免疫相关GTP酶M1在脓毒症诱导脑损伤小鼠皮质神经元细胞自噬中的作用

Role of immune-related GTPase M1 in cortical neurons autophagy of mice with sepsis-induced brain injury

目的:探讨免疫相关GTP酶M1(immune-related GTPase M1,IRGM1)在脓毒症诱导脑损伤(sepsis-induced brain injury,SIBI)小鼠皮质神经元细胞自噬中的作用。方法:将野生型和IRGM1基因敲除小鼠各60只分为野生型假手术(sham-operated wild-type,SWT)组、野生型模型(model wild-type,MWT)组、基因敲除假手术(sham-operated knockout,SKO)组和基因敲除模型(model knockout,MKO)组。模型组行小鼠盲肠结扎穿孔术(cecal ligation and puncture,CLP),CLP后6 h行神经行为学评分,如神经生物学低于6分,诊断为SIBI;假手术组只打开腹腔,未行CLP。采用HE染色观察小鼠大脑皮质区病理学改变;电子显微镜下观察小鼠皮质神经元细胞自噬的超微结构;实时定量PCR检测IRGM1和INF-γm RNA在小鼠大脑皮质组织中的表达;Western印迹检测小鼠大脑皮质组织微管相关蛋白1轻链3(LC3)-II,LC3-I,SQSTM1,IRGM1的蛋白相对表达量;免疫荧光法观察IRGM1在小鼠大脑皮质神经元中的表达。结果:电子显微镜显示,与SWT组比较,MWT组在CLP后12或24 h小鼠皮质神经元内质网扩张、线粒体肿胀、自噬体数量增加。Western印迹结果显示,CLP后6 h小鼠大脑皮质组织LC3-II表达开始增加,高表达维持至CLP后96 h,相反,SQSTM1在CLP后6 h开始下降。与SWT组比较,MWT组在CLP后12 h小鼠大脑皮质组织中IRGM1的m RNA和蛋白表达水平明显上调,同时,IFN-γ的m RNA表达也明显增加(P<0.05)。双色免疫荧光检测结果显示,CLP后24 h,与SWT组比较,MWT组小鼠皮质神经元中IRGM1表达明显上调。SWT组和SKO组小鼠大脑皮质细胞中自噬活性的基线水平相当低,几乎无LC3-II的表达,而SQSTM1表达均很高。但是,CLP后12 h,MWT组LC3-II表达明显升高,SQSTM1表达下调(P<0.05),而IRGM1基因敲除的MKO组小鼠大脑皮质组织中几乎无LC3-II,SQSTM1的表达明显上调。CLP后6 h,MWT组SIBI发生率90%(27/30),MKO组发生率96.67%(29/30)。CLP后12 h,MKO组小鼠神经生物学评分明显低于MWT组(4.97±0.71 vs 5.43±0.86;t=2.284,P=0.026)。小鼠大脑皮质HE染色显示,与MWT比较,MKO组小鼠脑皮质损伤严重,神经细胞数量明显减少。结论:SIBI时IRGM1表达对小鼠大脑皮质神经元损伤具有一定的保护作用,其机制可能与其调节小鼠大脑皮质神经元细胞自噬有关。

Objective： To investigate the role of immune-related GTPase M1（IRGM1） in cortical neurons autophagy in mice with sepsis induced brain injury（SIBI）.Methods： Sixty wild-type C57 BL/6 mice and sixty IRGM1 gene knockout C57 BL/6 mice were randomly divided into 4 groups： a sham-operated wild-type（SWT） group, a cecal ligation and puncture（CLP） model wild-type（MWT） group, a sham-operated knockout（SKO） group, and a CLP model knockout（MKO） group. Models of mice with sepsis were established by CLP. Six hours of after CLP, the neurobehavioral scores for mice were recorded. The mice were diagnosed with SIBI and enrolled for the studies in next step if the neurobehavioral score was less than 6 in the MWT and MKO groups. The sham operation group only opened the abdominal cavity without CLP. Pathological changes in mouse cerebral cortex were observed by HE staining. Electron microscope was used to observe the ultrastructure of autophagy in cortical neurons. The expression of IRGM1 and INF-γ mRNA in the cerebral cortex of mice were detected by Real time quantitative PCR. The protein expression of microtubule-associated protein 1 light chain 3（LC3）-II, LC3-I,sequestosome-1（SQSTM1） and IRGM1 were measured by Western blot. Immunofluorescence staining was used to examine the expression of IRGM1 in mouse cortical neurons.Results： In the MWT group, the cortical neurons showed dilated endoplasmic reticulum, swelling mitochondria, and increased number of autophagosomes after 6 or 24 h of CLP in contrast to the SWT group. At 6 h after CLP, the expression of LC3-II in the cerebral cortex began to up-regulate,and the up-regulation was maintained till 96 h after CLP; on the contrary, SQSTM1 began to decline after 6 h of CLP. Compared with SWT group, IRGM1 was strongly up-regulated in the cerebral cortex of mice at both mRNA and protein levels in the MWT group after 12 h of CLP,and the mRNA expression of IFN-γ was also increased significantly（P〈0.05）. At 24 h after CLP,the IRGM1 expression of cortical neurons in the MWT group was significantly higher than that in the SWT group. The baseline of autophagy activity was quite low in the cerebral cortex cells in the SWT and the SKO groups. There was almost no detected expression of LC3-II; conversely, the expression of SQSTM1 was very high after 12 h of CLP. However, the expression of LC3-II was significantly up-regulated and the expression of SQSTM1 was down-regulated in the MWT group（P〈0.05）. On the other hand, there was almost no detected LC3-II expression in cerebral cortex in the MKO group, and the expression of SQSTM1 was up-regulated. At 6 h after CLP, the incidence of SIBI was 90%（27/30） in the MWT group, and 96.67%（29/30） in the MKO group. At 12 h of CLP, the neurobehavioral scores in the MKO group was significantly lower than that in the MWT group（4.97±0.71 vs 5.43±0.86; t=2.284, P=0.026）. HE staining showed that mice in the MKO group suffered severe cerebral cortex injury, and the number of nerve cells was significantly reduced compared with that in the MWT group.Conclusion： The IRGM1 exerts a protective effect on the brain of the mice with SIBI, and its mechanism might be related to the regulation of autophagy in mouse cortical neurons.