| Objective: To investigate the interaction between the activation of Treg cells mediated by Kv1.3 channel gene and myocardial fibroblast(CFs)cells,and to clarify the key role of Kv1.3 channel gene in the activation of Treg cells and the promotion of myocardial fibrosis.Methods: the interference sequence were designed and ligated to Lentivirus vector,then the recombinant vector were transfected to Tregs of rat after,and Co-cultured with myocardial fibroblasts in vitro,and RT-qPCR and whole-cell patch-clamp methods were used to detect gene silencing of transfected Treg cells and ELISA method was used to detect cytokine secretion levels of Tregs and CFs cells.And Proliferation ability of CFs cells,and the mRNA levels of related genes Kv1.3,KCa3.1 and CRAC mRNA and Kv1.3 proteins levels were detected after co-culture the CFs with Treg cells by CCK-8,RT-qPCR and In cell western.Results: 1.The gene interfering lentivirus vector was successfully constructed and transfected into Tregs cells: Compared with normal group,The silent group expression of Kv1.3 mRNA was significantly decreased(P<0.01)and the inhibition rate reached 78%;Compared with normal group,the silent group of the peak current density of Kv1.3 potassium channel +40 mV was significantly decreased,the decreased value of 71.3%;2.CCK-8 experiment showed that compared with CFs group,the proliferation of CFs+Tregs group was the significantly increased(P<0.01);and compared with CFs+Tregs group,the proliferation of was the significantly increased(P<0.01);and CFs+Tregs+EPL group was significantly lower than CFs+Tregs group and CFs+RNAi-Tregs+EPL group showed no significant change.3.Co-culture system in ELISA method to detect show that compared with CFs group CFs+Tregs group type Ⅰ collagenase secreted,type Ⅲ collagenase,matrix metalloproteinases(MMP-2)significantly increased(P < 0.01),CFs+Tregs+EPL can inhibit the increase in vitro.Compared with the CFs group,there was no significant change the level of IL-10 of intracellular and extracellular and by CFs+Tregs group,but the TGF-β level was significantly increased(P<0.01).IL-10 level was decreased in the CFs+Tregs+EPL group(P<0.05),and TGF-β level was significantly reduced(P<0.01).EPL significantly inhibited TGF-β levels in both extracellular and intracellular of Tregs(P<0.05).Compared with the Tregs group,there was no significant change in IL-10 level in the RNAi-Tregs group and the TGF-β level was significantly reduced(P<0.01).The TGF-β levels in the CFs+RNAi-Tregs group were significantly lower than in the CFs+Tregs group(P<0.01).4.The Tregs+CFs group expressions of Kv1.3mRNA relative expression and KCa3.1 gene mRNA relative expression compared with Tregs group were significantly increased(P<0.01).However,RNAi-Tregs+EPL group Kv1.3 gene mRNA relative expression gene relative expression compared with Tregs group was significantly decreased(P<0.01).and RNAi-Tregs+CFs group and Tregs+CFs group of KCa3.1 gene mRNA relative expression compared with Tregs+CFs group was no changed;TheTregs group CRAC gene mRNA relative expression levels were no change.5.Compared with the Tregs group,the co-culture system Tregs+CFs group of Kv1.3 protein expression levels was significantly higher(P<0.01);and the Tregs+CFs+EPL group of Kv1.3 protein was significantly lower(P<0.01);RNAi-Tregs Kv1.3 protein significantly decreased(P<0.01).Compared with Tregs+CFs group,RNAi-Tregs+CFs group Kv1.3 protein was significantly reduced(P<0.01).However,there was no significant change in Kv1.3 protein expression between RNAi-Tregs+CFs groups and RNAi-Tregs+CFs+EPL groups.Conclusions Tregs and CFs were co-cultured in vitro,and Kv1.3 channel mediated the activation of Tregs and significantly induced the proliferation of CFs.By directly inhibiting the Kv1.3 channel,epridone can inhibit the activation of Tregs and reduce the secretion of TGF-β,thereby reducing the proliferation of CFs inhibiting myocardial fibrosis thereby inhibiting myocardial fibrosis suggesion: Kv1.3 may be a potential immunological target for the treatment of heart failure. |
PDF Full Text Request