金黄扶正散对免疫抑制小鼠的免疫调节作用及机制研究
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摘要
目的
为了探讨金黄扶正散对免疫抑制小鼠的免疫调节作用,本研究通过建立免疫抑制小鼠模型,观察金黄扶正散对免疫抑制小鼠免疫器官、抗应激能力、抗自由基水平和免疫细胞功能等方面的影响,并进行作用机制研究,为其临床应用提供依据。
方法
1 SPF级昆明种小鼠60只,14-18g,随机分为六组,分别为：正常对照组(生理盐水灌胃0.2mL/10g,连续10天)；环磷酰胺组(生理盐水灌胃0.2mL/10g,连续10天,同时隔天皮下注射环磷酰胺30mg/kg)；阳性对照组(每天灌胃左旋咪唑剂25 mg/kg,连续10天,给药后隔天皮下注射环磷酰胺30mg/kg)；金黄扶正茶高、中、低剂量组(每天分别灌胃金黄扶正茶5.08g/kg、2.54g/kg、1.27 g/kg,连续10天,给药后隔天皮下注射环磷酰胺30mg/kg),建立免疫功能低下小鼠模型。末次给药后小鼠禁食12h,处死,分别取胸腺,脾脏称重,计算胸腺、脾脏指数。将脾脏制成组织匀浆,离心,取上清,进行乳酸脱氢酶(LDH)活性测定和酸性磷酸酶(ACP)活性测定。
2造模及给药方法同1,分别进行负重游泳实验、耐高温实验、耐低温实验、常压耐缺氧实验和亚硝酸盐中毒性耐缺氧实验。游泳实验观察其游泳时间,其余各实验观察存活时间。
3造模及给药方法同1,取小鼠脾脏制成组织匀浆,离心,取上清,测定总抗氧化能力(T-AOC)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)和丙二醛(MDA)。
4造模及给药方法同1,采用四甲基偶氮唑蓝(MTT)法,测定刀豆蛋白A (ConA)诱导脾脏T淋巴细胞增殖和脂多糖(LPS)诱导B淋巴细胞增殖的影响,测定自然杀伤细胞(NK细胞)和淋巴因子激活的杀伤细胞(LAK细胞)活性,检测巨噬细胞吞噬中性红能力和诱导型一氧化氮合酶(iNOS)的活性。
5造模及给药方法同1.1,末次用药后小鼠禁食12h,各组小鼠尾静脉取血1mL,离心,取血清备用,采用细胞因子抗体芯片进行细胞因子定量检测。
6造模及给药方法同1.1,小鼠处死后,取脾,立即置液氮中,随后转移置-80℃低温冰箱保存,采用实时荧光定量PCR (FQ-PCR)方法检测脾脏T-bet、GATA mRNA的表达。
7造模及给药方法同1.1,小鼠处死后,取脾,4%多聚甲醛固定,常规脱水,石蜡包埋,切片,采用TUNEL染色法观察细胞凋亡情况。
8采用FQ-PCR方法检测脾脏Bcl-2、Bax、Fas和FasL mRNA的表达。
结果
1与正常对照组比较,环磷酰胺组胸腺指数,脾脏指数显著降低(P<0.05),给药后,金黄扶正散中、高剂量组可显著提高免疫抑制小鼠的胸腺指数、脾脏指数(P<0.01,P<0.05)。与正常对照组比较,环磷酰胺组可明显降低LDH、ACP的活性(P<0.01)；给药后,金黄扶正散低、中、高剂量组可明显提高免疫抑制小鼠的LDH活性和ACP活性(P<0.01,P<0.05)。
2与正常对照组比较,环磷酰胺组负重游泳时间明显减少(P<0.05),耐高温存活时间明显减少(P<0.01),耐低温存活时间明显减少(P<0.01),在常压缺氧条件下的存活时间明显减少(P<0.01),亚硝酸盐中毒性缺氧状态下存活时间明显减少(P<0.01)；给药后与模型组比较,金黄扶正散中、高剂量组能明显延长负重游泳时间(P<0.05),金黄扶正散低、中剂量组能明显延长耐高温存活时间(P<0.01),金黄扶正散低、中剂量组可显著提高小鼠的耐低温存活时间(P<0.05),金黄扶正散低、高剂量组能明显延长常压耐缺氧存活时间(P<0.01),金黄茶中剂量组能明显延长亚硝酸盐中毒性缺氧存活时间(P<0.01)。
3与正常对照组比较,环磷酰胺组可显著降低T-AOC能力(P<0.01),降低CAT、SOD、GSH-PX的活性(P<0.05,P<0.01)；明显提高MDA含量(P<0.05)。给药后,金黄扶正散中、高剂量组可明显提高免疫抑制小鼠的T-AOC能力(P<0.01)；金黄扶正散高剂量组可明显提高免疫抑制小鼠的CAT、SOD、GSH-PX的活性(P<0.05,P<0.01)；金黄扶正散中、高剂量组可明显降低免疫抑制小鼠的MDA含量(P<0.05,P<0.01)。
4与环磷酰胺组比较,金黄扶正散低、中、高剂量组均能显著提高免疫抑制小鼠的T、B淋巴细胞增殖能力(P<0.01)；金黄扶正散中、高剂量组能显著提高免疫抑制小鼠NK细胞、LAK细胞活性(P<0.01,P<0.05)；金黄扶正散高剂量组能显著性提高免疫抑制小鼠的CD3+淋巴细胞数(P<0.05),金黄扶正散中、高剂量组能显著性提高免疫抑制小鼠的CD4’淋巴细胞数(P<0.01),金黄扶正散低、中、高剂量组降低CD8+淋巴细胞数和提高CD4+/CD8+比值(P<0.01,P<0.05)；金黄扶正散中、高剂量组可提高腹腔巨噬细胞吞噬能力(P<0.05)；金黄扶正散低、中、高剂量组可提高巨噬细胞iNOS活性(P<0.01,P<0.05)。
5与正常对照组比较,环磷酰胺组的2种细胞因子IFN-Y和RANTES呈显著性降低(P<0.05),金黄扶正散低、中、高剂量组给药后IFN-γ有显著性提升,高剂量组的RANTES呈显著性升高(P<0.05)；与对照组比较,环磷酰胺组的5种细胞因子IL-5、IL-6、IL-9、IL-13和MCP-1呈显著性升高(P<0.01,P<0.05),给药组给药后有不同程度降低(P<0.01,P<0.05)；13种细胞因子GM-CSF、IL-1α、IL-1β、IL-2、IL-3、IL-4、IL-10、IL-12、IL-17、M-CSF、TNF-α、KC和VEGF无明显变化(P>0.05)。
6与环磷酰胺组比较,金黄扶正散低、中、高剂量组的T-bet mRNA表达量呈显著性提高而GATA mRNA表达量呈显著性降低(P<0.01)。
7与正常对照组比较,环磷酰胺组凋亡指数显著性提高(P<0.05)；金黄扶正散中、高剂量组凋亡指数与环磷酰胺组比较显著性下降(P<0.01,P<0.05)。金黄扶正散低剂量凋亡指数与环磷酰胺组比较无显著差异(P>0.05)。
8与环磷酰胺组比较,金黄扶正散中、高剂量组的bcl-2 mRNA表达量呈显著性提高(P<0.01),bcl-2/ bax mRNA比值呈显著性降低(P<0.01,P<0.05)。与环磷酰胺组比较,金黄扶正散中、高剂量组的Fas和FasL mRNA表达量均呈显著性降低(P<0.01,P<0.05)。
结论：
1采用免疫抑制剂环磷酰胺制造免疫抑制小鼠模型,造模后小鼠胸腺指数、脾指数下降,金黄扶正散可提高免疫抑制小鼠的胸腺指数、脾脏指数,提高免疫抑制小鼠的LDH和ACP活性,激活巨噬细胞的活化状态。
2金黄扶正散可延长免疫抑制小鼠负重游泳时间,提高其耐高温、低温、常压耐缺氧、亚硝酸盐中毒性缺氧的存活时间,提高免疫抑制小鼠抗应激的能力。
3金黄扶正散可显著提高免疫抑制小鼠CAT、T-SOD、GSH-PX的活性显著提高T-AOC能力,明显降低MDA含量,调节体内抗氧化酶的水平和自由基水平,改善机体的氧化还原状态。
4金黄扶正散能显著提高免疫抑制小鼠脾脏T淋巴细胞和B淋巴细胞增殖能力；提高免疫抑制小鼠NK细胞和LAK细胞杀伤活性；提高免疫抑制小鼠外周血CD3+和CD4+/CD8+比值；提高免疫抑制小鼠腹腔巨噬细胞吞噬活性及iNOS活力,从而提高免疫抑制小鼠的细胞免疫功能。
5小鼠血清中细胞因子的变化提示,免疫抑制小鼠的Th1细胞向Th2细胞偏移,Thl/Th2亚群失衡,金黄扶正散对免疫抑制小鼠Thl/Th2亚群的调节,可能是通过提高免疫抑制小鼠脾脏T-bet mRNA的表达和降低GATA mRNA的表达来实现的。
6金黄扶正散可抑制免疫抑制小鼠脾细胞凋亡,降低凋亡指数,其机制可能是通过促进bcl-2 mRNA的表达、提高bcl-2/bax的比值,抑制Fas与FasL mRNA的表达,影响线粒体、死亡受体依赖的凋亡途径来实现的。
Objective:To investigate the immune regulation effects of Jinhuang fuzheng Powders in immunosuppressive mice, the immunosuppressive mouse model was established to observe JH in immune organs, anti-stress ability, the levels of anti-free radical and immune cell function of immunosuppressive mice and to explore its mechanisms. This study provides a basis to clinical application.
Methods:
1 Mice were divided into 6 groups randomly. All of mice were administrated orally 10 days, at the same time, in addition to the control group. Mice were injected Cyclophosphamide (CTX) 30mg/kg every other day subcutaneously to establish the immunosuppressive mice model. These six group were Control group(ig NS 0.02mL/10g), CTX group(sc CTX 30mg/kg q.o.d), LMS group(ig LMS 25 mg/kg/d, 10d+sc CTX 30mg/kg q.o.d), JH-L group(ig JH 1.27 g/kg/d, 10d+sc CTX 30mg/kg q.o.d), JH-M group(ig JH 2.54g/kg/d, lOd+sc CTX 30mg/kg q.o.d) and JH-H group(ig JH 5.08g/kg/d, 10d+sc CTX 30mg/kg q.o.d) respectively. After the last administration, the mice were fasted more than 12h, the thymus and spleen were weighed to calculate the thymus index and spleen index, lactate dehydrogenase (LDH) activity and acid phosphatase (ACP) activity were measured in the spleen suspension.
2 After administration, swimming test, high temperature test, cold temperature test, normobaric hypoxia test and toxic hypoxia test were observed in mice.
3 Total antioxidant capacity(T-AOC), activities of catalase(CAT), superoxide dismutase(SOD), glutathione peroxidase(GSH-PX) and malondialdehyde (MDA) contents were measured in spleen of mice.
4 Using four methyl thiazolyl tetrazolium(MTT) method, T lymphocyte and B lymphocyte proliferation were measured in spleen cells induced by concanavalin A (ConA) and lipopolysaccharide(LPS) respectively. Activities of Natural killer cells(NK cells) and Lymphokine-activated killer cells(LAK cells) activities were measured. Peritoneal macrophages phagocytosis of neutral red ability and inducible Nitric oxide synthase (iNOS) activity were measured.
5 Cytokines were quantitative detectected by using cytokine antibody microarray in mice serum.
6 Expression of T-bet and GATA mRNA were detected by real time quantitative polymerase chain reaction (FQ-PCR) method in spleen of Mice.
7 After administration, mice were sacrificed and their spleen were fixed by 4% polyoxymethylene, dehydrated, embedded in paraffin and sliced, then apoptosis were observed by TUNEL method.
8 Expression of Bcl-2, Bax, Fas and FasL mRNA were detected by FQ-PCR method in spleen of Mice.
Results:
1 Compared with the control group, the thymus index and the spleen index of CTX group were decreased significantly (P＜0.05), after administration, the thymus index and the spleen index of middle dose group of JH(JH-M) and high dose group of JH(JH-H) were increased significantly (P＜0.01, P＜0.05); Compared with the control group, LDH and ACP activities of CTX group were decreased significantly (P＜0.01); after administration, LDH and ACP activities of JH-L, JH-M and JH-H groups were increased significantly (P＜0.01, P＜0.05).
2 Compared with the normal control group, the swimming time of CTX group was decreased significantly (P＜0.05), survival time in high temperature, low temperature, normobaric hypoxia and toxic hypoxia test were decreased significantly (P＜0.01); after administration, compared with CTX group, swimming time of JH-M and JH-H groups were prolonged significantly (P＜0.05), high temperature survival time of JH-L and JH-M groups were prolonged significantly (P＜0.01), cold temperature survival time of JH-L and JH-M group were prolonged significantly (P<0.05), normobaric hypoxia survival time of JH-L and JH-H groups were prolonged significantly (P＜0.01), toxic hypoxia survival time of JH-M group were prolonged significantly (P＜0 .01).
3 Compared with the control group, T-AOC capability and CAT, SOD, GSH-PX activities of CTX group were decreased significantly (P＜0.05, P＜0.01), MDA content of CTX group were increased significantly (P＜0.05). After administration, T-AOC capability of JH-M and JH-H groups were increased significantly (P＜0.01); CAT, SOD and GSH-PX activities of JH-H group were increased significantly (P＜0.05, P＜0.01); MDA content of JH-M and JH-H groups were decreased significantly (P＜0.05, P＜0.01).
4 Compared with CTX group, T and B lymphocyte proliferation ability of JH-L, JH-M and JH-H groups were improved significantly (P＜0.01). NK cells and LAK cells activities of JH-M and JH-H groups were increased significantly (P＜0.01, P＜0.05). CD3+ numbers of JH-H group were increased significantly (P＜0.05), CD4+ numbers of JH-M and JH-H groups were increased significantly (P＜0.01), CD8+ numbers of JH-L, JH-M and JH-H groups were decreased, CD4+/CD8+ ratio of JH-L, JH-M and JH-H groups were increased significantly (P＜0.01, P＜0.05). phagocytic ability of peritoneal macrophages of JH-M and JH-H groups were increased and iNOS activity of JH-L, JH-M and JH-H groups were increased significantly (P＜0.01, P＜0.05).
5 Compared with the control group, IFN-γand RANTES of CTX group decrease significantly (P＜0.05), after administration, IFN-γof JH-L, JH-M and JH-H groups and RANTES of JH-H group increase significantly (P＜0.05). Compared with the control group, IL-5, IL-6, IL-9, IL-13 and MCP-1 of CTX group increase significantly (P＜0.01, P＜0.05), after administration, IL-5, IL-6, IL-9, IL-13 and MCP-1 of JH-L, JH-M and JH-H groups were decreased to different degrees. The rest Cytokines of GM-CSF, IL-1 a, IL-10, IL-2, IL-3, IL-4, IL-10, IL-12, IL-17, M-CSF, TNF-α, KC and VEGF were no changes obviously(P＞0.05).
6 Compared with CTX group, expressions of T-bet mRNA of JH-L, JH-M and JH-H groups were increased but the expressions of GATA mRNA were decreased significantly(P＜0.01).
7 Compared with the control group, the apoptosis index of CTX group were increased significantly(P＜0.05). Compared with CTX group, the apoptosis index of JH-M and JH-H groups were decreased significantly(P＜0.01, P＜0.05). The apoptosis index of JH-L groups was no changes obviously(P＞0.05).
8 Compared with CTX group, the expressions of Bcl-2 mRNA and the ratio of Bcl-2/ Bax mRNA of JH-M and JH-H groups were increased significantly(P＜0.01, P＜0.05), but expressions of GATA mRNA were decreased significantly(P＜0.01). Compared with CTX group, expressions of Fas and FasL mRNA of JH-M and JH-H groups were decreased significantly(P＜0.01, P＜0.05).
Conclusions:
1 The immunosuppressive mice model is established by using CTX. The thymus index and the spleen index decrease in immunosuppressive mice. JH can enhance the thymus index and the spleen index and increase activities of LDH and ACP in immunosuppressive mice, antagonize the immune suppression state caused by CTX.
2 JH can prolong the swimming time of immunosuppressive mice, prolong survival time of high temperature, low temperature, normobaric hypoxia and toxic hypoxia, increase anti-stress ability in immunosuppressive mice.
3 JH can improve ability of T-AOC and increase activities of CAT, SOD and GSH-PX significantly, decrease content of MDA significantly in immunosuppressive mice, regulate antioxidant enzymes and free radicals levels in the body.
4 JH can enhance proliferative capacity of spleen T lymphocytes and B lymphocytes in immunosuppressive mice significantly; improve activities of NK cells and LAK cells in immunosuppressive mice; enhance CD3+ numbers and CD4+/CD8+ ratio in immunosuppressive mice; enhance phagocytic activity of peritoneal macrophage and activity of iNOS in immunosuppressive mice, enhance cellular immunological function in immunosuppressive mice.
5 Cytokine changes of serum suggest that Thl cells may be offset to Th2 cells in immunosuppressive mice, there are imbalance in Th1/Th2 subgroup. Regulation of JH on Thl/Th2 subgroup may be achieved by improving expression of T-bet mRNA and reducing expression of GATA mRNA in immunosuppressive mice.
6 JH can inhibit apoptosis of spleen cells, reduce the apoptotic index? the mechanism may be achieved by promoting expression of Bc1-2 mRNA and ratio of the Bcl-2/Bax, inhibiting the expression of Fas and FasL mRNA, affecting the mitochondrial-death receptor-dependent apoptosis pathway.
为了探讨金黄扶正散对免疫抑制小鼠的免疫调节作用,本研究通过建立免疫抑制小鼠模型,观察金黄扶正散对免疫抑制小鼠免疫器官、抗应激能力、抗自由基水平和免疫细胞功能等方面的影响,并进行作用机制研究,为其临床应用提供依据。
方法
1 SPF级昆明种小鼠60只,14-18g,随机分为六组,分别为：正常对照组(生理盐水灌胃0.2mL/10g,连续10天)；环磷酰胺组(生理盐水灌胃0.2mL/10g,连续10天,同时隔天皮下注射环磷酰胺30mg/kg)；阳性对照组(每天灌胃左旋咪唑剂25 mg/kg,连续10天,给药后隔天皮下注射环磷酰胺30mg/kg)；金黄扶正茶高、中、低剂量组(每天分别灌胃金黄扶正茶5.08g/kg、2.54g/kg、1.27 g/kg,连续10天,给药后隔天皮下注射环磷酰胺30mg/kg),建立免疫功能低下小鼠模型。末次给药后小鼠禁食12h,处死,分别取胸腺,脾脏称重,计算胸腺、脾脏指数。将脾脏制成组织匀浆,离心,取上清,进行乳酸脱氢酶(LDH)活性测定和酸性磷酸酶(ACP)活性测定。
2造模及给药方法同1,分别进行负重游泳实验、耐高温实验、耐低温实验、常压耐缺氧实验和亚硝酸盐中毒性耐缺氧实验。游泳实验观察其游泳时间,其余各实验观察存活时间。
3造模及给药方法同1,取小鼠脾脏制成组织匀浆,离心,取上清,测定总抗氧化能力(T-AOC)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)和丙二醛(MDA)。
4造模及给药方法同1,采用四甲基偶氮唑蓝(MTT)法,测定刀豆蛋白A (ConA)诱导脾脏T淋巴细胞增殖和脂多糖(LPS)诱导B淋巴细胞增殖的影响,测定自然杀伤细胞(NK细胞)和淋巴因子激活的杀伤细胞(LAK细胞)活性,检测巨噬细胞吞噬中性红能力和诱导型一氧化氮合酶(iNOS)的活性。
5造模及给药方法同1.1,末次用药后小鼠禁食12h,各组小鼠尾静脉取血1mL,离心,取血清备用,采用细胞因子抗体芯片进行细胞因子定量检测。
6造模及给药方法同1.1,小鼠处死后,取脾,立即置液氮中,随后转移置-80℃低温冰箱保存,采用实时荧光定量PCR (FQ-PCR)方法检测脾脏T-bet、GATA mRNA的表达。
7造模及给药方法同1.1,小鼠处死后,取脾,4%多聚甲醛固定,常规脱水,石蜡包埋,切片,采用TUNEL染色法观察细胞凋亡情况。
8采用FQ-PCR方法检测脾脏Bcl-2、Bax、Fas和FasL mRNA的表达。
结果
1与正常对照组比较,环磷酰胺组胸腺指数,脾脏指数显著降低(P<0.05),给药后,金黄扶正散中、高剂量组可显著提高免疫抑制小鼠的胸腺指数、脾脏指数(P<0.01,P<0.05)。与正常对照组比较,环磷酰胺组可明显降低LDH、ACP的活性(P<0.01)；给药后,金黄扶正散低、中、高剂量组可明显提高免疫抑制小鼠的LDH活性和ACP活性(P<0.01,P<0.05)。
2与正常对照组比较,环磷酰胺组负重游泳时间明显减少(P<0.05),耐高温存活时间明显减少(P<0.01),耐低温存活时间明显减少(P<0.01),在常压缺氧条件下的存活时间明显减少(P<0.01),亚硝酸盐中毒性缺氧状态下存活时间明显减少(P<0.01)；给药后与模型组比较,金黄扶正散中、高剂量组能明显延长负重游泳时间(P<0.05),金黄扶正散低、中剂量组能明显延长耐高温存活时间(P<0.01),金黄扶正散低、中剂量组可显著提高小鼠的耐低温存活时间(P<0.05),金黄扶正散低、高剂量组能明显延长常压耐缺氧存活时间(P<0.01),金黄茶中剂量组能明显延长亚硝酸盐中毒性缺氧存活时间(P<0.01)。
3与正常对照组比较,环磷酰胺组可显著降低T-AOC能力(P<0.01),降低CAT、SOD、GSH-PX的活性(P<0.05,P<0.01)；明显提高MDA含量(P<0.05)。给药后,金黄扶正散中、高剂量组可明显提高免疫抑制小鼠的T-AOC能力(P<0.01)；金黄扶正散高剂量组可明显提高免疫抑制小鼠的CAT、SOD、GSH-PX的活性(P<0.05,P<0.01)；金黄扶正散中、高剂量组可明显降低免疫抑制小鼠的MDA含量(P<0.05,P<0.01)。
4与环磷酰胺组比较,金黄扶正散低、中、高剂量组均能显著提高免疫抑制小鼠的T、B淋巴细胞增殖能力(P<0.01)；金黄扶正散中、高剂量组能显著提高免疫抑制小鼠NK细胞、LAK细胞活性(P<0.01,P<0.05)；金黄扶正散高剂量组能显著性提高免疫抑制小鼠的CD3+淋巴细胞数(P<0.05),金黄扶正散中、高剂量组能显著性提高免疫抑制小鼠的CD4’淋巴细胞数(P<0.01),金黄扶正散低、中、高剂量组降低CD8+淋巴细胞数和提高CD4+/CD8+比值(P<0.01,P<0.05)；金黄扶正散中、高剂量组可提高腹腔巨噬细胞吞噬能力(P<0.05)；金黄扶正散低、中、高剂量组可提高巨噬细胞iNOS活性(P<0.01,P<0.05)。
5与正常对照组比较,环磷酰胺组的2种细胞因子IFN-Y和RANTES呈显著性降低(P<0.05),金黄扶正散低、中、高剂量组给药后IFN-γ有显著性提升,高剂量组的RANTES呈显著性升高(P<0.05)；与对照组比较,环磷酰胺组的5种细胞因子IL-5、IL-6、IL-9、IL-13和MCP-1呈显著性升高(P<0.01,P<0.05),给药组给药后有不同程度降低(P<0.01,P<0.05)；13种细胞因子GM-CSF、IL-1α、IL-1β、IL-2、IL-3、IL-4、IL-10、IL-12、IL-17、M-CSF、TNF-α、KC和VEGF无明显变化(P>0.05)。
6与环磷酰胺组比较,金黄扶正散低、中、高剂量组的T-bet mRNA表达量呈显著性提高而GATA mRNA表达量呈显著性降低(P<0.01)。
7与正常对照组比较,环磷酰胺组凋亡指数显著性提高(P<0.05)；金黄扶正散中、高剂量组凋亡指数与环磷酰胺组比较显著性下降(P<0.01,P<0.05)。金黄扶正散低剂量凋亡指数与环磷酰胺组比较无显著差异(P>0.05)。
8与环磷酰胺组比较,金黄扶正散中、高剂量组的bcl-2 mRNA表达量呈显著性提高(P<0.01),bcl-2/ bax mRNA比值呈显著性降低(P<0.01,P<0.05)。与环磷酰胺组比较,金黄扶正散中、高剂量组的Fas和FasL mRNA表达量均呈显著性降低(P<0.01,P<0.05)。
结论：
1采用免疫抑制剂环磷酰胺制造免疫抑制小鼠模型,造模后小鼠胸腺指数、脾指数下降,金黄扶正散可提高免疫抑制小鼠的胸腺指数、脾脏指数,提高免疫抑制小鼠的LDH和ACP活性,激活巨噬细胞的活化状态。
2金黄扶正散可延长免疫抑制小鼠负重游泳时间,提高其耐高温、低温、常压耐缺氧、亚硝酸盐中毒性缺氧的存活时间,提高免疫抑制小鼠抗应激的能力。
3金黄扶正散可显著提高免疫抑制小鼠CAT、T-SOD、GSH-PX的活性显著提高T-AOC能力,明显降低MDA含量,调节体内抗氧化酶的水平和自由基水平,改善机体的氧化还原状态。
4金黄扶正散能显著提高免疫抑制小鼠脾脏T淋巴细胞和B淋巴细胞增殖能力；提高免疫抑制小鼠NK细胞和LAK细胞杀伤活性；提高免疫抑制小鼠外周血CD3+和CD4+/CD8+比值；提高免疫抑制小鼠腹腔巨噬细胞吞噬活性及iNOS活力,从而提高免疫抑制小鼠的细胞免疫功能。
5小鼠血清中细胞因子的变化提示,免疫抑制小鼠的Th1细胞向Th2细胞偏移,Thl/Th2亚群失衡,金黄扶正散对免疫抑制小鼠Thl/Th2亚群的调节,可能是通过提高免疫抑制小鼠脾脏T-bet mRNA的表达和降低GATA mRNA的表达来实现的。
6金黄扶正散可抑制免疫抑制小鼠脾细胞凋亡,降低凋亡指数,其机制可能是通过促进bcl-2 mRNA的表达、提高bcl-2/bax的比值,抑制Fas与FasL mRNA的表达,影响线粒体、死亡受体依赖的凋亡途径来实现的。
Objective:To investigate the immune regulation effects of Jinhuang fuzheng Powders in immunosuppressive mice, the immunosuppressive mouse model was established to observe JH in immune organs, anti-stress ability, the levels of anti-free radical and immune cell function of immunosuppressive mice and to explore its mechanisms. This study provides a basis to clinical application.
Methods:
1 Mice were divided into 6 groups randomly. All of mice were administrated orally 10 days, at the same time, in addition to the control group. Mice were injected Cyclophosphamide (CTX) 30mg/kg every other day subcutaneously to establish the immunosuppressive mice model. These six group were Control group(ig NS 0.02mL/10g), CTX group(sc CTX 30mg/kg q.o.d), LMS group(ig LMS 25 mg/kg/d, 10d+sc CTX 30mg/kg q.o.d), JH-L group(ig JH 1.27 g/kg/d, 10d+sc CTX 30mg/kg q.o.d), JH-M group(ig JH 2.54g/kg/d, lOd+sc CTX 30mg/kg q.o.d) and JH-H group(ig JH 5.08g/kg/d, 10d+sc CTX 30mg/kg q.o.d) respectively. After the last administration, the mice were fasted more than 12h, the thymus and spleen were weighed to calculate the thymus index and spleen index, lactate dehydrogenase (LDH) activity and acid phosphatase (ACP) activity were measured in the spleen suspension.
2 After administration, swimming test, high temperature test, cold temperature test, normobaric hypoxia test and toxic hypoxia test were observed in mice.
3 Total antioxidant capacity(T-AOC), activities of catalase(CAT), superoxide dismutase(SOD), glutathione peroxidase(GSH-PX) and malondialdehyde (MDA) contents were measured in spleen of mice.
4 Using four methyl thiazolyl tetrazolium(MTT) method, T lymphocyte and B lymphocyte proliferation were measured in spleen cells induced by concanavalin A (ConA) and lipopolysaccharide(LPS) respectively. Activities of Natural killer cells(NK cells) and Lymphokine-activated killer cells(LAK cells) activities were measured. Peritoneal macrophages phagocytosis of neutral red ability and inducible Nitric oxide synthase (iNOS) activity were measured.
5 Cytokines were quantitative detectected by using cytokine antibody microarray in mice serum.
6 Expression of T-bet and GATA mRNA were detected by real time quantitative polymerase chain reaction (FQ-PCR) method in spleen of Mice.
7 After administration, mice were sacrificed and their spleen were fixed by 4% polyoxymethylene, dehydrated, embedded in paraffin and sliced, then apoptosis were observed by TUNEL method.
8 Expression of Bcl-2, Bax, Fas and FasL mRNA were detected by FQ-PCR method in spleen of Mice.
Results:
1 Compared with the control group, the thymus index and the spleen index of CTX group were decreased significantly (P＜0.05), after administration, the thymus index and the spleen index of middle dose group of JH(JH-M) and high dose group of JH(JH-H) were increased significantly (P＜0.01, P＜0.05); Compared with the control group, LDH and ACP activities of CTX group were decreased significantly (P＜0.01); after administration, LDH and ACP activities of JH-L, JH-M and JH-H groups were increased significantly (P＜0.01, P＜0.05).
2 Compared with the normal control group, the swimming time of CTX group was decreased significantly (P＜0.05), survival time in high temperature, low temperature, normobaric hypoxia and toxic hypoxia test were decreased significantly (P＜0.01); after administration, compared with CTX group, swimming time of JH-M and JH-H groups were prolonged significantly (P＜0.05), high temperature survival time of JH-L and JH-M groups were prolonged significantly (P＜0.01), cold temperature survival time of JH-L and JH-M group were prolonged significantly (P<0.05), normobaric hypoxia survival time of JH-L and JH-H groups were prolonged significantly (P＜0.01), toxic hypoxia survival time of JH-M group were prolonged significantly (P＜0 .01).
3 Compared with the control group, T-AOC capability and CAT, SOD, GSH-PX activities of CTX group were decreased significantly (P＜0.05, P＜0.01), MDA content of CTX group were increased significantly (P＜0.05). After administration, T-AOC capability of JH-M and JH-H groups were increased significantly (P＜0.01); CAT, SOD and GSH-PX activities of JH-H group were increased significantly (P＜0.05, P＜0.01); MDA content of JH-M and JH-H groups were decreased significantly (P＜0.05, P＜0.01).
4 Compared with CTX group, T and B lymphocyte proliferation ability of JH-L, JH-M and JH-H groups were improved significantly (P＜0.01). NK cells and LAK cells activities of JH-M and JH-H groups were increased significantly (P＜0.01, P＜0.05). CD3+ numbers of JH-H group were increased significantly (P＜0.05), CD4+ numbers of JH-M and JH-H groups were increased significantly (P＜0.01), CD8+ numbers of JH-L, JH-M and JH-H groups were decreased, CD4+/CD8+ ratio of JH-L, JH-M and JH-H groups were increased significantly (P＜0.01, P＜0.05). phagocytic ability of peritoneal macrophages of JH-M and JH-H groups were increased and iNOS activity of JH-L, JH-M and JH-H groups were increased significantly (P＜0.01, P＜0.05).
5 Compared with the control group, IFN-γand RANTES of CTX group decrease significantly (P＜0.05), after administration, IFN-γof JH-L, JH-M and JH-H groups and RANTES of JH-H group increase significantly (P＜0.05). Compared with the control group, IL-5, IL-6, IL-9, IL-13 and MCP-1 of CTX group increase significantly (P＜0.01, P＜0.05), after administration, IL-5, IL-6, IL-9, IL-13 and MCP-1 of JH-L, JH-M and JH-H groups were decreased to different degrees. The rest Cytokines of GM-CSF, IL-1 a, IL-10, IL-2, IL-3, IL-4, IL-10, IL-12, IL-17, M-CSF, TNF-α, KC and VEGF were no changes obviously(P＞0.05).
6 Compared with CTX group, expressions of T-bet mRNA of JH-L, JH-M and JH-H groups were increased but the expressions of GATA mRNA were decreased significantly(P＜0.01).
7 Compared with the control group, the apoptosis index of CTX group were increased significantly(P＜0.05). Compared with CTX group, the apoptosis index of JH-M and JH-H groups were decreased significantly(P＜0.01, P＜0.05). The apoptosis index of JH-L groups was no changes obviously(P＞0.05).
8 Compared with CTX group, the expressions of Bcl-2 mRNA and the ratio of Bcl-2/ Bax mRNA of JH-M and JH-H groups were increased significantly(P＜0.01, P＜0.05), but expressions of GATA mRNA were decreased significantly(P＜0.01). Compared with CTX group, expressions of Fas and FasL mRNA of JH-M and JH-H groups were decreased significantly(P＜0.01, P＜0.05).
Conclusions:
1 The immunosuppressive mice model is established by using CTX. The thymus index and the spleen index decrease in immunosuppressive mice. JH can enhance the thymus index and the spleen index and increase activities of LDH and ACP in immunosuppressive mice, antagonize the immune suppression state caused by CTX.
2 JH can prolong the swimming time of immunosuppressive mice, prolong survival time of high temperature, low temperature, normobaric hypoxia and toxic hypoxia, increase anti-stress ability in immunosuppressive mice.
3 JH can improve ability of T-AOC and increase activities of CAT, SOD and GSH-PX significantly, decrease content of MDA significantly in immunosuppressive mice, regulate antioxidant enzymes and free radicals levels in the body.
4 JH can enhance proliferative capacity of spleen T lymphocytes and B lymphocytes in immunosuppressive mice significantly; improve activities of NK cells and LAK cells in immunosuppressive mice; enhance CD3+ numbers and CD4+/CD8+ ratio in immunosuppressive mice; enhance phagocytic activity of peritoneal macrophage and activity of iNOS in immunosuppressive mice, enhance cellular immunological function in immunosuppressive mice.
5 Cytokine changes of serum suggest that Thl cells may be offset to Th2 cells in immunosuppressive mice, there are imbalance in Th1/Th2 subgroup. Regulation of JH on Thl/Th2 subgroup may be achieved by improving expression of T-bet mRNA and reducing expression of GATA mRNA in immunosuppressive mice.
6 JH can inhibit apoptosis of spleen cells, reduce the apoptotic index? the mechanism may be achieved by promoting expression of Bc1-2 mRNA and ratio of the Bcl-2/Bax, inhibiting the expression of Fas and FasL mRNA, affecting the mitochondrial-death receptor-dependent apoptosis pathway.
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