Study On The Mechanism Of Eplerenone On Inhibition Of The Activation Of Tregs Mediated By Kv1.3 Channel And Reversing Cardiac Fibrosis

Objective:1.RT-PCR and Western Blotting were used to observe the expression of mRNA and protein in Kv1.3 plasma channel during chronic heart failure.To provide evidence for the immunological mechanism of chronic heart failure.2.To establish a co-incubation system between myocardial fibroblasts of neonatal SD rats and normal adult SD rats spleen CD4+CD25+ Tregs(hereinafter referred to as Tregs)in vitro,and to detect whether Tregs promotes the proliferation of CFs by molecular biological methods.To observe the effect of Eplerenone.The effect of it on the proliferation of two cells.Methods:1.Peripheral blood by immunomagnetic sorting method in patients with chronic heart failure and normal healthy volunteers of CD4+CD25+ Tregs.Grouped according to the training,the normal group,CHF group,CHF + EPL(30 ?M)group;Treg cells activity was detected by CCK-8,RT-PCR detected relative the expression of Kv1.3,KCa3.1 and CRAC channel mRNA level of Tregs,In-cell Western blotting was used to detect the relative expression level of Kv1.3 channel and KCa3.1 channel protein of Tregs.2.The spleen Tregs of normal adult SD rats were sorted by immunomagnetic bead sorting,and the myocardial fibroblasts of SD rats were isolated by differential adherence method.The experiment was conducted in the following groups:CFs,CFs + Tregs,CFs + Tregs + EPL and Tregs.The proliferation of CFs was detected by CCK-8 method.The expression levels of type ? collagen,type ? collagen and matrix metalloproteinase 2 secreted by CFs and TGF-??IL-10 secreted by Tregs were detected by ELISA.RT-PCR technique was used to detect the mRNA expression levels of Kv1.3,KCa3.1 and CRAC channel of Tregs.In-cell western blotting method detected Kv1.3 channel protein expression levels of Tregs.Results:1.Compared with the Ctrl group,the expression of Tregs Kv1.3,KCa3.1 and CRAC channels mRNA in chronic heart failure group was significantly higher(P<0.05).After it being incubated with EPL(30?M for 48 h)the relative expression of Kv1.3 channel and KCa3.1 channel protein and mRNA were significantly lower than those in chronic heart failure group.2.After 48 h incubation of the co-culture system,the cell proliferation was stable(CFs +Tregs vs CFs,P<0.05).EPL could inhibit its proliferation(P<0.05).The collagen of type ?,type ? and MMP2 secreted by CFs was increased(P<0.001).The expression levels of Kv1.3,KCa3.1 and CRAC channel mRNA in Tregs increased(CFs + Tregs vs Tregs,P<0.001),and EPL decreased the mRNA level of each channel(CFs + Tregs +EPLvs CFs + Tregs,P<0.01),the decrease of Kvl.3 channel was the most significant(P<0.001).The Kv1.3 channel protein of Tregs increased(CFs + Tregs vs Tregs,P<0.001),and EPL could inhibit it(P<0.001).The expression levels of Kvl.3 channel and KCa3.1 channel mRNA and protein in Tregs increased(normal vs CHF,P<0.05),and EPL could inhibit it(P<0.01).Conclution:At the time of chronic heart failure,although the number of Tregs was descend,but the expression of three ion channels mRNA of Tregs and protein of Kv1.3 and KCa3.1 channels of Tregs were all overexpressed.These results showed that Tregs were activated during heart failure.EPL could significantly inhibit the expression of mRNA in three ion channels and two channels protein Kvl.3 and KCa3.1 channels of Tregs.After co-incubation of Tregs and CFs in vitro,it was found that Tregs could promote the proliferation of both kinds of two cells,and the proliferation of CFs was more obvious than that of Tregs.By detecting the inhibitory effect of EPL on Kvl.3 channel,it is concluded that the function of Tregs increases through Kv1.3.TGF-? is strongly mediated and autocrine to activate its own function.TGF-?,as an important fibrogenic factor,promotes the proliferation of CFs,which may lead to the immunological mechanism of occurrence and development of heart failure.