| Objective: To investigate the immune regulation effects of Radix Millettia Speciosa (RMSF) in immunosuppressive mice, the immunosuppressive mice model was established to observe the impact on Immune System of RMSF in immune organs, cell immunity, humoral immunity and mononuclear-scavenger system of immunosuppressive mice, and to compare the immunoregulatory effects and mechanisms of RMSF and total flavonoids of Radix Millettia Championi (RMCF) on immunosuppressed mice. This study provides a theoretical basis for its clinicalapplication.Methods: 1. The content of RMSF was determined by NaNO2-=Al(NO3)3-NaOH colorimetric with Rutin as a standard sample. Four kinds of resins for purifying RMSF were compared by static adsorption and elution test, and the separation and purification process parameters were optimized by the single factor experiment.2. Kunming mice were randomly divided into normal control group (NS 10 mL/kg), CTX group (NS 10 mL/kg), LMS+CTX group (20mg/kg), RMSF+CTX group (40, 20, 10 mg/kg) and RMCF+CTX group (40, 20, 10 mg/kg). Each group was intragastric administration once a day for 21 days. Mice were injected intraperitoneally with CTX (80 mg/kg) to establish immunosuppressed model mice on days 1, 2, 8.3. The changes of body weight, thymus and spleen index, WBC, the content of serum hemolysin, DTH and the phagocytic function of monocyte-macrophage of mice were determined to investigate the immune regulation effects of RMSF on the immune regulation of immunosuppressive mice.4. The number of WBC, thymus and spleen index; the specific cellular immunity, such as DTH and T lymphocyte proliferation assay; the specific humoral immunity, such as B lymphocyte proliferation assay; the nonspecific immune, such as indexes of the clearance rate of carbon, the activity of ACP and LDH in immunosuppressed mice were determined to compare RMSF and RMCF in terms of efficacy.5. The pathological morphological changes of spleen and thymus were observed by HE staining.6. The expression level of IL-2, TNF-a and IFN-y in serum of each group were determined.7. Expression of IFN-?, T-bet and Gata-3 mRNA were detected by real time quantitative polymerase chain reaction (FQ-PCR) method in spleen of Mice.Results: 1. With rutin as the control, the content of RMSF was 2.38mg/g by UV spectrophotometry at the wavelength of 510 nm. The optimum total flavonoids preparation process of RMSF was carried out by single factor compared, Clarification technology and purification technology were selected as follows: the concentrated sample was purified by Macroporous resin AB-8,sample concentration of 1.12 mg/mL at pH 5.5, sample velocity of 1.0 mL/min,and the optimal desorption conditions were achieved by using 4 BV of 80%ethanol asdesorption solvent at a flow rate of 1.0 mL/min. Under these conditions, the recovery rate of RMSF was 52.5% with the purity of 64.6%.2. A variety of commonly used index to reflect the innate and adaptive immunity had declined on CTX group mice(P<0.01 or P<0.05), indicating that the immunosuppressive mice models were successful.3. Compared with the CTX group, a certain dose of RMSF could significantly increase the body weight, the index of spleen and thymus (P<0.01 or P<0.05), the number of WBC (P<0.01), the degree of DTH (P<0.01), the content of serum hemolysin (P<0.05) and the phagocytic function of monocyte-macrophage in immunosuppressive mice (P<0.01 oP<0.05).4. Histological and morphological observation indicates that: Compared with the CTX group, RMSF (40?0 mg/kg) and RMCF (40?20 mg/kg) could significantly increased the thickness of thymic cortex and thymic epithelial cells;RMSF (40 ? 20 ? 10 mg/kg) and RMCF (40 mg/kg) had clear boundaries between the red pulp and white pulp, germinal center of splenic nodule was obvious and the density of cortical lymphocytes increased. Indicating that a certain dose RMSF and RMCF could repair the splenic and thymus tissue injury from CTX induced, so as to achieve the protection of immune organs.5. Compared with CTX group, RMSF and RMCF at different dose could significantly increase the WBC number (P<0.01); the index of spleen and thymus in immunosuppressive mice were also improved at different levels(P<0.01 or P<0.05); the proliferation ability of T and B lymphocyte in immunesuppression mice were promoted (P<0.01 or P<0.05), and the degree of DTH was increased (P<0.01); the phagocytic function of monocyte-macrophage in immunesuppression mice was improved (P<0.01 or P<0.05); the spleen of ACP and LDH activity were strengthen in immunosuppressive mice (P<0.01 or P<0.05); the cytokines of IL-2, TNF-a and IFN-y in serum were improved at different levels (P<0.01 or P<0.05). RMCF (40, 20, 10 mg/kg) were weaker than the corresponding RMSF group in improving the WBC number (P<0.01 or P<0.05); RMCF (40, 20 mg/kg) were weaker than the corresponding RMSF group in improving the ability of T lymphocyte proliferation and the degree of DTH (P<0.01 or P<0.05); RMCF (40, 20 mg/kg) were weaker than the corresponding RMSF group in raising the level of IFN-? (P<0.05); RMCF (40 mg/kg) was stronger than RMSF (40 mg/kg) in improving the ability of expurgation index (P<0.05). For other indicators, there was no statistically difference between RMCF and RMSF (P>0.05).6. Compared with CTX group, RMSF (40, 20, 10 mg/kg) could raised the level of IFN-y and T-bet in immunosuppressive mice (P<0.01 or P<0.05);RMCF (40, 20 mg/kg) can also make a significant increase at the expression level of T-bet (P<0.05), and RMSF had an obvious effect (P<0.01). The amount of GATA-3 mRNA expression was significantly decreased in RMCF (40, 20, 10 mg/kg) and RMSF (40, 20 mg/kg) (P<0.01 or P<0.05), and there were no statistically significant difference between them (P>0.05).Conclusion: 1. RMSF presented significant dose-response immune regulation for specific and nonspecific immune function in immunosuppressed mice.2. RMSF and RMCF had a certain effect on the immune function in the CTX-induced immunosuppressed mice, which had a similar role in immune regulation. RMCF was weaker than RMSF in improving cellular immune function.3. The immune regulation mechanism of RMSF might be achieved by promoting the expression of T-bet, IFN-y mRNA and inhibiting the expression of GATA mRNA in the spleen of immmunosuppressed mice, which could maintain the balance of Th1/Th2, and achieve the bidirectional regulation of immune function. |
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