抑制3型脱碘酶表达能通过上调过氧化物酶体增殖物激活受体γ共激活因子1α改善脓毒症骨骼肌线粒体功能  

Inhibition of type 3 deiodinase expression can improve mitochondrial function in skeletal muscle of sepsis by up-regulating peroxisome proliferator-activated receptor-γcoactivator-1α

作　　者：王刚 段剑锋 曹科[1,2] 高涛 蒋安琪 许芸 朱章华 虞文魁[1,2] Wang Gang;Duan Jianfeng;Cao Ke;Gao Tao;Jiang Anqi;Xu Yun;Zhu Zhanghua;Yu Wenkui(Department of Critical Care Medicine,Nanjing Drum Tower Hospital,Affiliated Hospital of Medical School,Nanjing University,Nanjing 210008,Jiangsu,China;The State Key Laboratory of Pharmaceutical Biotechnology,Nanjing 210008,Jiangsu,China)

机构地区：[1]南京大学医学院附属鼓楼医院重症医学科,江苏南京210008 [2]医药生物技术国家重点实验室,江苏南京210008

出　　处：《中华危重病急救医学》2024年第8期841-847,共7页Chinese Critical Care Medicine

基　　金：国家自然科学基金(82002082,82302442)。

摘　　要：目的探讨靶向抑制3型脱碘酶(Dio3)对脓毒症骨骼肌线粒体的保护作用及其机制。方法①体内实验:通过盲肠结扎穿孔术(CLP)构建脓毒症大鼠模型;利用腺相关病毒靶向干扰大鼠胫骨前肌Dio3的表达。按随机数字表法将雄性SD大鼠分为阴性敲除假手术组(shNC+Sham组)、阳性敲除假手术组(shD3+Sham组)、阴性敲除CLP组(shNC+CLP组)和阳性敲除CLP组(shD3+CLP组),每组8只。制模后取胫骨前肌,用蛋白质免疫印迹试验(Western blotting)检测Dio3、过氧化物酶体增殖物激活受体γ共激活因子1α(PGC1α)、沉默信息调节因子1(SIRT1)等蛋白表达;实时荧光定量聚合酶链反应(RT-qPCR)检测甲状腺激素受体(THRα、THRβ)、单羧酸转运蛋白10(MCT10)等T3调控基因,线粒体DNA(mtDNA),线粒体生物合成相关基因PGC1α的mRNA表达;透射电镜下观察线粒体形态。②体外实验:体外培养小鼠成肌样细胞C2C12,利用慢病毒干扰Dio3表达,并用脂多糖(LPS)构建内毒素细胞模型,并分为shNC组、shD3组、shNC+LPS组和shD3+LPS组。免疫荧光染色分析PGC1α胞内分布。免疫共沉淀联合Western blotting检测PGC1α乙酰化水平。结果①体内实验:与shNC+Sham组相比,shNC+CLP组骨骼肌内Dio3蛋白表达明显升高(Dio3/β-Tubulin:3.32±0.70比1.00±0.49,P<0.05),而shD3+Sham组无差异;shD3+CLP组Dio3蛋白表达较shNC+CLP组明显降低(Dio3/β-Tubulin:1.42±0.54比3.32±0.70,P<0.05)。与shNC+CLP组相比,shD3+CLP组T3调控基因的表达明显上调〔THRαmRNA(2^(-ΔΔCt)):0.67±0.05比0.33±0.01,THRβmRNA(2^(-ΔΔCt)):0.94±0.05比0.67±0.02,MCT10 mRNA(2^(-ΔΔCt)):0.65±0.03比0.57±0.02,均P<0.05〕。电镜结果提示,shNC+CLP组骨骼肌线粒体损伤明显,而shD3+CLP组线粒体形态保持完整;与shNC+Sham组相比,shNC+CLP组线粒体数量明显减少(个/HP:10.375±1.375比13.750±2.063,P<0.05),而shD3+CLP组线粒体数量较shNC+CLP组明显增多(个/HP:11.250±2.063比10.375±1.375,P<0.05);shNC+CLP组mtDNA表达�Objective To investigate the protective effects and mechanisms of targeted inhibition of type 3 deiodinase(Dio3)on skeletal muscle mitochondria in sepsis.Methods①In vivo experiments:adeno-associated virus(AAV)was employed to specifically target Dio3 expression in the anterior tibial muscle of rats,and a septic rat model was generated using cecal ligation and puncture(CLP).The male Sprague-Dawley(SD)rats were divided into shNC+Sham group,shD3+Sham group,shNC+CLP group,and shD3+CLP group by random number table method,with 8 rats in each group.After CLP modeling,tibial samples were collected and Western blotting analysis was conducted to assess the protein levels of Dio3,peroxisome proliferator-activated receptor-γcoactivator-1α(PGC1α),and silence-regulatory protein 1(SIRT1).Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was utilized to examine mRNA expression of genes including thyroid hormone receptors(THRα,THRβ),monocarboxylate transporter 10(MCT10),mitochondrial DNA(mtDNA),and PGC1α.Transmission electron microscopy was employed to investigate mitochondrial morphology.②In vitro experiments:involved culturing C2C12 myoblasts,interfering with Dio3 expression using lentivirus,and constructing an endotoxin cell model by treating cells with lipopolysaccharide(LPS).C2C12 cells were divided into shNC group,shD3 group,shNC+LPS group,and shD3+LPS group.Immunofluorescence colocalization analysis was performed to determine the intracellular distribution of PGC1α.Co-immunoprecipitation assay coupled with Western blotting was carried out to evaluate the acetylation level of PGC1α.Results①In vivo experiments:compared with the shNC+Sham group,the expression of Dio3 protein in skeletal muscle of the shNC+CLP group was significantly increased(Dio3/β-Tubulin:3.32±0.70 vs.1.00±0.49,P<0.05),however,there was no significant difference in the shD3+Sham group.Dio3 expression in the shD3+CLP group was markedly reduced relative to the shNC+CLP group(Dio3/β-Tubulin:1.42±0.54 vs.3.32±0.70,P<0.05).

关 键 词：脓毒症 3型脱碘酶 线粒体损伤 过氧化物酶体增殖物激活受体γ共激活因子1α 代谢复苏 

分 类 号：R459.7[医药卫生—急诊医学]