GSDMD通过激活caspase-9-caspase-3凋亡信号通路参与脓毒症相关获得性衰弱  

GSDMD mediates sepsis-related acquired weakness by activating caspase9-caspase-3 apoptotic signaling pathway

作　　者：王德琼 周喆 张黎 张钟健 李霜 杨大春 WANG Deqiong;ZHOU Zhe;ZHANG Li;ZHANG Zhongjian;LI Shuang;YANG Dachun(College of Medicine,Southwest Jiaotong University,Chengdu 610031,China;Department of Cardiovascular Medicine,General Hospital of Western Theater Command,Chengdu 610083,China)

机构地区：[1]西南交通大学医学院,四川成都610031 [2]西部战区总医院心内科,四川成都610083

出　　处：《中国病理生理杂志》2023年第4期694-704,共11页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金青年科学基金项目(No.81800338);西部战区总医院院管课题面上项目(No.417311V2Q)。

摘　　要：目的:探讨gasdermin D(GSDMD)-胱天蛋白酶9(caspase-9)-caspase-3在脓毒症相关获得性衰弱发生发展中的机制。方法:诱导C2C12小鼠成肌细胞分化为肌管,设置3个组:control组,加入与脂多糖(LPS)组等体积的无菌磷酸盐缓冲液(PBS);LPS组:加入LPS,使LPS在培养液中的终浓度为1 mg/L,干预12 h;LPS+腺嘌呤核苷三磷酸(ATP)组:加入LPS,使LPS在培养液中的终浓度为1 mg/L,干预12 h后加入ATP,使ATP在培养液中的终浓度为5 mmol/L,干预0.5 h。光镜观察肌管形态;Western blotting检测肌球蛋白重链(MHC)、成肌蛋白(myogenin)、caspase-1、caspase-11、GSDMD、caspase-9、cleaved caspase-9、caspase-3、cleaved caspase-3、肌肉环指蛋白1(MuRF-1)和atrogin-1蛋白水平;腺病毒转染敲减GSDMD;TUNEL染色检测细胞凋亡情况;碘化丙啶(PI)染色观察细胞损伤情况;JC-1染色检测线粒体膜电位。结果:诱导5~7 d后,光镜观察到细胞由多角形分化为管状;Western blotting结果显示,与成肌细胞相比,肌管中肌管标志蛋白MHC和myogenin表达显著升高,证明肌管诱导成功。Western blotting结果显示,与control组比较,LPS组和LPS+ATP组caspase-1、caspase-11、GSDMD、caspase-9、cleaved caspase-9、caspase-3、cleaved caspase-3、MuRF-1和atrogin-1蛋白水平均显著升高;与si-NC+LPS组比较,si-GSDMD+LPS组caspase9、cleaved caspase-9、caspase-3、cleaved caspase-3、MuRF-1和atrogin-1蛋白水平显著降低;与si-NC+LPS+ATP组比较,si-GSDMD+LPS+ATP组caspase-9、cleaved caspase-9、caspase-3、cleaved caspase-3、MuRF-1和atrogin-1蛋白水平显著降低。Western blotting结果显示,与control组比较,si-NC组GSDMD表达无显著差异,si-GSDMD组GSDMD表达显著降低,说明腺病毒转染敲减肌管GSDMD表达成功。TUNEL染色结果显示,与control组比较,LPS组和LPS+ATP组细胞凋亡水平均显著升高;与si-NC+LPS组比较,si-GSDMD+LPS组细胞凋亡水平显著降低;与si-NC+LPS+ATP组比较,si-GSDMD+LPS+ATP组细胞凋亡水平显著降低。PI染AIM:To explore the effect of gasdermin D(GSDMD)on the occurrence and development of sepsis-related acquired weakness.METHODS:Mouse skeletal muscle myoblasts(C2C12)were induced to differentiate into myotubes and were divided into 3 groups:the cells in control group were treated with the same volume of sterile phosphatebuffered saline(PBS)as lipopolysaccharide(LPS)group;the cells in LPS group were treated with LPS at a final concentration of 1 mg/L for 12 h;the cells in LPS+adenosine triphosphate(ATP)group were treated with LPS at a final concentration of 1 mg/L for 12 h and then with ATP at a final concentration of 5 mmol/L for 0.5 h.The morphology of myotubes was observed under a light microscope.Western blotting was performed to detect myosin heavy chain(MHC),myogenin,caspase-1,caspase-11,GSDMD,caspase-9,cleaved caspase-9,caspase-3,cleaved caspase-3,muscle RING-finger protein-1(MuRF-1),and atrogin-1.Adenoviruses were used to knock down GSDMD.TUNEL staining was used to detect cell apoptosis.Propidium iodide(PI)staining was used to observe myotube injury.JC-1 staining was used to detect myotube mitochondrial membrane potential.RESULTS:After induction for 5 to 7 d,the cells were observed to differentiate from polygonal to tubular shape under a light microscope.Western blotting results showed that,compared with myoblasts,the relative expression levels of MHC and myogenin were significantly increased in myotubes,which verified that the induction of myotubes was successful.Western blotting results showed that compared with control group,the protein levels of caspase-1,caspase-11,GSDMD,caspase-9,cleaved caspase-9,caspase-3,cleaved caspase-3,MuRF-1 and atrogin-1 were significantly increased in LPS group and LPS+ATP group.Compared with si-NC+LPS group,the protein levels of caspase-9,cleaved caspase-9,caspase-3,cleaved caspase-3,MuRF-1 and atrogin-1 were decreased significantly in si-GSDMD+LPS group.Compared with si-NC+LPS+ATP group,the protein levels of caspase-9,cleaved caspase-9,caspase-3,cleaved caspase-3,MuRF-1 and a

关 键 词：重症监护室获得性衰弱 脓毒症 gasdermin D 线粒体 胱天蛋白酶9 胱天蛋白酶3 

分 类 号：R685[医药卫生—骨科学] R363.2[医药卫生—外科学]