摘要
目的探讨脓毒症心肌损伤时心肌细胞及线粒体自噬水平的变化。方法雄性C57BL/J小鼠随机分为空白对照组(NC)、LPS处理6、12、24、36 h组。LPS处理组小鼠腹腔注射10 mg/kg LPS建立脓毒症模型,空白对照组注射等量生理盐水。分别于以上时间点处死小鼠,收集血液及心脏组织,提取心肌组织胞质蛋白、线粒体及线粒体蛋白,另取心肌组织进行冰冻切片;应用ELISA试剂盒测定血清心肌肌钙蛋白I(c Tn I)含量变化;JC-1染色结合荧光酶标仪检测LPS刺激后不同时间点线粒体膜电位变化;Western blot法检测自噬相关蛋白微管相关蛋白1轻链3(LC3)、PTEN诱导激酶1(pink1)、E3泛素连接酶帕金森病蛋白(parkin)水平;免疫荧光组织化学染色检测心肌组织LC3、pink1/parkin蛋白的定位及其在不同处理组的表达差异。结果与对照组相比,LPS诱导的脓毒症小鼠血清c Tn I在6 h即开始明显升高;LPS处理组线粒体膜电位下降,在LPS 12 h组最低;细胞自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ在腹腔注射LPS 12 h明显升高,然后逐渐下降;pink1/parkin在腹腔注射LPS 6 h明显升高,然后逐渐下降。结论脓毒症心肌损伤时心肌细胞及线粒体自噬水平升高,自噬应激可能更早发于心肌线粒体。
Objective To investigate the alterations in the level of myocardial cells and mitochondrial autophagy during myocardial injury in lipopolysaccharide（ LPS）-induced septic animal models. Methods Male C57 BL/J mice were randomly divided into negative control group（ NC）,LPS treatment groups（ 6,12,24,36 hours）. The LPS treatment groups were subjected to LPS（ 10 mg/kg） injection intraperitoneally,and the NC group was injected intraperitoneally with the same amount of saline. The mice were sacrificed at the above time points to collect blood and heart tissues. Cytoplasmic protein,mitochondria and mitochondrial proteins were extracted from the myocardial tissue,and other myocardial tissue was frozen for next analysis. Cardiac troponin I（ c Tn I） levels in sera were evaluated by ELISA; mitochondrial membrane potential（ MMP） was tested by JC-1 staining and fluorescence cytometry at different time points after LPS intraperitoneal injection.Furthermore,the levels of autophagy-related proteins such as microtubule-associated protein 1 light chain 3（ LC3）,PTEN-induced kinase 1（ pink1）,E3-ubiquitin ligase parkin were measured by Western blotting and fluorescent immunohistochemistry.Results Compared with the control group,the serum levels of c Tn I induced by LPS were significantly higher as 6 hours,while the MMP was significantly lower in the LPS treatment groups,and the lowest was in the 12-hour group. The expression of autophagy-related protein LC3-Ⅱ/LC3-Ⅰsignificantly increased in the LPS 12-hour treatment group,pink1/parkin was significantly elevated in the LPS 6-hour treatment group,and they then gradual y decreased. Conclusion The autophagy stress is activated in myocardium during myocardial injury induced by LPS treatment and it probably happens earlier at myocardial mitochondria.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2016年第2期177-181,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81470414)
