| 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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