Studying The Immunoregulatory Activity Of Codonopsis Pilosula Polysaccharide In SD Rats

Objective: The aim of this study is to investigate the immunomodulatory activity of Codonopsis pilosula Polysaccharid(CPP)and explore the underlying cellular mechanism.We detected the effect of CPP on the proliferation of rat splenic lymphocytes and peritoneal macrophages in vitro and as well its effect on immunocompromised rat modelin vivo.Our findings provide the experimental basis for the development and utilization of CPP as a potential immunoenhancer.Methods: Splenic lymphocytes and peritoneal macrophages were derived from eight-week-old SD rats,and used as target cells to test CPP effect on immune cell activity.CCK-8 kit was adopted to examine the effects of CPP on the proliferation of rat splenic lymphocytes and peritoneal macrophages.Lactate dehydrogenase(LDH)kit,acid phosphatase(ACP),interleukin-2(IL-2),interleukin-4(IL-4)and interleukin-6(IL-6)were used to detect the effects of CPP on rat spleen lymphocytes and peritoneal macrophages.The effect of CPP on the spleen index and thymus index of the immunocompromised rat was measured by weighing.CPP’s effect on rat spleen and thymus morphology were observed by HE staining.The changes of cytokines(C3,C4,IgG,IgM,IgA)secretion in serum were detected by ELISA.The effect of CPP on serum hormones(GH,T3,T4,INS)was detected by RIA.The expression of Bax,Bcl-2 and NF-κBp-p65 in rat spleen and thymus was detected by immunohistochemistry and Western-blot.Results: Cell experiments⑴Compared with the control group,CPP groups(50μg/mL,100 μg/m L,200 μg/mL)showed enhanced growth of rat splenic lymphocytes and peritoneal macrophages,(P<0.05).The optimal treatment was 200 μg/mL CPP with the treatment time of 24h(P<0.01).⑵Compared to the control group,no obvious cytotoxicity was observed in CPP-treated groups(P>0.05).⑶Compared to the control group,the phagocytosis of macrophage in different concentrations of CPP was significantly enhanced(P<0.01).⑷Compared to the control group,increased secretion of TNF-α,IL-2,IL-4 and IL-6 in different concentrations of CPP.Animal experiments⑴CPP-treated immunosuppressive rats induced by cyclophosphamide showed increased spleen and thymus index compared to the control immunosuppressive ones(P<0.05-0.01).The effect of CPP was in a dose-dependent manner(P<0.01).⑵Serum C3,C4,IgG,IgM,IgA,GH levels were increased in different concentrations of CPP groups(50 mg/kg,100 mg/kg,200 mg/kg(P<0.05-0.01).⑶The protein expression of Bax and NF-κBp-p65 were decreased while the expression of Bcl-2 protein was increased in spleen and thymus of CPP-treated immunosuppressive rats(P<0.05 compared to the controls).Conclusion: ⑴CPP can enhance the proliferation and phagocytosis of rat splenic lymphocytes and peritoneal macrophages and the secretion of related cytokines TNF-α,IL-2,IL-4 and IL-6,so as to improve the immuno activity of rat spleen lymphocytes and peritoneal macrophages.⑵CPP enhances the spleen index and thymus index in immunosuppressive rat model,indicating that CPP protects immune tissues and organs.⑶CPP increases the concentration of cytokines C3,C4,IgG,IgM and IgA secreted by spleen cells in rat model,indicating that CPP has immunomodulatory activities.⑷CPP may stimulate the proliferation of rat’s spleen lymphocytes and peritoneal macrophages by increasing the body’s hormone concentrations of GH.⑸CPP enhances the immune capacity of immunocompromised rats,and its mechanism may be related to the inhibition of Bax and NF-κBp-p65 expression and meanwhile the up-regulation of Bcl-2 expression,an antiapoptotic protein in immune organs.