SIRT5通过促进IDH2...小鼠肺上皮细胞的抗氧化能力_蒙婷.pdf

doi:1011659/jjssx08E022099基础研究SIT5 通过促进 IDH2 去琥珀酰化增加 COPD 小鼠肺上皮细胞的抗氧化能力蒙婷1，2，米叶斯尔买买提艾力2，徐丹3，李争3，李风森1，3(1 新疆医科大学中医学院，新疆 乌鲁木齐 830011;2 新疆维吾尔自治区第八人民医院内科，新疆 乌鲁木齐 830000;3 新疆医科大学第四附属医院国家中医临床研究基地，新疆乌鲁木齐 830000)摘要 目的探讨去乙酰化酶 5(SIT5)对慢性阻塞性肺病(COPD)中肺上皮细胞抗氧化能力的调节作用及机制。方法体外实验:肺上皮细胞 MLE-12 经 H2O2、SIT5 过表达质粒(SIT5-OE)或空载体(EV)处理后分为 control 组、H2O2组、SIT5-OE 组、EV 组、H2O2+SIT5-OE 组、H2O2+EV 组。在体实验:用烟草烟雾(CS)和脂多糖(LPS)诱导小鼠 COPD 模型，并分为正常组(小鼠不进行任何处理)、CS 组(模型小鼠)、CS+saline 组(生理盐水静脉注射到模型小鼠中)、CS+rhSIT5 组(将 rhSIT5 静脉注射到模型小鼠中)、CS+rhSIT5+SIT5 inhibitor 1 组(将 rhSIT5 和 SIT5 抑制剂共同注射到模型小鼠中)。按照说明书检测各组肺细胞/肺组织中丙二醛(MDA)、活性氧(OS)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的水平;流式细胞术检测细胞凋亡水平。免疫沉淀法和蛋白免疫印迹法检测柠檬酸脱氢酶 2(IDH2)的琥珀酰化水平和 SIT5 蛋白表达。结果与 EV 组相比，SIT5-OE 组抑制 IDH2 的琥珀酰化水平降低，SIT5 蛋白表达增加，差异均有统计学意义(P 0 05)。与 control 组相比，H2O2组中IDH2 的琥珀酰化、凋亡率、MDA、OS 的水平都增加/高(P 0 05)。与 H2O2+EV 组相比，H2O2+SIT5-OE 组的琥珀酰化和细胞凋亡率降低，且 SOD 和 GSH-Px 水平增加，差异均有统计学意义(P 0 05)。与正常组相比，CS 组肺组织中 IDH2 的琥珀酰化、MDA、OS 的水平均增加/高(P 0 05)。与 CS+saline 组相比，CS+rhSIT5 组中 IDH2 的琥珀酰化减少，而 SOD 和 GSH-Px 水平均增加，差异均有统计学意义(P 0 05)。与 CS+rhSIT5 组相比，CS+rhSIT5+SIT5 inhibitor 1 组中 MDA、OS 的水平均增加，SOD、GSH-Px 水平明显降低，差异均有统计学意义(P 0 05)。结论SIT5 通过促进 IDH2 去琥珀酰化增加 COPD 小鼠肺上皮细胞的抗氧化能力。关键词去琥珀酰化;去乙酰化酶 5;慢性阻塞性肺病;小鼠;肺上皮细胞;柠檬酸脱氢酶 2 中图分类号563 3 文献标识码A 收稿日期2022-08-17 基金项目新疆维吾尔自治区自然科学基金(2021D01A143);2022 年新疆维吾尔自治区青年岐黄学者项目 通信作者李风森，E-mail:fengsen602163 comSIT5 increasing the antioxidant capacity of COPD mouse lung epithelial cells by promoting IDH2 desuccinylationMENG Ting1，2，Miyesier Maimaiti Aili2，XU Dan3，LI Zheng3，LI Feng-sen1，3(1 College of Traditional Chinese Medicine，Xinjiang Medical University，Urumqi Xinjiang 830000，China;2 Department of Internal Medicine，Eighth People s Hospital of Xinjiang UygurAutonomous egion，Urumqi Xinjiang 830000，China;3 National Clinical esearch Base of Traditional Chinese Medicine，Fourth AffiliatedHospital of Xinjiang Medical University，Urumqi Xinjiang 830000，China)Abstract:ObjectiveTo investigate the regulatory effect of sirtuin 5(SIT5)on the antioxidant capacity of lung epithelial cells inchronic obstructive pulmonary disease(COPD)and its mechanism MethodsIn vitro experiment:lung epithelial cells MLE-12 were treatedwith H2O2，SIT5 over-expression(SIT5-OE)or empty vector(EV)and then divided into the control group，H2O2group，SIT5-OEgroup，EV group，H2O2+SIT5-OE group and H2O2+EV group In vivo experiment:the mice were treated by cigarette smoke(CS)andlipopolysaccharide(LPS)for inducing the establishment of COPD model，and they were divided into the normal group(mice without anytreatment)，CS group(model mice)，CS+saline group(injected saline intravenously after modeling)，CS+rhSIT5 group(injected rhSIT5intravenously after modeling)and CS+rhSIT5+SIT5 inhibitor 1 group(co-injected rhSIT5 and SIT5 inhibitor after modeling)Thelevels of malondialdehyde(MDA)，reactive oxygen species(OS)，superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)inlung cells/tissues of each group were detected according to the instructions The level of apoptosis was detected by flow cytometry The succi-nylation level of isocitrate dehydrogenase 2(IDH2)and expression of SIT5 protein were detected by immunoprecipitation and Western blotesultsCompared with the EV group，the inhibitation of the succinylation level of IDH2 in the SIT5-OE group was decreased，and theexpression of SIT5 protein were increased，with statistically significant difference(P 0 05)Compared with the control group，the levels ofIDH2 succinylation，apoptosis rate，MDA and OS in the H2O2group were all increased(P 0 05)Compared with the H2O2+EV group，the succinylation and apoptosis rate of the H2O2+SIT5-OE group were decreased，and the levels of SOD and GSH-Px were increased，with112局解手术学杂志J EG ANAT OPE SUG2023，32(3)http:/www jjssxzz cnstatistically significant differences(P 0 05)Compared with the normal group，the levels of IDH2 succinylation，MDA and OS in the lungtissue in the CS group were all increased(P 0 05)Compared with the CS+saline group，the level of IDH2 succinylation was decreased inthe CS+rhSIT5 group，while the levels of SOD and GSH-Px were increased，with statistically significant differences(P 0 05)Comparedwith the CS+rhSIT5 group，the levels of MDA and OS in the CS+rhSIT5+SIT5 inhibitor 1 group were all increased，with statisticallysignificant differences(P 0 05)ConclusionSIT5 increases the antioxidant capacity of lung epithelial cells in COPD mice by promo-ting IDH2 desuccinylationKeywords:desuccinylation;sirtuin 5;chronic obstructive pulmonary disease;mouse;lung epithelial cell;isocitrate dehydrogenase 2慢性阻塞性肺疾病(chronic obstructive pulmonarydisease，COPD)通常是由吸烟引起的一种肺部疾病，其特征是肺功能不可逆损伤，并伴随慢性炎症反应1。越来越多的证据表明，与健康人群相比，COPD 患者肺组织中有明显的内皮细胞和上皮细胞凋亡及氧化损伤2。去乙酰化酶 5(sirtuin 5，SIT5)是 sirtuins 家族的重要成员，其在线粒体中被发现，可参与细胞生理动态调节，SIT5 的去乙酰化酶活性低，而去琥珀酰化的活性较高3，并在细胞抗氧化中发挥重要作用4。线粒体异柠檬酸脱氢酶 2(isocitrate dehydrogenase 2，IDH2)通过产生 NADPH 调节线粒体氧化还原平衡，并减少氧化应激诱导的细胞损伤，这主要是受 SIT5 的去琥珀酰化调控4-5。然而，COPD 肺上皮细胞的氧化损伤是否受 SIT5 调控及 SIT5 调节 IDH2 去琥珀酰化在 COPD 肺上皮细胞氧化损伤中的作用和价值尚未明确。因此，本文拟通过在体内外研究 SIT5 对COPD 肺上皮细胞氧化损伤的影响并基于 SIT5 介导的去琥珀酰化探讨