:
1. With DEAE-Sepharose fast flow method, three fractions were obtained, designated TCBPⅠ,TCBPⅡ,and TCBPⅢ. The highest content of the fractions was observed for TCBPⅠ, followed by TCBPⅡ, and finally TCBPⅢ. TCBPⅠ(50,100,200μg/ml) had no effect on splenocyte metabolic activity(P=0.597, P=0.398,P=0.073). TCBPⅢat concentrations of 50 and 100μg/ml did not enhance the splenocyte metabolic activity (P=0.497,P=0.398), however, significantly enhancing activity was found at the concentration of 200μg/ml (P=0.034). TCBPⅡwith the highest activity was subjet to further study.
2. Compared with control group, TCBPⅡ(50,100,200μg/ml) significantly enhanced the splenocyte metabolic activity (P=0.048,P=0.001,P=0.000) and the production of TNF-αcytokine (P=0.016,P=0.000,P=0.000) in a dose-dependent manner in a certain concentration range, but it could not enhance IFN-γand IL-2 in normal mice in vitro. The TCBPⅡ(50,100,200μg/ml) markedly promoted the metabolic activity of mouse peritoneal exudate cells (P=0.004,P=0.000, P=0.000) and production of TNF-αby the cells in normal mice in vitro (P=0.044,P=0.001,P=0.000). TCBPⅡ(50,100,200 mg/kg) could not affect the swelling degree in DTH and the level of serum hemolysin induced by HRBC immunization in normal mice.
3. TCBPⅡ(100,200 mg/kg) remarkably increased the swelling degree in DTH in immunosuppressed mice induced by CTX in vivo (P=0.014,P=0.006) and enhanced celluar immunity function. TCBPⅡ(100,200 mg/kg) remarkably promoted the serum clearance of carbon particles in immunosuppressed mice induced by CTX in vivo (P=0.017,P=0.012), indicating an increase in phagocytic function of macrophages. TCBPⅡ(50,100,200 mg/kg) increased the level of serum hemolysin induced by HRBC immunization in immunosuppressed mice (P=0.011,P=0.001, P=0.000), indicating an enhance in humoral immunity.
4. TCBPⅡ(50,100,200 mg/kg) could inhibit the growth of S180 solid tumor (P =0.000,P=0.000,P=0.000), with the inhibition rate of 26.7%,32.5%, and 40.7%. And TCBPⅡ(100,200 mg/kg) increased the serum levels of TNF-αin S180 solid tumor mice (P=0.039,P=0.001). The effects were dose-dependent in a certain concentration range. TCBPⅡ(100,200 mg/kg) remarkably potentiated the rate and index of phagocytosis of CRBC by peritoneal macrophages in the tumor-bearing mice in vivo respectively (P=0.005, P=0.000), (P=0.011, P=0.003)
Conclusion:
1. The fraction TCBPⅡ(Tupistra chinensis Baker polysaccharidesⅡ) is the highest in activity and with considerable content.
2. TCBPⅡcan significantly enhance immunocyte metabolic activity and promote secretion of TNF-αby immunocytes in normal mice in vitro, but can not affect IFN-γand IL-2. TCBPⅡcan not enhance the swelling degree in DTH and the level of serum hemolysin in normal mice.
3. The experimental model of immunosuppressed mice induced by cyclophosphamide (CTX) was established successfully.
4. TCBPⅡhas the capacity of enhancement of immune function in immunosuppressed mice induced by CTX in vivo. TCBPⅡcan markedly potentiate DTH, elevate the corrected phagocytic index (α), promote the phagocytic function of macrophages, and increase the level of serum hemolysin in immunosuppressed mice. It is suggested that TCBPⅡis able to enhance the function of cellular and humoral immunity in immunosuppressed mice induced by CTX in vivo.
5. The experimental model of S180 solid tumor-bearing mice was established successfully.
6. TCBPⅡcan inhibit the growth of S180 solid tumor, increase the serum level of TNF-a, and potentiate the rate and index of phagocytosis of CRBC by peritoneal macrophages in the tumor-bearing mice in vivo.
7. The preliminary experimental results demonstrate that TCBPⅡis a sort of effective immunomodulator, and the underling mechanism may be related with its potentiation of the macrophages phagocytosis and the production of TNF-a in mice.
开口箭(Tupistra chinensis Baker)又名牛尾七、竹根七等,主要分布于我国西南地区,民间主要用于治疗咽喉肿痛、风湿痹痛、跌打损伤、胃痛、毒蛇狂犬咬伤、蚊虫叮咬等。开口箭属植物主要活性成分为皂苷,从该属植物根茎中已分离提纯出多种皂苷成份。研究发现,开口箭具有良好的抗炎作用,有良好的开发利用价值。皂苷是其抗炎的重要物质基础之一,抑制炎症介质PGE2、NO的产生是其作用机理；开口箭皂苷体外对HL-60、Caski、251肿瘤细胞具有明显的抑制作用,体内外对S180肉瘤细胞也有很强的抑制作用；同时开口箭皂苷还能够抑制血小板的聚集与活化。也有研究报道,开口箭水提物能明显抑制炎症早期的水肿和渗出,具有抗炎镇痛的功效。我们在活性筛选时发现,开口箭水提物具有一定的免疫活性,从而引起了我们的研究兴趣。本文对分离得到的开口箭多糖组分TCBPⅡ(Tupistra chinensis Baker polysaccharidesⅡ, TCBPⅡ)的免疫调节作用及其机制进行了初步研究。
目的：
1.提取分离开口箭粗多糖并筛选出活性成分。
2.研究开口箭多糖(TCBPⅡ)体外对正常小鼠脾细胞代谢MTT活力及产生肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)、白细胞介素-2(IL-2)的影响；体外对正常小鼠腹腔渗出细胞代谢MTT活力及产生TNF-α的影响；体内对正常小鼠迟发型超敏反应(DTH)、血清溶血素水平的影响。
3.研究开口箭多糖(TCBPⅡ)体内对环磷酰胺诱导免疫抑制小鼠的DTH、单核巨噬细胞吞噬功能、血清溶血素水平的影响。
4.研究开口箭多糖(TCBPⅡ)对荷S180实体瘤小鼠抑瘤作用、血清TNF-α、IFN-γ含量及腹腔单核巨噬细胞吞噬功能的影响。
方法：
1.采用热水提取、乙醇沉淀法从开口箭植物的根茎中提取粗制多糖,再用Sevage法去蛋白。经DEAE-Sepharose fast flow阴离子交换柱对粗制多糖进行分离纯化,得到不同组分开口箭多糖。通过测定不同组分多糖对正常小鼠脾脏淋巴细胞增殖作用筛选出活性成分,命名TCBPⅡ
2.取正常小鼠脾细胞,加入开口箭多糖(TCBPⅡ),在37℃下培养48h,收集培养上清,用ELISA方法检测其中TNF-α、IFN-γ、IL-2的含量,或者用MTT比色法检测细胞代谢MTT活力。
3.取正常小鼠腹腔渗出细胞,加入开口箭多糖(TCBPⅡ),在37℃下培养10h,收集培养上清,用ELISA方法检测其中TNF-α的含量,或者用MTT比色法检测细胞代谢MTT活力。
4.正常小鼠50只,随机分为5组,生理盐水对照组、阳性药物(灵芝多糖100mg/kg)组、TCBPⅡ低、中、高剂量(50,100,200mg/kg)组,给药组小鼠分别连续腹腔注射(ip)干预药物7d。于给药后第1日用1%二硝基氟苯(dinitro-fluorobenzene, DNFB)腹部致敏,末次给药后将1%DNFB涂抹于5组实验小鼠右侧耳廓,24h后处死动物,用打孔器取相同面积两侧耳片称重,比较给药组与对照组耳廓肿胀程度。
5.正常小鼠50只,随机分为5组,生理盐水对照组、阳性药物(灵芝多糖100mg/kg)组、TCBPⅡ低、中、高剂量(50,100,200mg/kg)组,给药组小鼠分别连续ip干预药物7d。给药后第2天上午,各鼠腹腔注射20%(V/V)的生理盐水兔红细胞0.2 ml进行免疫,同日下午继续给药,免疫后d6(免疫当天为d0),动物摘眼球取血,分离和收集血清用于血清溶血素水平测定。
6.皮下注射环磷酰胺(CTX)复制免疫功能低下的动物模型。小鼠50只,随机分为5组,正常对照组、CTX模型组、TCBPⅡ低、中、高剂量(50,100,200mg/kg)+CTX组,采用DNFB诱导迟发型变态反应(delayed-type hypersensitivity, DTH)模型,观察小鼠细胞免疫功能；碳廓清法检测单核巨噬细胞的吞噬功能；兔红细胞免疫法检测体液免疫功能。
7.建立荷S180实体瘤小鼠动物模型,生理盐水对照组：每天腹腔注射(ip)无菌生理盐水0.1ml/10g体重,其他组给药体积与此相同；阳性对照组：每天ip灵芝多糖100mg/kg; TCBPⅡ低、中、高剂量组：每天分别ip TCBPⅡ50、100、200mg/kg;连续给药7d后次日上午,分离血清用于TNF-α和IFN-γ的含量测定。取血后,分离瘤体,称重,并计算抑瘤率。
8.取荷S180实体瘤小鼠50只,随机分为生理盐水对照组、阳性药物(灵芝多糖100mg/kg)组、TCBPⅡ低、中、高剂量(50,100,200mg/kg)组,每组10只,腹腔注射给药,每天1次,连续7天。停药24h后,每只小鼠腹腔注射10%鸡红细胞悬液1ml,30min后,脱颈椎处死动物,检测腹腔巨噬细胞的吞噬活力。
9.统计分析采用SPSS 13.0统计软件,两样本均数比较,用独立样本t检验(Independent-Sample T Test)。多个样本均数比较,经方差齐性检验后进行单因素方差分析(One-Way ANOVA),方差齐时,采用LSD方法进行组间两两比较；方差不齐时,作Welch检验,再用DunnettT3方法进行组间两两比较,显著性水准取a=0.05,以P<0.05时,组间差异判断为具有统计学意义。
结果：
1.洗脱得三个多糖组分,分别命名为TCBPⅠ、TCBPⅡ、TCBPⅢ；TCBPⅠ含量最多,TCBPⅡ次之,TCBPⅢ最少。TCBPⅠ低、中、高浓度(50、100、200μg/ml)均无促进脾细胞代谢MTT活力作用(P=0.597,P=0.398,P=0.073),TCBPⅢ低、中浓度(50、100μg/ml)无促进脾细胞代谢MTT活力(P=0.497,P=0.398),高浓度(200μg/ml)可显著促进脾细胞代谢MTT活力(P=0.034)。因此,TCBPⅡ活性最高,用于后续研究。
2. TCBPⅡ低、中、高浓度(50,100,200μg/ml)均显著促进正常小鼠脾细胞代谢MTT活力(P=0.048,P=0.001,P=0.000),促进正常小鼠脾细胞分泌细胞因子TNF-α(P=0.016, P=0.000, P=0.000),且在一定的浓度范围内具有剂量依赖趋势,但对IFN-γ、IL-2的分泌无促进作用；TCBPⅡ低、中、高浓度(50,100,200μg/ml)显著增强正常小鼠腹腔渗出细胞代谢MTT活力(P=0.004,P=0.000,P=0.000),促进腹腔渗出细胞分泌TNF-α(P=0.044, P=0.001, P=0.000); TCBPⅡ低、中、高剂量(50,100,200mg/kg)对正常小鼠DTH反应耳廓肿胀度和兔红细胞免疫所致血清溶血素水平均无影响。
3. TCBPⅡ中、高剂量(100,200mg/kg)可显著提高环磷酰胺诱导免疫抑制小鼠DTH反应耳廓肿胀度(P=0.014,P=0.006),提高免疫抑制小鼠细胞免疫功能；TCBPⅡ中、高剂量(100,200mg/kg)可显著提高环磷酰胺诱导免疫抑制小鼠体内碳粒廓清能力(P=0.017,P=0.012),提高免疫抑制小鼠腹腔巨噬细胞吞噬功能；TCBPⅡ低、中、高剂量(50,100,200mg/kg)均可显著提高兔红细胞所致环磷酰胺诱导免疫抑制小鼠血清溶血素水平(P=0.011,P=0.001,P=0.000),提高免疫抑制小鼠体液免疫功能。
4. TCBPⅡ低、中、高剂量(50,100,200mg/kg)对小鼠S180实体瘤生长均有显著的抑制作用(P=0.000,P=0.000,P=0.000),抑瘤率分别为26.7%,32.5%,40.7%；同时,TCBPⅡ中、高剂量(100,200mg/kg)可显著提高荷S180实体瘤小鼠血清中TNF-α的水平(P=0.039,P=0.001),但对IFN-γ的水平无影响。TCBPⅡ中、高剂量(100,200mg/kg)可显著促进荷S180实体瘤小鼠腹腔巨噬细胞吞噬鸡红细胞(CRBC)的吞噬率(P=0.005,P=0.000)和吞噬指数(P=0.011,P=0.003)。
结论：
1.开口箭多糖(TCBPⅡ)是筛选的含量较多且活性最强部位。
2. TCBPⅡ体外可以显著增强正常小鼠免疫细胞代谢活力,促进正常小鼠免疫细胞分泌细胞因子TNF-α,但对IFN-γ、IL-2的分泌无促进作用。TCBPⅡ整体水平上无促进正常小鼠DTH和血清溶血素抗体生成作用。
3.采用环磷酰胺成功建立免疫功能低下的小鼠实验模型。
4. TCBPⅡ能显著增强环磷酰胺诱导的免疫抑制小鼠的DTH,增加校正碳粒廓清指数(α),提高吞噬活性,促进血清溶血素抗体生成,提示TCBPⅡ能够显著促进环磷酰胺诱导的免疫功能抑制小鼠的非特异性和特异性(体液免疫和细胞免疫)免疫功能。
5.成功建立了荷S180实体瘤小鼠动物模型。
6. TCBPⅡ能显著抑制荷S180小鼠实体瘤的生长,提高荷S180实体瘤小鼠血清中TNF-α的水平,增强其腹腔巨噬细胞吞噬鸡红细胞的吞噬率和吞噬指数,提示TCBPⅡ能够显著改善荷S180实体瘤小鼠的免疫功能。
7.初步证明开口箭多糖是一种良好的免疫调节剂,其免疫调节机制可能与活化或增强巨噬细胞的功能、促进TNF-α的产生或分泌有关。
Polysaccharides, the macromolecules widely existing in variable organisms ranging from mammals, higher plants, bacteria, viruses, fungi and many other lower organisms, exhibit unusual biological properties. While many kinds of polysaccharides have been isolated and purified from all kinds of organisms, it has been thought that polysaccharides might be good material for the development of new therapeutic agents. The polysaccharides can be used as vaccines against infection of pathogenic bacteria and are also useful against diseases caused by aberrant and abnormal cell-cell interaction, such as cancer metastasis and inflammation. Polysaccharides immunomodulators were first discovered 60 years ago. Although very few has been rigorously studied, certain polysaccharides immunomodulators have been identified that have profound effects in the regulation of immune responses during the progression of infections diseases, and studies have begun to define structural aspects of these molecules that govern their function and interaction with cells of the host immune system.
Tupistra chinensis Baker, also called niuweiqi, zhugenqi and so on in Chinese, is endemic in south-western regions of the People's Republic of China. As a Chinese folk medicine, this species has usually been used for treatment of rheumatic diseases, snake-bite and so on. Many steroidal saponins were isolated from rhizomes of Tupistra chinensis Baker, which was the main active component. It was found that Tupistra chinensis Baker had good anti-inflammatory activity and favorable exploitation value. And saponin is a very important substance for treatment of inflammation. The mechanism involves decreasing the quantity of mediators of inflammation like prostaglandin E2 and nitrogen monoxidum. Saponins of Tupistra chinensis Baker had significant cytotoxicity on HL-60, Caski and U251 tumor cells, strong inhibitory effect on S180 sarcoma cells in vivo and in vitro, and inhibitory effect on the platelets'aggregation and activation induced by TNBS. Also, it was reported that water extraction of Tupistra chinensis Baker could inhibit the early inflammatory exudation and edema, and had anti-inflammatory and analgesic effect. In the screening for immune active substances, we found that Tupistra chinensis Baker polysaccharides had immunological activity, which attracted our interest to study further. This paper reports the immunomodulatory effects of Tupistra chinensis Baker polysaccharidesⅡ.
Objectives:
1. To isolate the crude polysaccharides of Tupistra chinensis Baker and identify the active fraction.
2. To study the effect of Tupistra chinensis Baker polysaccharides (TCBPⅡ) on the production of tumor necrosis factor a (TNF-α), interferon y (INF-y) and interleukin 2 (IL-2) by murine spleen cells, and the effect on spleen cells metabolic activity with methylthiazolyl tetrazolium(MTT) colorimetry assay. To investigate the effect of Tupistra chinensis Baker polysaccharides (TCBPⅡ) on the production of tumor necrosis factor a (TNF-a) by murine peritoneal exudate cells and the effect on peritoneal exudate cells metabolic activity with methylthiazolyl tetrazolium (MTT) colorimetry assay. To study the effect of Tupistra chinensis Baker polysaccharides (TCBPⅡ) on the swelling degree in DTH and the level of serum hemolysin induced by RRBC immunization in normal mice in vivo.
3. To study the effect of Tupistra chinensis Baker polysaccharides (TCBPⅡ) on the swelling degree in DTH, the phagocytic function of macrophages and the level of serum hemolysin induced by RRBC immunization in immunosuppressed mice induced by CTX in vivo.
4. To study the effect of Tupistra chinensis Baker polysaccharides (TCBPⅡ) on the serum amount of tumor necrosis factorα(TNF-α) and interferonγ(INF-γ), the tumor growth inhibition rate, and the phagocytic function of macrophages in S180 tumor-bearing mice.
Methods:
1. Hot water extraction and ethanol precipitation were used to isolate the crude polysaccharides from the rhizomes of Tupistra chinensis, then Sevage method was adopted for deproteination. After that, further separation and purification was carried out by anion-exchange chromatography with DEAE-Sepharose fast flow.
2. Splenocytes from normal mice were incubated at 37℃in the presence of Tupistra chinensis Baker polysaccharides (TCBPⅡ) for 48h. ELISA was employed to determine the amount of TNF-α, IFN-γ, and IL-2 in the culture supernatants. And MTT colorimetry was used for the assay of cell metabolic activity.
3. Peritoneal exudate cells from normal mice were incubated at 37℃in the presence of Tupistra chinensis Baker polysaccharides (TCBPⅡ) for 10h. ELISA was employed to determine the amount of TNF-αin the culture supernatants. And MTT colorimetry was used for the assay of cell metabolic activity.
4.50 normal mice were randomly divided into 5 groups, including normal saline group, positive control group and TCBPⅡ(50,100, and 200mg/kg) groups, and the treatment was given once daily for 7 consecutive days. Mouse model for DTH was established by priming the mice with 1% dinitrofluorobenzene(DNFB) applied to the shaved abdomen skin and attacking on the right ear 7 days later. Twenty four hours after attack, the weight of double ears was weighed to evaluate the influence of TCBPⅡon the swelling of the ear tissues.
5.50 normal mice were randomly divided into 5 groups, including normal saline group, positive control group and TCBPⅡ(50,100, and 200mg/kg) groups, and the treatment was given once daily for 7 consecutive days. The next day after treatment, each mouse was then administered intraperitoneally with 20% RRBCs in a total volume of 0.2 ml.The sixth day after the immunization, the serum samples were collected for haemolysin assay.
6. The immunosuppressed mice were induced by cyclophosphamide (CTX). Cellular immunity was assessed with the mouse delayed type hypersensitivity (DTH) model induced by DNFB, Phagocytosis of mononuclear macrophage was determined by the method of carbon particle clearance test, and humoral immunity was evaluated with the production of specific antibody (haemolysin) against rabbit red blood cells.
7. Mouse models bearing S180 solid tumor were established. The control group was then administered intraperitoneally with sterile physiological saline solution at 0.1ml/10g body weight. The administered volume of other groups was the same as that of control. The positive control group was administered with Ganoderma lucidum polysaccharides(GLP), at 100 mg/kg intraperitoneally and the low, median and high dose groups of Tupistra chinensis Baker polysaccharides (TCBPⅡ)were administered at 50,100, and 200mg/kg intraperitoneally. After 7 days, the serum samples were collected. Then TNF-αand IFN-γlevels in the samples were measured by using enzyme-linked immunosorbent assay (ELISA). Then the implanted tumors and spleens were removed and weighed, and the tumor growth inhibition rate.
8.50 normal mice were randomly divided into 5 groups, including normal saline group, Ganoderma lucidum polysaccharides(GLP100mg/kg) and TCBPⅡ (50,100, and 200mg/kg) groups, and the treatment was given once daily for 7 consecutive days. Phagocytosis of macrophages in S180 tumor-bearing mice was evaluated with percentage of phagocytized chicken red blood cells(CRBC).
Results:
1. With DEAE-Sepharose fast flow method, three fractions were obtained, designated TCBPⅠ,TCBPⅡ,and TCBPⅢ. The highest content of the fractions was observed for TCBPⅠ, followed by TCBPⅡ, and finally TCBPⅢ. TCBPⅠ(50,100,200μg/ml) had no effect on splenocyte metabolic activity(P=0.597, P=0.398,P=0.073). TCBPⅢat concentrations of 50 and 100μg/ml did not enhance the splenocyte metabolic activity (P=0.497,P=0.398), however, significantly enhancing activity was found at the concentration of 200μg/ml (P=0.034). TCBPⅡwith the highest activity was subjet to further study.
2. Compared with control group, TCBPⅡ(50,100,200μg/ml) significantly enhanced the splenocyte metabolic activity (P=0.048,P=0.001,P=0.000) and the production of TNF-αcytokine (P=0.016,P=0.000,P=0.000) in a dose-dependent manner in a certain concentration range, but it could not enhance IFN-γand IL-2 in normal mice in vitro. The TCBPⅡ(50,100,200μg/ml) markedly promoted the metabolic activity of mouse peritoneal exudate cells (P=0.004,P=0.000, P=0.000) and production of TNF-αby the cells in normal mice in vitro (P=0.044,P=0.001,P=0.000). TCBPⅡ(50,100,200 mg/kg) could not affect the swelling degree in DTH and the level of serum hemolysin induced by HRBC immunization in normal mice.
3. TCBPⅡ(100,200 mg/kg) remarkably increased the swelling degree in DTH in immunosuppressed mice induced by CTX in vivo (P=0.014,P=0.006) and enhanced celluar immunity function. TCBPⅡ(100,200 mg/kg) remarkably promoted the serum clearance of carbon particles in immunosuppressed mice induced by CTX in vivo (P=0.017,P=0.012), indicating an increase in phagocytic function of macrophages. TCBPⅡ(50,100,200 mg/kg) increased the level of serum hemolysin induced by HRBC immunization in immunosuppressed mice (P=0.011,P=0.001, P=0.000), indicating an enhance in humoral immunity.
4. TCBPⅡ(50,100,200 mg/kg) could inhibit the growth of S180 solid tumor (P =0.000,P=0.000,P=0.000), with the inhibition rate of 26.7%,32.5%, and 40.7%. And TCBPⅡ(100,200 mg/kg) increased the serum levels of TNF-αin S180 solid tumor mice (P=0.039,P=0.001). The effects were dose-dependent in a certain concentration range. TCBPⅡ(100,200 mg/kg) remarkably potentiated the rate and index of phagocytosis of CRBC by peritoneal macrophages in the tumor-bearing mice in vivo respectively (P=0.005, P=0.000), (P=0.011, P=0.003)
Conclusion:
1. The fraction TCBPⅡ(Tupistra chinensis Baker polysaccharidesⅡ) is the highest in activity and with considerable content.
2. TCBPⅡcan significantly enhance immunocyte metabolic activity and promote secretion of TNF-αby immunocytes in normal mice in vitro, but can not affect IFN-γand IL-2. TCBPⅡcan not enhance the swelling degree in DTH and the level of serum hemolysin in normal mice.
3. The experimental model of immunosuppressed mice induced by cyclophosphamide (CTX) was established successfully.
4. TCBPⅡhas the capacity of enhancement of immune function in immunosuppressed mice induced by CTX in vivo. TCBPⅡcan markedly potentiate DTH, elevate the corrected phagocytic index (α), promote the phagocytic function of macrophages, and increase the level of serum hemolysin in immunosuppressed mice. It is suggested that TCBPⅡis able to enhance the function of cellular and humoral immunity in immunosuppressed mice induced by CTX in vivo.
5. The experimental model of S180 solid tumor-bearing mice was established successfully.
6. TCBPⅡcan inhibit the growth of S180 solid tumor, increase the serum level of TNF-a, and potentiate the rate and index of phagocytosis of CRBC by peritoneal macrophages in the tumor-bearing mice in vivo.
7. The preliminary experimental results demonstrate that TCBPⅡis a sort of effective immunomodulator, and the underling mechanism may be related with its potentiation of the macrophages phagocytosis and the production of TNF-a in mice.
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[22]吕晓英,刘新华,李由,等.红毛五加多糖对胃癌细胞的诱导凋亡作用[J].中国癌症杂志,2001,11(2)：137-142.
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[24]沈平,王三龙,杨崇仁,等.弯蕊开口箭的多羟基甾体皂苷元[J].植物学报,2003,45(5)：626-629.
[25]Pauli GF. Conballariaceae, (Part Ⅱ). The cardenolides of sepirantha convallarioides[J].Planta Med,1995,61(1):162-162.
[26]杨帆,沈平,杨崇仁,等.来自弯蕊开口箭的新甾体配糖体[J].云南植物研究,2001,23(3)：373-380.
[27]PanWB, Chang FR, Wu YC. Spirostanol sapogenins from the underground parts of Tupistra chinensis[J]. Chem Pharm Bull (Tokyo),2000,48(9):1350-1353.
[28]杨仁州,朱小琪,寸富兰.心不甘中甾体皂苷元的分离和结构鉴定(2)[J].云南植物研究,1987,9(2)：217-217.
[29]杨仁州,朱小琪,寸富兰.心不甘中甾体皂苷元的分离和结构鉴定(3)[J].云南植物研究,1987,9(2)：223-223.
[30]杨仁州,王德祖,付坚.十个甾体皂苷元的13CNMR谱[J].云南植物研究,1997,9(3)：371-371.
[31]王一飞,李兴从.弯蕊开口箭中一个新的甾体皂苷元[J].云南植物研究,1987,9(3)：341-344.
[32]濮社班,钱士辉,袁昌齐,等.五种开口箭属植物根状茎中甾体皂苷及甾体皂苷元的初步研究[J].中国野生植物资源,1999,19(5)：59-61.
[33]黄丽,廖全斌,邹坤,等.开口箭中甾体皂苷元含量的测定[J].三峡大学学报(自然科学版),2003,25(6)：562-564.
[34]乐开礼,杨德馨,周云线.须瓣开口箭的药理研究[J].药学学报,1963,10(1)：39-39.
[35]Cai J, Zhu ZG, Yu CL, et al. Saponin from Tupistra chinensis Baker inhibits mouse sarcoma S180 cell proliferation in vitro and implanted solid tumor growth in mice[J].南方医科大学学报,2007,27(2)：188-190,194.
[36]Zou K, Wang J, DuM, et al. A pair of diastereoisomerc steroidal saponins from cytotoxic extracts of Tupistra chinensis rhizomes[J]. Chem Pharm Bull(Tokyo) (Tokyo),2006,54(10):14402-14421.
[37]朱正光,余传林,蔡晶,等.开口箭提取物的抗肿瘤作用研究[J].中药材,2006,29(3)：277-279.
[38]杨春艳,杨兴海,刘英,等.开口箭祛痰、抗炎及抑菌实验研究[J].中国民族民间医药杂志,2005,(73)：103-106.
[39]李小莉,张迎庆,洪蓓蓓.民间草药开口箭的抗炎镇痛作用的研究[J].湖北中医学院学报,2005,7(4)：28-29.
[40]邱 教,董卫国.开口箭提取物对大鼠实验性结肠炎的治疗机制研究[J].山东医药,2005,45(26)：4-6.
[41]吴光旭,刘爱媛,陈维信.开口箭提取物对荔枝霜疫霉菌的抑制作用及其对荔枝过时的贮藏效果[J].中国农业科学,2006,39(8)：1703-1708.
[42]汤子春,邹坤,汪鋆植,等.开口箭与筒鞘蛇菰提取物醒酒作用的实验研究[J]. 时珍国医国药,2006,17(11)：2163-2165.
[43]瞿绍双.土家族药开口箭漱口液治疗咽喉炎45例疗效观察[J].中国民族民间医药杂志,1999,(38)：140-140.
[44]徐叔云.药理实验方法学[M].北京：人民卫生出版社,2002：1420-1455.
[45]刘民,马华,李柏青.MTT法检测小鼠淋巴细胞增殖性反应探讨[J].中国实验动物学杂志,1999,9(3)：146-149
[46]李建军,雷林生,余传林.灵芝多糖抗肿瘤作用的免疫学相关性研究[J].中药材,2007,30(1)：71-73.
[47]Mizukoshi S, Tsukamoto M, Tanaka H, et al. Anti-inflammatory and immunosuppressive effects of 1,6-anhydro-3,4-dideoxy-2-furfuryl-beta-D-threo-3-enopyranose (MT2221) a novel anhydro-enopyranose derivative, on experimental animal models [J]. Biol Pharm Bull,1994,17(8):1070-1074.
[48]仇成凤,雷林生,吴玉英,等.以兔红细胞作为抗原建立小鼠体液免疫反应动物模型[J].南方医科大学学报,2009,29(12)：2473-2476.
[49]李越中.药物微生物技术[M].北京：化学工业出版社,2004：127-129.
[50]Stites DP, Terr AI, Parslow TG. Basic and clinical immunology [M]. Norwalk:Appleton and Lange,1994:20-87.
[51]Bin-Hafeez B, Haque R, Parvez S, et al. Immunomodulatory effects of fenugreek(Trigonella foenum graecum L.) extract in mice [J]. Int Immunopharmacol,2003,3(2):257-265.
[52]张华,曲震寰,王莹等.小鼠腹腔巨噬细胞的分离培养与吞噬功能测定[J].黑龙江医药科学,2007,30(5)：9-10.
[53]Pfeffer K. Biological functions of tumor necrosis factor cytokines and their receptors[J]. Cytokine Growth Factor Rev,2003,14(3-4):185-191.
[54]籍海虹,程玉英.宫颈癌病人血清IFN-γ及IL-4水平的变化及其临床意义[J].齐鲁医学杂志,2004,19(6)：528-528.
[55]姜维洁,李培成,张金福.肺癌患者IFN-γ和IL-10水平的变化[J].上海医学检验杂志,2000,15(3)：157-158
[56]王冠庭,朱金水,徐家裕.晚期胃癌患者外周血T细胞亚群、NK细胞和干扰素活性检测[J].上海医学,1995,18(2)：109-110.
[57]盛学岐,林存智,朱新红.CDla分子和IFN-γ因子对良恶性胸腔积液诊断价值[J].青岛大学医学院学报,2005,41(4)：300-301.
[58]甘雪婷,白宪光,冯志华.血清转化生长因子β1和干扰素-γ在肝硬化及肝癌中的表达及其临床意义[J].临床内科杂志,2003,20(9)：484-486.
[59]周振英,吴晓柳,朱月清.恶性肿瘤患者外周血细胞TNF-α和IFN-γ检测的临床意义[J].河南肿瘤学杂志,2003,16(3)：159-161.
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