过表达Sirt3通过Keap1/Nrf2通路调控高糖应激诱导的肾小管上皮细胞衰老  

Overexpression of Sirt3 regulates stress-induced senescence in renal tu⁃bular epithelial cells through Keap1/Nrf2 pathway

作　　者：王自强[1,2] 王颖 赵坤霄[1] 王保兴[1] 李英 Wang Ziqiang;Wang Ying;Zhao Kunxiao;Wang Baoxing;Li Ying(Department of Nephrology,The Third Hospital of Hebei Medical University,Shijiazhuang 051000,China;Department of Nephrology,The First Affiliated Hospital of Hainan Medical University,Haikou 570102,China)

机构地区：[1]河北医科大学第三医院肾内科,河北石家庄051000 [2]海南医学院第一附属医院肾内科,海南海口570102

出　　处：《中国病理生理杂志》2023年第11期2020-2026,共7页Chinese Journal of Pathophysiology

基　　金：海南省自然科学基金资助项目(No.821QN406);海南省卫生健康行业科研项目(No.22A200045)。

摘　　要：目的:本研究拟观察过表达沉默信息调节因子3(silent information regulator 3,Sirt3)对高糖(high glucose,HG)诱导的肾小管上皮细胞应激性衰老的影响,并进一步探究过表达Sirt3对Kelch样ECH关联蛋白1(Kelch-like ECH-associated protein 1,Keap1)/核因子E2相关因子2(nuclear factor E2-related factor 2,Nrf2)通路活性的影响,探讨其可能的作用机制。方法:本研究以HK-2细胞为模型,应用质粒转染过表达Sirt3,采用ML385(Nrf2特异性抑制剂)阻断Keap1/Nrf2通路,按照预定细胞分组给予不同刺激,Western blot法检测Sirt3、Keap1、Nrf2、衰老相关蛋白P16和P21蛋白的表达,DCHF-DA标记法检测细胞内活性氧(reactive oxygen species,ROS)的积聚,衰老相关β-半乳糖苷酶(senescence-associatedβ-galactosidase,SA-β-Gal)染色法检测细胞衰老状况。结果:(1)与正常糖对照组相比,高糖刺激48 h后,衰老相关蛋白P16和P21的水平显著升高(P<0.05);与高糖+空载质粒转染对照组相比,过表达Sirt3后,P16和P21的表达明显降低(P<0.05),SA-β-Gal阳性细胞减少,且显著逆转高糖环境下HK-2细胞内ROS的蓄积。(2)与正常糖对照组相比,高糖环境下HK-2细胞中Nrf2、醌氧化还原酶1(NADPH quinone oxidore-ductase 1,NQO1)蛋白水平降低,Keap1蛋白表达水平升高(P<0.05);与高糖+空载质粒转染对照组相比,转染Sirt3质粒后,Nrf2及其下游蛋白NQO1的表达增加,同时核Nrf2的表达显著增加(P<0.05)。(3)进一步应用ML385抑制Keap1/Nrf2通路活性,结果显示,与高糖+转染Sirt3质粒组相比,加入ML385后Nrf2、NQO1和血红素加氧酶1(heme oxygenase-1,HO-1)蛋白表达降低,衰老相关蛋白P16和P21的表达明显升高(P<0.05)。结论:高糖环境下,过表达Sirt3可通过上调Keap1/Nrf2通路的活性,促进Nrf2向细胞核易位,减少细胞内ROS蓄积,从而减轻应激性衰老。AIM:The aim of this study was to examine the effect of silent information regulator 3(Sirt3)overexpression on stress-induced senescence in renal tubular epithelial cells stimulated by high glucose(HG).The effect of Sirt3 on the activity of Kelch-like ECH-associated protein 1(Keap1)/nuclear factor E2-related factor 2(Nrf2)pathway was also observed,and the possible mechanism was discussed.METHODS:HK-2 cells were used in this study.Sirt3 was overexpressed by plasmid transfection.The Keap1/Nrf2 pathway was blocked by ML385(Nrf2 specifically inhibitor).The HK-2 cells received different stimulation depending on group assignment.The expression of the Sirt3,Keap1,Nrf2,and the senescence-related proteins P16,P21 proteins was detected by Western blot.Intracellular reactive oxygen species(ROS)generation was examined by DCHF-DA.Senescence-associatedβ-galactosidase(SA-β-Gal)staining was used to detect senescent cells.RESULTS:(1)Compared with the normal glucose(NG)group,the levels of the senescence-related proteins P16 and P21 were significantly increased after 48 h of HG stimulation(P<0.05).Compared with the HG+vector group,the levels of P16 and P21 were significantly decreased,SA-β-Gal positive cells were decreased,ROS accu‑mulation was significantly reduced in HK-2 cells under a HG environment after overexpression of Sirt3(P<0.05).(2)Compared with the NG group,the levels of the Nrf2 and NADPH quinone oxidoreductase 1(NQO1)proteins were de-creased,and the expression of the Keap1 protein was increased in HK-2 cells under a HG environment(P<0.05).Com-pared with the HG+vector group,the expression of Nrf2 and its downstream protein NQO1 was increased,while the ex-pression of Nrf2 was significantly increased after transfection of the Sirt3 plasmid(P<0.05).(3)ML385 was further ap-plied to inhibit the activity of Keap1/Nrf2 pathway.Compared with the HG+Sirt3 group,the expression levels of the Nrf2,NQO1 and heme oxygenase 1(HO-1)proteins were decreased,the expression levels of P16 and P21 were significantly in-creased after ML3

关 键 词：沉默信息调节因子3 高糖 肾小管上皮细胞 应激性衰老 Keap1/Nrf2信号通路 

分 类 号：R587.2[医药卫生—内分泌] R363[医药卫生—内科学]