Effect Of Astragaloprotein On The Balance Of Th17/Treg Cells In CIA Model Mice And Its Mechanism

ObjectiveRheumatoid arthritis is a kind of chronic autoimmune disease characterized by the multi-joint synovitis,bone and cartilage destruction.The early-stage disease is mainly manifested as the red,swollen,hot,pain and dysfunction of joint,while the late-stage patients may suffer from the joint deformity in different degrees with a high morbidity.Astragalus membranaceus glycoprotein(AmGP)is a kind of homogeneous component of the glycoprotein isolated from the dried roots of Astragalus membranacus via water extraction,and then a kind of 16kD natural glycoprotein active molecule was isolated and purified from AmGP and named AmRP10-16.Th17 cell and Treg cell are two subtypes of CD4+T cells.Th17/Treg cell balance plays an important role in maintaining the immune homeostasis of the cells.The latest stndies have shown that Th17/Treg cell imbalance is the key link in the pathogenesis of RA.Previous studies of the research group showed that AmGP had a certain immunosuppressive effect in vitro,and the in-vivo administration could improve the joint inflammation of rats with adjuvant arthritis(AA)and mice with collagen-induced arthritis(CIA).However,the immune activity of AmRP10-16 isolated from AmGP and its effect and mechanism on CIA mice remain unclear.In the in-vitro experiment,the splenic lymphocytes of mice were separated conventionally,and the effect of AmGP on the proliferation of spleen T and B lymphocytes in mice was detected via MTT assay to investigate the in-vitro immunological activity of AmGP.In the in-vivo experiment,CIA mice model was established with the intraperitoneal injection of AmGP;the effect and possible mechanism of AmGP on CIA mice were investigated with Th17/Treg cell balance as an entry point,so as to provide experimental basis for clinical treatment of RA with Astragalus membranaceus.Methods1.MTT assay was used to detect the effect of AmGP on the proliferation of spleen T and B lymphocytes in mice.2.CIA mice model was established,and the mice with successful modeling were randomlydivided into the model group,hydrocortisone(HC)positive control group and high-dose,medium-dose and low-dose AmGP group with 10 mice in each group.At 42d after the fundamental immunity,the drug was administrated for treatment.The mice in HC group were injected with 10mg/kg HC intraperitoneally.The mice in high-dose,medium-dose and low-dose AmGP group were injected with 0.5mg/kg,1.0mg/kg and 2.0mg/kg AmGP intraperitoneally.The mice in normal group and model group were injected intraperitoneally with equal volume of normal saline(0.4ml/20g,once per day).After two weeks of treatment,the general conditions,arthritis index score and pathological changes in knee joint of mice in each group were observed to evaluate the severity of illness of CIA mice.3.The proportion of Th17/Treg cells in peripheral blood in mice was detected via flow cytometry.4.The protein expressions of RORyt,Foxp3,P-STAT3 and P-STAT5 in spleen tissues of mice were detected via Western blot.5.The positive protein expressions of RORyt,Foxp3,P-STAT3 and P-STAT5 in spleen tissues of mice were detected via immunohistochemical method.Results1.In-vitro immunological activity of AmGPAmGP in different concentrations significantly inhibited proliferation of spleen T and B lymphocytes in mice in vitro(P<0.01).AmRP10-16 in different concentrations had the immunosuppressive activity on the in-vitro proliferation of spleen T and B lymphocytes in mice under the stimulation of ConA and LPS.2.Preparation and morphological observation of CIA mice modelAmGP in different concentrations could not only improve the diet and mental state of CIA mice,increase the activity and reduce the arthritis index score of CIA mice,but also improve the pathological state of joint synovium and reduce the degree of damage to articular cartilage and bone,suggesting that AmGP can improve the arthritis of CIA mice model,which has the pharmacological inhibition effect on CIA model.3.Effect and mechanism of AmGP on Th17/Treg cell balance in CIA mice(1)Flow cytometry showed that the proportion of Thl 7 cells in peripheral blood of mice in model group was increased,while that of Treg cells was decreased(P<0.01).Compared with those in model group,the proportion of Th17 cells in HC group and each AmGP group was decreased,while that of Treg cells was increased,and the differences were statistically significant(P<0.05,P<0.01).(2)Western blot and immunohistochemistry showed that compared with those in normal group,the protein expression quantities of RORyt and P-STAT3 in the spleen tissues of mice in model group were increased,while those of Foxp3 and P-STAT5 were decreased(P<0.01);compared with those in model group,the protein expression quantities of RORyt and P-STAT3 in the spleen tissues of mice in HC group and each AmGP group were decreased(P<0.05,P<0.01),while those of Foxp3 and P-STAT5 were significantly increased(P<0.05,P<0.01).Conclusions1.AmGP has the immunosuppressive activity on in-vitro proliferation of spleen T and B lymphocytes in mice.2.AmGP can reduce the arthritis index and articular swelling of CIA mice,suggesting that AmGP can improve the arthritis of CIA mice to a certain extent and inhibit the progression of disease.3.AmGP can reduce the levels of Th17 cells in peripheral blood and its transcription factors,RORyt and P-STAT3,in CIA mice,and up-regulate the levels of Treg cells and its transcription factors,Foxp3 and P-STAT5,suggesting that AmGP can decrease the RORyt expression through effectively inhibiting the activation of JAK-STAT3 signaling pathway,promote the activation of JAK-STAT5 signaling pathway and up-regulate the Foxp3 expression,thus effectively recovering Th17/Treg cell balance.