| ObjectiveThis study was aimed at investigating the effect of Rhizoma Drynaria Flavonoids (RDF) on regulation of immune functions in cyclophosphamide-induced immunosuppressive mice and mechanism, which would provide experimental evidence for researching and developing RDF as an immunomodulator agent.Methods60 kunming mice (SPF) were randomly divided into 6 groups:normal control(NC) group, CTX model group, Levamisole (LMS) group, and high-, medium-, low-dose RDF treated groups (0.8,0.4,0.2 g/kg). Except the normal control group, the mice in the other groups were intraperitoneally injected (ip) with CTX to establish immunosuppression model. After 20 days of administration, all mice were killed, blood samples, hepatic tissues, spleens and thymuses were taken. The thymus index and the spleen index, clearance index K and phagocytic index a, delayed type hypersensitivity and serum hemolysin were detected. The contents of WBC, RBC, HGB, LYMPH%, PLT, HCT, MCHC, RDW-CV, RDW-SD were counted by hematology analyzer. IL-2, TNF-a, IFN-y levels in serum were measured by ELISA. The activities of LDH and ACP in spleen tissues were determined by colorimetry. Lymphocyte proliferation capacity in spleen was detected by MTT. T-lymphocyte subsets CD4+, CD8+T cells in peripheral blood were detected by FACS. The expressions of T-bet, GATA-3, IFN-y and IL-4 mRNA in spleen tissues were analyzed by RT-PCR.Results1. Effects of RDF on non-specific immune function in immunosuppressive miceIn this study, compared to the normal control group, the thymus index and the spleen index, clearance index K and phagocytic index a of CTX model group were significantly reduced (P<0.01). Compared to the CTX model group, the spleen index in the LMS group and low-, medium-, high-dose RDF treated groups were significantly elevated. Moreover, the thymus index in the LMS group and medium-, high-dose RDF groups were significantly increased, clearance index K and phagocytic index a in the LMS group and low-, medium-, high-dose RDF groups were significantly enhanced (P<0.05, P<0.01).2. Effects of RDF on the contents of blood in immunosuppressive miceCompared to the normal control group, the contents of WBC, RBC, HGB, LYMPH%, PLT, HCT in blood of CTX model group were significantly reduced, while the content of RDW-SD was significantly increased (P<0.01), there was no obvious difference in the contents of MCHC, RDW-CV (P>0.05). Compared to the CTX model group, the levels of WBC, RBC, HGB, LYMPH%, PLT, HCT in the LMS group and low-, medium-, high-dose RDF groups were significantly increased (P<0.05, P<0.01). However, there was no obvious difference in the levels of MCHC, RDW-SD, RDW-CV in among the treatment group.3. Effects of RDF on serum hemolysin, delayed type hypersensitivity, the activities of LDH and ACP in immunosuppressive miceThe results showed that, ear swellings, serum hemolysin, LDH and ACP activities of CTX model group were significantly decreased compared with normal control group (P<0.01). Compared to the CTX model group, ear swellings of LMS group and high-dose RDF group were elevated obviously, the level of serum hemolysin of LMS group and medium-, high-dose RDF groups were obviously increased. LDH activity in the LMS group and low-, medium-, high-dose RDF groups were significantly increased, ACP activity of LMS group and medium-, high-dose RDF groups were obviously elevated (P<0.05, P<0.01).4. Effects of RDF on cytokines in immunosuppressive miceThe ELISA kit results showed that, the levels of serum IL-2, TNF-a, IFN-y of CTX model group were lower than that of the normal control group (P<0.01). Compared to the CTX model group, serum IL-2 and IFN-y levels in the LMS group and medium-, high-dose RDF groups were significantly increased, while the level of TNF-a was significantly increased (P<0.05, P<0.01).5.Effects of RDF on immunocytes in immunosuppressive miceCompared to the normal control group, CD4+T cells, CD4+/CD8+ratio, ConA-induced T lymphocyte proliferation capacity and LPS-induced B lymphocyte proliferation ability in the CTX model group were obviously decreased, while the number of CD8+T cells was obviously increased (P<0.05, P<0.01). Compared to the CTX model group, CD4+T cells, ConA-induced T lymphocyte proliferation capacity of LMS group and low-, medium-, high-dose RDF groups were obviously increased, CD4+/CD8+ ratio in the LMS group and medium-, high-dose RDF groups were obviously elevated, the number of CD8+ T cells in the LMS group and low-, medium-, high-dose RDF groups were significantly decreased (P<0.05, P<0.01). However, there is no statistical significance in index of B-lymphocyte proliferation capacity among the RDF treatment group.6. Effects of RDF on T-bet, GATA-3, IFN-γ and IL-4 mRNA expressions of spleens in immunosuppressive miceThe results showed that, T-bet, IFN-γ mRNA expressions and T-bet/GATA-3, IFN-γ/IL-4 ratios in the CTX model group were lower than that of the normal control group, while GATA-3, IL-4 mRNA expressions were much higher than that of the normal control group (P<0.01). After administration, compared to the CTX model group, T-bet mRNA expression and T-bet/GATA-3 ratio in the LMS group and medium-, high-dose RDF treated groups were significantly increased, IFN-γ mRNA expression in the LMS group and low-medium-, high-dose RDF groups were significantly increased, GATA-3, IL-4 mRNA expressions were significantly decreased, IFN-γ/IL-4 ratio in the LMS group and low-, medium-, high-dose RDF groups were significantly enhanced (P<0.05, P<0.01).ConclusionsRDF enhanced the thymus index, the spleen index, macrophage phagocytic function, activities of LDH and ACP of spleens in immunosuppressive mice. It also increased the contents of WBC, RBC, HGB, LYMPH%, PLT, HCT, delayed type hypersensitivity and serum hemolysin, increased the levels of serum IL-2, TNF-α, IFN-γ, T lymphocytes proliferative capacity of spleens and CD4+T cells, CD4+/CD8+ratio in peripheral blood in immunosuppressive mice. RDF regulated the Th1/Th2 subgroup by increasing T-bet, IFN-γ mRNA expressions while reducing GATA-3 and IL-4 mRNA levels in immunosuppressive mice. This study showed that, RDF improved the immunity of the mice by protecting or enhancing the non-specific immunity, humoral and cellular immune functions in CTX-induced immunosuppressive mice, and antagonize the immunosuppressive status that induced by CTX. |
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