山奈酚调控AMPK/NOX4通路抑制高糖诱导的肾小球系膜细胞氧化应激和胞外基质积聚

Kaempferol inhibited high glucose-induced oxidative stress and extracellular matrix accumulation in glomerular mesangial cells through regulating AMPK/NOX4 pathway

本文研究山奈酚(kaempferol,KAE)对高糖诱导的肾小球系膜细胞(GMCs)增殖、氧化应激的保护作用及其机制。建立GMCs体外高糖模型,对细胞增殖、ROS、NADPH氧化酶、超氧化物歧化酶(SOD)、丙二醛(MDA)含量进行检测。qRT-PCR和Western blot检测GMCs中相关因子表达。运用siNOX4、sip22phox和compound C研究KAE对高糖诱导的细胞内信号通路影响。采用分子对接方法研究KAE与Sestrin2、AMPK之间相互作用。结果表明KAE显著抑制高糖诱导的GMCs细胞增殖、ROS产生、NOX活性和MDA水平,增强细胞内SOD活性。qRT-PCR和Western blot结果显示,KAE可以抑制细胞中TGF-β1、Col IV、NOX4和p22phox的表达,并且增加Sestrin2和AMPK细胞因子的表达。siNOX4和sip22phox抑制HG诱导的ROS、TGF-β1和Col IV的产生,提示其可能是由NOX4/p22phox信号通路介导的。Compound C显著逆转KAE对HG诱导的细胞增殖、ROS、NOX4、TGF-β1和Col IV的保护作用。KAE能够减弱高糖诱导的GMCs细胞氧化应激和ECM积聚,其作用机制可能是通过调控AMPK/NOX4信号通路。

The purpose of the present research was to discover the function of kaempferol(KAE)in high glucose(HG)-induced glomerular mesangial cells(GMCs)and the underlying mechanism.The HG model of GMCs was established in vitro.Cell proliferation,reactive oxygen species(ROS)generation,NADPH oxidase(NOX),superoxide dismutase(SOD),malondialdehyde(MDA)were studied using commercial kits.The expression of related factors in GMCs were detected by qRT-PCR and Western blot.siNOX4,sip22phox and compound C were detected the effects of KAE on GMCs intracellular signaling pathway induced by HG with Western blot.The interaction of KAE with Sestrin2 and AMPK wered studied by molecular docking method.Results demonstrated that KAE significantly alleviated cell proliferation,reduced ROS,NOX,and MDA levels,meanwhile enhance SOD activity in HG-induced GMCs.Besides,the production of TGF-β1,Collagen IV(Col IV),NOX4,and p22phox were also inhibited by KAE.In addition,KAE elevated the expression levels of Sestrin2 and AMPK in HG-induced GMCs.siNOX4 and sip22phox suppressed HG-induced ROS,TGF-β1,and Col IV production,indicating that it might be mediated by NOX4/p22phox signaling.Compound C reversed the protective effects of KAE towards HG-induced cell proliferation,ROS,NOX4,TGF-β1,and Col IV in GMCs.These findings revealed that KAE inhibited HG-induced OS and ECM accumulation in GMCs,which is partially mediated via AMPK/NOX4 pathway.