首页|烟草烟雾暴露通过TNF-α/Caspase-3/GSDME通路介导细胞焦亡促进骨骼肌萎缩

烟草烟雾暴露通过TNF-α/Caspase-3/GSDME通路介导细胞焦亡促进骨骼肌萎缩

Cigarette smoking induces skeletal muscle atrophy through TNF-α/Caspase-3/GSDME-mediated pyroptosis

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目的 探讨半胱氨酸天冬氨酸蛋白酶3(cysteinyl aspartate specific proteinase-3,Caspase-3)/Gasdermin-E(GSDME)介导的细胞焦亡在烟草烟雾(CS)暴露诱导的骨骼肌萎缩中的作用.方法 将C57BL/6小鼠暴露于烟草烟雾24周,构建肺气肿小鼠模型.HE染色观察腓肠肌组织的形态学变化;免疫组化检测腓肠肌中焦亡相关蛋白表达.用烟草烟雾提取物(CSE)处理C2C12小鼠骨骼肌细胞,建立体外骨骼肌萎缩模型.进一步采用Caspase-3抑制剂Z-DEVD-FMK和GSDME抑制剂Dimethyl fumarate(DMF)处理C2C12细胞.观察抑制Caspase-3/GSDME后对CSE诱导的骨骼肌萎缩的影响.用肿瘤坏死因子α(TNF-α)刺激骨骼肌细胞,观察TNF-α对Caspase-3/GSDME蛋白表达和肌管的影响.Western blot用于测定Caspase-3和GSDME蛋白的表达水平;Hoechst33342/Propidium Iodide(PI)双染色检测PI阳性细胞率;乳酸脱氢酶(LDH)释放实验检测C2C12细胞的LDH释放量;免疫荧光检测C2C12肌管直径变化.结果 CS诱导小鼠腓肠肌萎缩的同时伴随着GSDME介导的焦亡增强(P＜0.05).体外实验结果表明,与对照组相比,CSE组C2C12细胞cleaved-caspase-3、GSDME-N的蛋白表达显著增高,PI阳性率和LDH释放率增加,肌管直径减小(P＜0.05).分别抑制Caspase-3和GSDME均可明显改善CSE诱导的骨骼肌细胞焦亡和肌管萎缩(P＜0.05).TNF-α促进肌管萎缩和骨骼肌细胞cleaved-caspase-3、GSDME-N蛋白的表达.结论 CS可以通过TNF-α激活caspase-3/GSDME通路介导的焦亡,促进骨骼肌萎缩.
Objective To investigate the role of cysteinyl aspartate specific proteinase-3(Caspase-3)/gasdermin-E(GSDME)-mediated pyroptosis in skeletal muscle atrophy induced by cigarette smoke in mice.Methods To construct a mouse model of COPD,C57BL/6 mice were exposed to cigarette smoke(CS)for 24 weeks.HE staining was used to observe the changes in the morphology of the gastrocnemius muscle in mice.Immunohistochemistry was used to detect the expression of pyroptosis-related proteins in gastrocnemius muscle.To construct a model of skeletal muscle cell atrophy in vitro,C2C12 myoblasts were induced to differentiate into skeletal muscle cells with 2％horse serum,and then skeletal muscle cells were treated with cigarette smoke extract(CSE).Skeletal muscle cells were further treated with the caspase-3 inhibitor Z-DEVD-FMK and the GSDME inhibitor Dimethyl fumarate(DMF)to explore the effects of inhibition of caspase-3/GSDME on CSE-induced skeletal muscle cell atrophy.To observe the effects of TNF-α on the expression of caspase-3 and GSDME proteins as well as the impact on myotubes,skeletal muscle cells were stimulated with tumor necrosis factor-alpha(TNF-α).Western blotting was applied to detect protein expression levels of caspase-3 and GSDME in skeletal muscle cells.Hoechst 33 342/Hoechst33342/Propidium Iodide(PI)staining was applied to detect the PI-positive rate of skeletal muscle cells.The lactate dehydrogenase(LDH)release of C2C12 myotubes was measured by LDH release test.Immunofluorescence was used to detect changes in myotube diameter.Results CS-induced skeletal muscle atrophy was observed in mice,accompanied by increased pyroptosis-associated proteins(c-caspase-3 and GSDME-N)(P＜0.05).CSE also induced elevated c-caspase-3 and GSDME-N expression in C2C12 cells,resulting in increased LDH release,positive ratio of PI,along with reduced myotube diameter(P＜0.05).In addition,TNF-α promotes myotube atrophy and the expression of cleaved-caspase-3 and GSDME-N proteins in skeletal muscle cells.Conclusion CS can induce skeletal muscle atrophy through activated TNF-α/Caspase-3/GSDME-mediated pyroptosis.

Cigarette smokechronic obstructive pulmonary diseaseskeletal muscle atrophycysteinyl aspartate specific proteinase-3gasdermin-Epyroptosis

谭玉芬、熊国林、叶园园、黄翠碧、韦鑫燕、李杰、黄丽华、宾雁飞

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广西医科大学第二附属医院呼吸与危重症医学科(广西南宁 530007)

广西医科大学公共卫生学院(广西南宁 530021)

烟草烟雾 慢性阻塞性肺疾病 骨骼肌萎缩与功能障碍 半胱氨酸天冬氨酸蛋白酶3 Gasdermin-E 焦亡

广西自然科学基金广西科技计划项目广西医科大学第二附属医院院内国家自然科学基金培育项目

2023GXNSFAA026303桂科AD22035221GJPY2023010

2024

10.7507/1671-6205.202408075

中国呼吸与危重监护杂志
四川大学华西医学中心,四川大学华西医院

中国呼吸与危重监护杂志

CSTPCD
影响因子：1.306
ISSN：1671-6205
年,卷(期)：2024.23(10)