Study On Mechanism Of PD-1 Mediated Regulation Of T Cell Overactivation In RA With Moxibustion

Objective : In the view of the explicit anti-inflammatory and immunoregulation function of moxibustion for RA,our research selected PD-1 as the entry point and“PD-1/PD-L1 pathway—T cell overactivation”as the main line to explore the effects of moxibustion on T cell overactivation in treating RA,the research aims to revealing the mechanism of moxibustion treatment in a further and deep-going way.Methods:Experiment one we select 45 SD rats,random divided into three groups:normal control,RA model,moxibustion treatment.We established AA model by subcutaneous of rats right foot with Complete Freunds' Adjuvant(CFA)from the 1th day,Moxa-cone moxibustion treatment was given once a day to “ Shenshu ”,“ Zusanli”points from the 8th day,each course sustain six day a week,all treatment Carry out for 3 weeks,expressions of mPD-1 and mPD-L1 in synovial tissues were detected by immunohistochemistry and levels of sPD-1 and sPD-L1 in serum were examined by ELISA.Experiment two we select 30 SD rats,random divided into two groups : normal control,PD-1 RNAi group.We used RNAi to get PD-1 gene interference model from 2th day,no treatment is provided.Expression of PD-1mRNA in serum was examined by RT-PCR and levels of PD-1 in serum was examined by ELISA.Experiment three?four?five we selected 60 SD rats,random divided into four groups:normal control,RA model,moxibustion treatment and PD-1RNAi group,each group had 15 SD rats.We established AA model by subcutaneous of rats right foot with Complete Freunds' Adjuvant(CFA),and used RNAi to get PD-1gene interference model,Moxa-cone moxibustion treatment was given once a day to“Shenshu”,“Zusanli”points from the 8th day,each course sustain six day a week,all treatment Carry out for 3 weeks.All rats' general condition were observed,foot swelling degree were observed regularly;pathological morphology of synovial tissue and levels of IL-4,IL-10 and IFN-?were detected by HE and ELISA;Lck and Fyn smaples of splenic tissues were examined by immunohistochemistry,CD25,CD69,CD71 smaples of splenic tissue and serum were examined by FCM.Results:1.Test of mPD-1 ? mPD-L1 levels in synovial tissues showed that:Compared with control group,levels of mPD-1 and mPD-L1 in model group elevated significantly(P<0.01);levels of mPD-1?mPD-L1 from moxibustion group increased significantly comparing with those from model group(P<0.05).2.Test of sPD-1 ? sPD-L1 levels in serum showed that:Compared with control group,levels of sPD-1 and sPD-L1 from model group significantly elevated(P<0.01);comparing with the model group,levels from moxibustion group dramatically reduced(P<0.05).3.Test of PD-1 levels in synovial tissues showed that:Compared with control group,levels of PD-1 in RNAi group reduced significantly(P<0.01).4.Test of PD-1 mRNA levels in serum showed that:Compared with control group,levels of PD-1 mRNA in RNAi group reduced significantly(P<0.01).5.Moxibustion could significantly improve the experimental RA animals' overall condition and pathological morphology of inflammative synovium,reduce their joint perimeters.The role significantly reduced in the case of PD-1 disturbed.6.Test of Lck,Fyn levels in spleen tissues shows:As compared with those from control group,the levels of Lck,Fyn from the RA models elevated significantly(P<0.01);compared with the model group,levels from moxibustion group dramatically reduced(P<0.01);compared with the moxibustion group,the content of Lck in PD-1 RNAi reduced is less than the effect of moxibustion group.7.Test of CD69,CD71,CD25 levels in spleen tissues and serum T cell shows:As compared with those from control group,the levels of CD69,CD71,CD25 from spleen tissues and the levels of CD69,CD71 from serum in RA models elevated significantly(P<0.01)and the levels of CD25 from serum in RA models had no significant difference;compared with the model group,levels of CD69,CD71,from moxibustion group dramatically reduced(P<0.01),and the levels of CD25 from serum had no significant difference;compared with the moxibustion group,the content of CD25 in PD-1 RNAi reduced is less than the effect of moxibustion group.8.Test of serum levels of inflammatory cytokines shows:As compared with those from control group,the levels of IFN-?from the RA models elevated significantly(P<0.01),the levels of IL-10 reduced(P<0.05)and the levels of IL-4 showed no significant difference(p>0.05);compared with the model group,moxibustion group's IFN-? levels dramatically reduced(P<0.01);IL-10 and IL-4 levels elevated significantly(P<0.01);compared with the moxibustion group,the content of IFN-??IL-10 and IL-4 in PD-1 RNAi changed are less than the effect of moxibustion group.Conclusions:1.Moxibustion could promote expression of mPD-1?mPD-L1 as well as inhibit expression of sPD-1?sPD-L1 to strengthen the signal of PD-1/PD-L1 pathway thus regulate the over-expression of T cells.2.Moxibustion could restoring the balance of Th1/Th2 cytokine secretion and have explicit anti-inflammatory effects in RA,which are closely related with PD-1's negative regulation on T cell activation.