NF-κB对纤溶酶调控小鼠集合管M-1细胞γ-ENaC表达的影响  

Effect of NF-κB on plasmin-regulatedγ-ENaC expression in mouse collecting duct M-1 cells

作　　者：张文馨 李蕾 葛皖粤 许在平 王运来 许钒 ZHANG Wenxin;LI Lei;GE Wanyue;XU Zaiping;WANG Yunlai;XU Fan(School of Pharmacy,Anhui University of Chinese Medicine,Hefei 230012,China;Anhui Provincial Key Laboratory of Chinese Medicinal Formula,Hefei 230012,China)

机构地区：[1]安徽中医药大学药学院,安徽合肥230012 [2]安徽省中药复方重点实验室,安徽合肥230012

出　　处：《中国病理生理杂志》2025年第4期723-731,共9页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金面上项目(No.82274375,No.81974536);安徽省科技重大专项项目(No.202203a07020006);安徽省高校科研重大项目(No.2024AH040142)。

摘　　要：目的:探讨纤溶酶对小鼠肾皮质集合管上皮细胞(M-1细胞)上皮钠离子通道γ亚基(epithelial sodium channel γ-subunit, γ-ENaC)激活的影响及核因子κB(nuclear factor-κB, NF-κB)的作用。方法:本研究以小鼠M-1细胞为对象,设计三个实验部分:第一部分将细胞分为对照组、纤溶酶组、纤溶酶+BAY 11-7082(NF-κB抑制剂)组以探讨纤溶酶对炎症因子及γ-ENaC的作用;第二部分分为对照组、BAY 11-7082组、BAY 11-7082+纤溶酶组,用BAY 11-7082预先处理M-1细胞,以探讨纤溶酶调控γ-ENaC表达中NF-κB的作用;第三部分分为对照组、纤溶酶组、白细胞介素6(interleukin-6, IL-6)组,用纤溶酶或IL-6处理M-1细胞,以探讨NF-κB下游的IL-6对γ-ENaC的调控作用。药物干预后,ELISA法检测各组细胞培养上清液中IL-6、肿瘤坏死因子α(tumor necrosis factor-α, TNF-α)、单核细胞趋化蛋白1(monocyte chemoattractant protein-1, MCP-1)和IL-1β水平,Western blot检测磷酸化核因子κB抑制蛋白α (phosphorylated inhibitor of nuclear factor-κB α,p-IκBα)/IκBα、p-NF-κB p65/NF-κB p65和γ-ENaC蛋白水平,免疫荧光染色检测γ-ENaC膜上分布情况。结果:与对照组相比,10 mg/L纤溶酶作用24 h后细胞上清液IL-6、TNF-α、MCP-1和IL-1β含量上升(n=3,P<0.01),p-IκBα/IκBα、p-NF-κB p65/NF-κB p65和γ-ENaC蛋白表达升高(n=5,P<0.01),膜上γ-ENaC分布增加。与纤溶酶组相比,纤溶酶与BAY 11-7082共培养后各组细胞IL-6、TNF-α、MCP-1和IL-1β含量下降(n=3,P<0.05或P<0.01),p-IκBα/IκBα、p-NF-κB p65/NF-κB p65和γ-ENaC蛋白表达降低(n=5,P<0.05或P<0.01),膜上γ-ENaC分布减少。单纯给予BAY 11-7082组与对照组相比,p-NF-κB p65/NF-κB p65表达降低(n=5,P<0.05或P<0.01),γ-ENaC没有明显变化,再给予纤溶酶刺激后,p-IκBα/IκBα、p-NF-κB p65/NF-κB p65及γ-ENaC表达升高(n=5,P<0.05),膜上分布增加。NF-κB下游中的IL-6干预24 h后细胞γ-ENaC蛋白表达增加(n=5,P<0.05),γ-ENaC膜上分布增�AIM:To investigate the effect of plasmin on epithelial sodium channelγ-subunit(γ-ENaC)activation in mouse M-1 cells and the role of nuclear factor-κB(NF-κB).METHODS:Mouse M-1 cells were divided into three experimental parts.In the first part,the cells were divided into a control group,a plasmin group,and a plasmin+BAY 11-7082(NF-κB inhibitor)group to explore the effects of plasmin on inflammatory factors andγ-ENaC.In the second part,the cells were divided into a control group,a BAY 11-7082 group and a BAY 11-7082+plasmin group.M-1 cells were pretreated with BAY 11-7082 to explore the role of NF-κB in the regulation ofγ-ENaC expression by plasmin.In the third part,the cells were divided into a control group,a plasmin group and an interleukin-6(IL-6)group.M-1 cells were treated with plasmin or IL-6 to explore the regulatory effect of IL-6 downstream of NF-κB onγ-ENaC.After drug intervention,the levels of IL-6,tumor necrosis factor-α(TNF-α),monocyte chemoattractant protein-1(MCP-1)and IL-1βin cell culture supernatants were detected by ELISA.The protein levels of phosphorylated inhibitor of nuclear factor-κBα(p-IκBα)/IκBα,p-NF-κB p65/NF-κB p65,andγ-ENaC was detected by Western blot.The distribution ofγ-ENaC on the membrane was detected by immunofluorescence staining.RESULTS:Compared with control group,the levels of IL-6,TNF-α,MCP-1 and IL-1βin the supernatants of cells treated with 10 mg/L plasmin for 24 h were increased(n=3,P<0.01),the protein levels of p-IκBα/IκBα,p-NF-κB p65/NF-κB p65 andγ-ENaC were increased(n=5,P<0.01),and the distribution ofγ-ENaC on the membrane was increased.Compared with plasmin group,the levels of IL-6,TNF-α,MCP-1,IL-1β,p-IκBα/IκBα,p-NF-κB p65/NF-κB p65 andγ-ENaC,and theγ-ENaC distribution on the membrane were lowered in plasmin and BAY 11-7082 coculture group(n=3,P<0.05 or P<0.01).Compared with control group,the ratio of p-NF-κB p65/NF-κB p65 decreased in BAY 11-7082 group(n=5,P<0.05),whereas there was no significant change inγ-ENaC.After stimu

关 键 词：上皮钠离子通道γ亚基 纤溶酶 核因子ΚB 炎症因子 

分 类 号：R364.5[医药卫生—病理学] R692[医药卫生—基础医学] Q556[生物学—生物化学]