双重打击致急性肺损伤大鼠膈肌中泛素-蛋白酶体途径变化的研究

摘要

Objective To study the role of ubiquitin-proteasome pathway in a double-hit model (hemorrhagic shock followed by mimicked infection) rat diaphragmatic muscle. Methods 24 SD rats were randomly divided into two groups, control group with no surgery performed, and double-hit model group. A double-hit model of hemorrhagic shock with endotoxemia was utilized. The protein levels of E2-14k and MuRF1 in diaphragms were measured by Western blot. The mRNA levels of ubiquitin and proteasome subunit C2 in diaphragms were measured by means of real time polymerase chain reaction (RT-PCR). The lung Pathological changes of rats were observed, and the diaphragmatic ultrastructure was also measured by electron microscopy. Results The protein expression levels of E2-14k, MuRF1 of diaphragmatic tissues in HS group was 1.65 ± 0.38, 1.88±0.32, respectively, which were significantly higher than those 1.17±0.25, 1.21±0.24 in the group ,P<0.01. The mRNA expression of ubiquitin and C2 subunit of proteasome mRNA level of diaphragmatic tissues in HS group was 1.75 ± 0.36, 1.61 ± 0.41, respectively, which were significantly higher than those 1.22 ± 0.23,1.01 ± 0.25 in the control group ,P<0.01. Ultra structural examination showed mitochondria derangement in HS group, including swollen mitochondria with abnormal cristae, and disrupted external membrane of mitochondria. Conclusions The mRNA and protein expressions of ubiquitin-proteasome pathway in diaphragm increased significantly in HS rats, suggesting that the activity of ubiquitin-proteasome pathway increased which led to an increase of protein degradation.%目的：观察大鼠失血性休克(HS)+内毒素二次打击后膈肌组织内蛋白降解途径之一的泛素-蛋白酶体系统组成成分的变化。方法24只SD大鼠随机分为两组：假手术对照组(C组)和二次打击组(HS组)，每组12只。建立“失血性休克-内毒素”二次打击大鼠模型。蛋白免疫印记(Western blot)方法测定大鼠膈肌内E2-14k、MuRF1的蛋白表达；逆转录-聚合酶链式反应(RT-PCR)技术检测膈肌内泛素和蛋白酶体C2亚基的mRNA表达；采用苏木素-伊红(HE)染色在光镜下观察肺组织病理学改变；另外取膈肌标本固定后做电镜检测。结果蛋白免疫印记结果显示，HS组大鼠膈肌组织中E2-14k及MuRF1的蛋白表达分别为(1.65±0.38、1.88±0.32),与C组（分别为1.17±0.25,1.21±0.24）比较明显增加，差异有统计学意义，P<0.01。RT-PCR分析结果显示，HS组大鼠膈肌组织中泛素及蛋白酶体C2亚基的mRNA表达分别为(0.81±0.28、0.50±0.25，与C组（分别为0.89±0.20比0.50±0.15）比较明显增加，差异有统计学意义，P<0.05。HS组电镜下显示膈肌间线粒体肿胀、嵴减少，外膜模糊、变形，部分溶解破坏等超微结构的改变。结论二次打击大鼠膈肌组织内泛素-蛋白酶体降解途径被激活，可能是引起蛋白降解的重要因素之一。