几丁质酶-3样蛋白1对脂多糖诱导小鼠骨骼肌卫星细胞炎症损伤相关分子表达的影响  

Effects of chitinase-3-like protein 1 on the expression of inflammatory damage-related molecules in mouse skeletal muscle satellite cells induced by lipopolysaccharide

作　　者：李福星[1] 盛志勇[1] 周仪华[1] 李婧滢 许建宁[1] Li Fuxing;Sheng Zhiyong;Zhou Yihua;Li Jingying;Xu Jianning(Department of Intensive Care Unit,the Second Affiliated Hospital of Nanchang University,Nanchang 330006,Jiangxi,China)

机构地区：[1]南昌大学第二附属医院重症医学科,江西南昌330006

出　　处：《中华危重病急救医学》2021年第12期1428-1433,共6页Chinese Critical Care Medicine

基　　金：江西省自然科学基金面上项目(20202BAB206058)。

摘　　要：目的探讨在脓毒症模型中几丁质酶-3样蛋白1(CHI3L1)介导骨骼肌干细胞损伤的潜在机制。方法用6种浓度的脂多糖(LPS)刺激体外培养的小鼠骨骼肌卫星细胞,采用酶联免疫吸附试验(ELISA)和细胞增殖与毒性试剂盒(CCK-8)筛选出细胞处理最佳浓度。构建CHI3L1过表达和干扰载体转染骨骼肌卫星细胞,采用聚合酶链反应(PCR)、蛋白质免疫印迹试验(Western blotting)验证转染效率。将细胞按随机数字表法分为空白对照组(不加任何干预的细胞)、模型组(LPS刺激未转染的细胞)、过表达CHI3L1组(LPS刺激转染了CHI3L1过表达质粒的细胞)、过表达CHI3L1对照组(LPS刺激转染了空载质粒的细胞)、干扰CHI3L1组(LPS刺激转染了CHI3L1干扰序列的细胞)、干扰CHI3L1对照组(LPS刺激转染了CHI3L1干扰序列阴性对照的细胞)。采用ELISA检测细胞外天冬氨酸特异性半胱氨酸蛋白酶1(caspase-1)和白细胞介素-1β(IL-1β)的含量;采用Western blotting检测细胞中IL-1β、信号转导和转录激活因子3(STAT3)、蛋白激酶B(Akt)、磷酸化Akt(p-Akt)的蛋白表达水平。结果根据CCK-8和ELISA检测结果筛选出最佳浓度为5 mg/L的LPS进行后续实验,且PCR、Western blotting证实转染有效。与空白对照组比较,模型组细胞外IL-1β、caspase-1含量和细胞中Akt、p-Akt、IL-1β的蛋白表达水平均明显升高〔IL-1β(ng/L):11.22±0.55比8.63±0.63,caspase-1(pmol/L):9.47±0.22比8.65±0.15,Akt/GAPDH:1.36±0.12比1.06±0.15,p-Akt/GAPDH:0.78±0.07比0.09±0.01,IL-1β/GAPDH:1.38±0.12比0.18±0.03,均P<0.05〕。与模型组、过表达CHI3L1对照组比较,过表达CHI3L1组细胞外IL-1β、caspase-1含量和细胞中p-Akt、IL-1β的蛋白表达均明显升高〔IL-1β(ng/L):14.93±0.97比11.22±0.55、9.38±0.40,caspase-1(pmol/L):10.35±0.03比9.47±0.22、8.46±0.24,p-Akt/GAPDH:1.21±0.04比0.78±0.07、0.63±0.04,IL-1β/GAPDH:1.87±0.08比1.38±0.12、1.51±0.17,均P<0.05〕。与模型组、干扰CHI3L1对照�Objective To explore the potential mechanism of chitinase-3-like protein 1(CHI3L1)involved in skeletal muscle stem cell injury induced by sepsis.Methods Six different concentrations of lipopolysaccharide(LPS)were used to stimulate mouse skeletal muscle satellite cells cultured in vitro.Enzyme linked immunosorbent assay(ELISA)and cell counting kit-8(CCK-8)were used to determine the optimal concentration.The overexpression and interference vectors of CHI3L1 were constructed to transfect skeletal muscle satellite cells,and the transfection efficiency was verified by polymerase chain reaction(PCR)and Western blotting.The cells were randomly divided into blank control group(cells without any intervention),model group(LPS-stimulated untransfected cells),overexpressing CHI3L1 group(LPS-stimulated cells transfected with CHI3L1 plasmid),overexpressing CHI3L1 control group[LPS-stimulated cells transfected with negative control(NC)plasmid],CHI3L1 interference group[LPS-stimulated cells transfected with CHI3L1 small interfering RNA(siRNA)],CHI3L1 interference control group(LPS-stimulated cells transfected with CHI3L1-siRNA NC).The levels of extracellular caspase-1 and interleukin-1β(IL-1β)were detected by ELISA.The protein expressions of intracellular IL-1β,signal transducer and activator of transcription 3(STAT3),protein kinase B(Akt)and phosphorylated Akt(p-Akt)were detected by Western blotting.Results According to the results of CCK-8 and ELISA,the best concentration of 5 mg/L LPS was selected for the subsequent experiment.The transfection was validated by PCR and Western blotting.Compared with the blank control group,the levels of extracellular IL-1β,caspase-1 and the protein expressions of intracellular Akt,p-Akt,and IL-1βwere significantly increased in the model group[IL-1β(ng/L):11.22±0.55 vs.8.63±0.63,caspase-1(pmol/L):9.47±0.22 vs.8.65±0.15,Akt/GAPDH:1.36±0.12 vs.1.06±0.15,p-Akt/GAPDH:0.78±0.07 vs.0.09±0.01,IL-1β/GAPDH:1.38±0.12 vs.0.18±0.03,all P<0.05].Compared with the model group and the overexpr

关 键 词：几丁质酶-3样蛋白1 脓毒症 骨骼肌干细胞 白细胞介素-1β 天冬氨酸特异性半胱氨酸蛋白酶1 

分 类 号：R459.7[医药卫生—急诊医学]