醛固酮受体拮抗剂基于慢性心衰时T细胞Kv1.3通道的Th17/Treg平衡免疫调节机制

After being confirmed of reduction of chronic heart failure (CHF) mortality by Aldosterone antagonists (AAs), its anti-CHF machanism studies are undergoing deeply and gradually, especially more and more evidences imply AAs maybe exert the effect through regulating immune activation/inactivation during CHF, however it is still unclear. Based on the critical role of Kv1.3 channels in activating of T lymphocytes (in previous electrophysiology and protein/gene test, we found high expression and functional enhancement of the channel on the membrane of T lymphocytes in hypertension and myocardial infarction patients, and drugs could reverse it) and imbalance of Th17/Treg during CHF, adopt immuno- fluorescence and magnetic bead sorting/flow cytometry techniques to separate, purify and proliferate peripheral plasma CD4+CD25+Foxp3+ Treg cells from CHF rat model and patients, and utilize molecular biology, whole-cell patch clamp, biochemistry, and fluorescence probe approaches to detect Kv1.3 current, functional changes and the expression of its protein/gene; calcium signaling regulation etc. before and after AAs administration. Illustrate the electrophysiological mechanism of AAs and the basis of calcium signaling pathway in developing immunodisorder during CHF to exploit new idea in treatment and study of heart failure, which clinical application value would be higher.

自醛固酮(ALD)受体拮抗剂进一步降低慢性心衰的病死率得到肯定后，其抗心衰机制的研究逐渐深入，尤其越来越多的证据提示其可能通过调节心衰时的免疫激活/失活过程发挥效应，但其机制仍不清楚。基于Kv1.3钾通道在T淋巴细胞活化中的重要作用（我们前期电生理及蛋白基因检测发现高血压及心梗患者该通道在T淋巴细胞高表达并功能性增强，药物可对抗之）和心衰时T细胞亚群Th17/Treg的失衡表现，本项目拟利用免疫荧光，磁珠分选/流式检测等手段，分离纯化增殖慢性心衰大鼠及患者外周血CD4+CD25+Foxp3+ Treg细胞，通过分子生物学，膜片钳，生物化学及荧光探针等方法，检测给予ALD受体拮抗剂前后的Kv1.3电流及其蛋白基因的表达和功能改变；及钙信号的变化等，阐明ALD受体拮抗剂改善慢性心衰时免疫系统紊乱的电生理学机制及钙信号转导本质，开拓心衰治疗及研究的新思路，临床应用价值较高。

调节性T淋巴细胞（Tregs）作为重要的抗炎细胞分泌TGFβ和IL-10，对心肌纤维化作用仍存争议（认为TGFβ致纤维化而IL-10抗纤维化）。心衰时Tregs增殖减少，但其功能是否增强未见报道。我们发现心衰II、III期患者Th17/Treg比例较正常人增加近3倍，Th17明显增加而Tregs明显减少；多种细胞因子分泌均显著高于正常人，患者血浆TGF-β增加（2.89倍）明显高于IL-10的增加（1.65倍），患者外周血CD4+T淋巴细胞及Treg细胞Kv1.3等通道电流密度、mRNA水平及蛋白表达均明显高于正常人，细胞内Ca2+介导的细胞功能增强；心衰模型大鼠多种免疫因子也均不同程度地明显增加，Tregs的Kv1.3通道电流密度为空白组的3-4倍，提示心衰后期免疫应答为主要表现，Tregs增殖能力虽然减弱，但Kv1.3介导的Tregs功能活化增强，主要引起细胞因子TGF-β的分泌增加。依普利酮能够抑制多种淋巴细胞Kv1.3等离子通道。心肌成纤维细胞（CFs）与Treg细胞共孵育后两者互相促进增殖（CFs增殖1.6倍，Tregs增殖53.6%），Tregs的Kv1.3通道mRNA和蛋白表达均明显上调，胞内外TGFβ水平显著增加，明显高于IL-10水平的增加。依普利酮可能通过抑制Kv1.3通道介导的Treg细胞活化减少致纤维化因子TGF-β的分泌而抑制成纤维细胞增殖，从而有利于心衰。