间隙连接蛋白43调控PI3K/Akt/mTOR通路介导腹膜间皮细胞EMT机制及健脾益气方干预效应

Impairment of peritoneal mesothelial cells is generally considered to be initiators during peritoneal fibrosis.To protect peritoneal mesothelial cells is an important target for preventing peritoneal fibrosis.Connexin43 has been demonstrated playing an important role in the injury course of peritoneal mesothelial cells caused by high glucose,but its molecular mechanisms is still unknown.This study is based on Cx43 is functional impairment marker of peritoneal mesothelial cells and can regulate epithelial-to-mesenchymal transition of peritoneal fibrosis via PI3K/Akt/mTOR signaling pathway.Piqi insufficiency is an early keystone of peritoneal dialysis-related peritoneal fibrosis.Choose Jianpi Yiqi Decoction as object of study,by replicating peritoneal fibrosis rats model and culturing epithelial-to-mesenchymal human peritoneal mesothelium cells,observing effects on morphology and mobility and gap junction intercellular communication of peritoneal mesothelial cells and detecting Cx43 and major protein of PI3K/Akt/mTOR signaling pathway by using methods of gene transfection,western blotting,real-time polymerase chain reaction and transmission electron microscopy.Elucidating molecular mechanisms of Jianpi Yiqi Decoction intervention epithelial-to-mesenchymal transition of peritoneal fibrosis is mediated via PI3K/Akt/mTOR signaling pathway by regulated of Cx43,which can provide a new suepects in the lineup and methods with Chinese medicine for treating peritoneal dialysis-related peritoneal fibrosis .

腹膜间皮细胞(PMC)损伤是腹膜纤维化(PF)的始动因素，针对PMC的保护是预防PF的重要靶点。已有研究证实间隙连接蛋白43(Cx43)在高糖导致的PMC损伤过程起着重要作用，但其中的分子机制尚未明了。本项目基于Cx43是PMC功能损伤的主要物质基础，Cx43调控PI3K/Akt/mTOR通路介导的PMC EMT进程，结合脾气亏虚为PD相关的腹膜早期病变的病机关键，选用“健脾益气方”为研究载体，通过复制PD大鼠PF模型及HPMC EMT模型，应用基因转染、RT-PCR、Western blot、透射电镜等方法，观察健脾益气方对PMC EMT时形态学、胞间通讯功能、迁移率、PI3K/Akt/mTOR通路关键分子及Cx43表达的影响，阐明健脾益气方通过影响Cx43的表达，调控PI3K/Akt/mTOR通路介导EMT的分子机制，为PD相关性PF发病机制研究及中医药治疗提供新的思路和方法。

腹膜间皮细胞损伤是腹膜纤维化的始动因素，间皮细胞转分化（EMT）是与PD相关的腹膜早期病变的关键。本项目在中医理论指导下，紧密结合脾气亏虚是PD相关的腹膜早期病变的主要病机，针对EMT是腹膜纤维化的起始环节和可逆阶段，通过开展体内与体外实验研究健脾益气方调控Cx43表达，干预PI3K/Akt/mTOR信号通路介导的腹膜间皮细胞EMT进程，起到防治腹膜损伤的效果。体内实验通过复制PD大鼠腹膜纤维化模型（空白组大鼠除外），随机分模型组、健脾益气方高剂量组、健脾益气方低剂量组、阿托伐他汀组，每天药物灌胃一次，连用30 d。结果发现，与空白组相比，模型组Cx43、E-cadherin蛋白表达水平明显降低（P＜0.05），腹膜致密层厚度及p-AKT、p-mTOR、α-SMA表达水平明显升高（P＜0.05）；予健脾益气方干预后，大鼠腹膜纤维化病理及腹膜间皮细胞损伤程度得到明显改善，腹膜致密层厚度及Cx43、E-cadherin蛋白表达明显升高（P＜0.05），p-AKT、p-mTOR、α-SMA蛋白表达明显降低（P＜0.05）。体外实验通过体外培养培养人腹膜间皮细胞（HPMC），TGF-β1（6ng/ml）诱导下，制备HPMC EMT模型，制备健脾益气方、阿托伐他汀含药血清；研究表明：Cx43蛋白在正常腹膜间皮细胞间有较高表达。用慢病毒感染调控Cx43表达，发现腹膜间皮细胞PI3K/Akt/mTOR通路活化程度及EMT进程受Cx43表达水平影响。予PI3K/Akt/mTOR通路抑制剂Rap阻断PI3K/Akt/mTOR通路，发现CX43下降水平及HPMC细胞转分化程度均有所逆转。CCK-8法筛选健脾益气方的安全浓度，确定10 μg/ml、80 μg/ml为健脾益气方的低、高干预浓度，予不同浓度健脾益气方干预后，Cx43、E-cadherin表达量升高（P＜0.01）。α-SMA、vimentin、p-AKT/AKT及p-mTOR/mTOR表达量明显降低（P＜0.01）。本项目研究表明，健脾益气方能有效保护腹膜间皮细胞、防治腹透相关性腹膜损伤的作用，其机制可能是通过调控Cx43表达，干预PI3K/Akt/mTOR信号通路介导的腹膜间皮细胞EMT进程。为中医药防治PD纤维化，保护腹膜间皮细胞功能，防治腹膜损伤提供新思路和新方法。