线粒体靶向抗氧化剂Mito-Tempo对脓毒症小鼠急性肝损伤的保护作用

Protective effect of the mitochondria-targeted antioxidant Mito-Tempo against acute liver injury in mice with sepsis

摘要

目的 探讨线粒体靶向抗氧化剂Mito-Tempo对脓毒症小鼠急性肝损伤的影响及可能机制。 方法 将C57BL/6小鼠随机分为对照组（Control组）、盲肠结扎穿刺组（CLP组）、Mito-Tempo治疗组（CLP+Mito-Tempo组）。采用盲肠结扎穿刺术（cecal ligation and puncture，CLP）构建小鼠脓毒症急性肝损伤模型，CLP+Mito-Tempo组在CLP处理前1 h使用Mito-Tempo腹腔注射进行预处理。24 h后麻醉并处死小鼠，取小鼠肝脏组织进行HE染色观察肝组织病理损伤；ELISA法监测血清中炎症因子，免疫荧光染色检测肝组织中活性氧（reactive oxygen species，ROS）的水平；电镜观察线粒体形态，Western blot法检测细胞焦亡相关蛋白剪切型含半胱氨酸的天冬氨酸蛋白水解酶-1（cleaved cysteinyl aspartate specific proteinase 1，cleaved-Caspases-1）、剪切型焦孔素D（cleaved-gasdermin D，GSDMD）、白细胞介素-1α（interleukin 1α，IL-1α）、白细胞介素-1β（interleukin 1β，IL-1β）的表达水平。 结果 与Control组相比，CLP组肝损伤加重，ROS水平增加，线粒体形态出现破坏，细胞焦亡相关蛋白cleaved-Caspase-1、cleaved-GSDMD、IL-1α、IL-1β的表达水平增加；与CLP组相比，CLP+Mito-Tempo组肝组织损伤程度减轻，ROS水平降低，线粒体形态部分恢复，细胞焦亡相关蛋白cleaved-Caspase-1、cleaved-GSDMD、IL-1α、IL-1β的表达水平降低。 结论 Mito-Tempo可以减轻脓毒症小鼠急性肝损伤，其机制可能与降低氧化应激水平抑制细胞焦亡的发生有关。

Abstract

Objective To investigate the effect of the mitochondria-targeted antioxidant Mito-Tempo on acute liver injury in mice with sepsis and its possible mechanisms. Methods C57BL/6 mice were randomly divided into control group（Control group），cecal ligation puncture group（CLP group），and Mito-Tempo treatment group（CLP+Mito-Tempo group）. Cecal ligation and puncture（CLP） was used to establish a mouse model of sepsis and acute liver injury，and the mice in the CLP+Mito-Tempo group were pretreated with intraperitoneal injection of Mito-Tempo at 1 hour before CLP treatment. After 24 hours，the mice were anesthetized and sacrificed，and HE staining was used to observe liver histopathological injury；ELISA was used to measure the serum levels of inflammatory factors，and immunofluorescent staining was used to measure the level of reactive oxygen species（ROS） in liver tissue；an electron microscope was used to observe the morphology of mitochondria；Western blot was used to measure the expression levels of cleaved cysteinyl aspartate specific proteinase 1（cleaved caspase-1），cleaved gasdermin D（GSDMD），interleukin-1α（IL-1α），and interleukin-1β（IL-1β）. Results Compared with the Control group，the CLP group had aggravation of liver injury，an increase in ROS level，destroyed mitochondrial morphology，and increases in the expression levels of the pyroptosis-related proteins cleaved caspase-1，cleaved GSDMD，IL-1α，and IL-1β. Compared with the CLP group，the CLP+Mito-Tempo group had alleviation of liver injury，a reduction in ROS level，partial restoration of mitochondrial morphology，and significant reductions in the expression levels of the pyroptosis-related proteins cleaved caspase-1，cleaved GSDMD，IL-1α，and IL-1β. Conclusion Mito-Tempo can alleviate acute liver injury in mice with sepsis，possibly by reducing the level of oxidative stress and inhibiting pyroptosis.

关键词

脓毒症 / 急性肝损伤 / 氧化应激 / 细胞焦亡

Key words

sepsis / acute liver injury / oxidative stress / pyroptosis

基金资助

重庆市科卫联合重点资助项目(2023ZDXM004)