线粒体自噬在脓毒症相关性脑病大鼠认知功能障碍中的作用

Role of mitophagy in cognitive dysfunction in rats with sepsis-associated encephalopathy

目的评价线粒体自噬在脓毒症相关性脑病大鼠认知功能障碍中的作用。方法清洁级健康雄性SD大鼠24只,13~14周龄,体重230~250 g,采用随机数字表法分为3组(n=8):假手术组(Sham组)、脓毒症相关性脑病组(SAE组)和脓毒症相关性脑病+自噬抑制剂3-甲基腺嘌呤(3-MA)组。采用盲肠结扎穿孔法制备大鼠脓毒症相关性脑病模型,3-MA组于模型制备后30 min时腹腔注射3-MA 10 mg/kg。术后24 h采用Morris水迷宫实验测试认知功能,记录逃避潜伏期和靶象限活动时间比率。分别于水迷宫测试结束后,处死大鼠取海马组织,HE染色,行病理学损伤评分;采用Western blot法测定自噬相关蛋白LC3、Beclin1和p62的表达,计算LC3Ⅱ/LC3Ⅰ比值,分离海马线粒体,采用分光光度法测定线粒体膜电位(MMP)、ATP含量和ATP酶活性。结果与Sham组比较,SAE组和3-MA组逃避潜伏期延长,靶象限活动时间比率降低,海马病理学损伤评分降低,MMP、ATP含量和ATP酶活性降低,SAE组海马LC3Ⅱ/LC3Ⅰ比值升高,Beclin1表达上调,p62表达下调,3-MA组海马LC3Ⅱ/LC3Ⅰ比值降低,Beclin1和p62表达上调(P<0.05);与SAE组比较,3-MA组逃避潜伏期延长,靶象限活动时间比率降低,海马病理学损伤评分降低,LC3Ⅱ/LC3Ⅰ比值降低,Beclin1表达下调,p62表达上调,MMP、ATP含量和ATP酶活性降低(P<0.05)。结论海马线粒体自噬参与了脓毒症相关性脑病大鼠认知功能障碍的过程。

Objective To evaluate the role of mitophagy in cognitive dysfunction in rats with sepsis-associated encephalopathy(SAE).Methods Twenty-four clean-grade healthy male Sprague-Dawley rats,aged 13-14 weeks,weighing 230-250 g,were divided into 3 groups(n=8 each)using a random number table method:sham operation group(Sham group),SAE group and SAE+autophagy inhibitor 3-methyladenine(3-MA)group(3-MA group).The SAE models were developed by cecal ligation and puncture in anesthetized animals.3-MA 10 mg/kg was intraperitoneally injected at 30 min after developing the model in 3-MA group.Cognitive function was assessed by Morris water maze test,and the escape latency and ratio of the time of staying at the target quadrant were recorded.After the end of Morris water maze test,the rats were sacrificed and hippocampal tissues were collected for microscopic examination of the pathological changes which were scored after hematoxylin-eosin staining and for determination of the expression of autophagy-related proteins LC3,Beclin1 and p62(by Western blot).The ratio of LC3Ⅱ/LC3Ⅰwas calculated.The hippocampal mitochondria were isolated to measure mitochondrial membrane potential(MMP),ATP content and ATPase activity by spectrophotometry.Results Compared with Sham group,the escape latency was significantly prolonged,the ratio of the time of staying at the target quadrant was decreased,the pathological score of hippocampus was decreased,and the contents of MMP and ATP and ATPase activity were decreased in SAE and 3-MA groups,the ratio of LC3Ⅱ/LC3Ⅰwas significantly increased,the expression of Beclin1 was up-regulated,and the expression of p62 was down-regulated in SAE group,and the ratio of LC3Ⅱ/LC3Ⅰwas significantly decreased,and the expression of Beclin1 and p62 was up-regulated in 3-MA group(P<0.05).Compared with SAE group,the escape latency was significantly prolonged,the ratio of the time of staying at the target quadrant was decreased,the pathological score of hippocampus was decreased,the ratio of LC3/LC3Ⅰwas decreased,the expression of Beclin1 was down-regulated,the expression of p62 was up-regulated,and the contents of MMP and ATP and ATPase activity were decreased in 3-MA group(P<0.05).Conclusions Hippocampal mitophagy is involved in cognitive dysfunction in the rats with SAE.