Antagonization of eplerenone on proliferation of cardiac fibroblasts induced by co-cultured Treg cells based on inhibition of Kv1.3 channel of Tregs / 中国药理学通报

ABSTRACT

Aim To establish a co-incubation system in cardiac fibroblasts of SD neonatal rats and spleen CD4+ CD25 + regulatory T lymphocytes (Tregs) of normal adult SD rats,and to investigate the effects of eplerenone(EPL) on the interaction of two cells and the relationship with the Kvl.3 channel on Tregs cell membrane.Methods 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 groupsCFs,CFs + Tregs,CFs + Tregs + EPL,Tregs.The proliferation of CFs was detected by CCK-8 method.The expression levels of type Ⅰ collagen,type m collagen and matrix metalloproteinase 2 (MMP-2) secreted by CFs were detected by ELISA.The mRNA expression levels of Kv1.3,KCa3.1 on Tregs cell membrane and intracellular CRAC channel were detected by RT-qPCR technique.Tregs cell membrane Kvl.3 channel protein expression levels were determined by In-Cell Western blot.Results After 48 h incubation of the co-culture system,the cell proliferation was stable.CFs proliferation was marked(P ＜0.01),which could be inhibited by EPL(P ＜0.01).The type Ⅰ,type Ⅲ collagen and MMP-2 secreted by CFs increased (P ＜ 0.01).The expression levels of Kv1.3,KCa3.1 and CRAC channel mRNA in Tregs increased by 6.95,1.99 and 1.53 fold (CFs + Tregs vs Tregs,P ＜0.01),EPL decreased the mRNA level of each channel (CFs +Tregs + EPLvs CFs + Tregs,P＜0.01),and the decrease of Kv1.3 channel was the most significant (P ＜ 0.01).The Kv1.3 channel protein of Tregs increased by 67.9％ (CFs + Tregs vs Tregs,P ＜0.01),which could be inhibited by EPL(P ＜ 0.01).Conclusions Tregs cultured with CFs after 48 h can significantly promote the proliferation of CFs,and EPL can down-regulate the Kv1.3 channel expression on the Tregs membrane and inhibit the activation/proliferation of Tregs,indirectly inhibiting myocardial fibrosis.