氢气对脓毒症相关性脑病小鼠海马线粒体动力学的影响

Effect of hydrogen on mitochondrial dynamics in hippocampus of mice with sepsis-associated encephalopathy

目的评价氢气对脓毒症相关性脑病小鼠海马线粒体动力学的影响。方法清洁级健康雄性C57小鼠224只,体重20~25 g,6~8周龄,采用随机数字表法分为4组(n=56):假手术组(Sham组)、假手术+氢气组(Sham+H_(2)组)、脓毒症相关性脑病组(SAE组)和脓毒症相关性脑病+氢气组(SAE+H_(2)组)。采用盲肠结扎穿孔法制备脓毒症相关性脑病模型。Sham+H_(2)组和SAE+H_(2)组小鼠分别于术后1和6 h时吸入2%氢气1 h。记录术后7 d生存情况。术后24 h时处死小鼠,光镜下观察海马组织CA1区病理学结果;采用荧光分光光度法检测海马组织线粒体膜电位(MMP),采用荧光素-荧光酶发光法检测线粒体ATP含量。分别于术后6、12和24 h时处死小鼠,采用Western blot法测定海马组织线粒体动力相关蛋白1(Drp1)和线粒体融合蛋白2(Mfn2)的表达。结果与Sham组比较,SAE组和SAE+H_(2)组术后7 d生存率下降,海马组织MMP和线粒体ATP含量降低,Drp1表达上调,Mfn2表达下调(P<0.05),海马CA1区病理学损伤明显,Sham+H_(2)组上述指标差异无统计学意义(P>0.05)。与SAE组比较,SAE+H_(2)组术后7 d生存率提高,海马组织MMP和线粒体ATP含量升高,Drp1表达下调,Mfn2表达上调(P<0.05),海马CA1区病理学损伤减轻。结论氢气改善脓毒症相关性脑病小鼠海马线粒体功能的机制可能与其促进线粒体融合,抑制线粒体分裂有关。

Objective To evaluate the effect of hydrogen on mitochondrial dynamics in hippocampus of mice with sepsis-associated encephalopathy(SAE).Methods A total of 224 clean-grade healthy male C57 mice,weighing 20-25g,aged 6-8 weeks,were divided into 4 groups(n=56 each)using a random number table method:sham operation group(group Sham),sham operation+hydrogen group(group Sham+H_(2)),SAE group and SAE+hydrogen group(group SAE+H_(2)).Sepsis was produced by cecum ligation and puncture(CLP).Sham+H_(2)and SAE+H_(2) groups inhaled 2%hydrogen for 1 h starting from 1 and 6 h after CLP,respectively.The postoperative 7-day survival rate was recorded.Brain tissues were obtained at 24 h after operation for examination of the pathological changes in hippocampal CA1 region(with a light microscope)and for determination of mitochondrial membrane potential(MMP)(by fluorescence spectrophotometry)and ATP content(by a bioluminescence assay)in hippocampal tissues.At 6,12 and 24 h after operation,hippocampal mitochondria were isolated for determination of the expression of dynamin-related protein 1(Drp1)and the mitochondrial fusion proteins mitofusin 2(Mfn2)(by Western blot).Results Compared with group Sham,the postoperative 7-day survival rate was significantly decreased,the contents of MMP and ATP were decreased,the expression of Drp1 was up-regulated,and the expression of Mfn2 was down-regulated(P<0.05),the pathological changes were aggravated in hippocampal CA1 region in SAE and SAE+H_(2) groups,and no significant change was found in the parameters mentioned above in group Sham+H_(2)(P>0.05).Compared with group SAE,the postoperative 7-day survival rate was significantly increased,the contents of MMP and ATP were increased,the expression of Drp1 was down-regulated,and the expression of Mfn2 was up-regulated(P<0.05),the pathological changes were attenuated in hippocampal CA1 region in group SAE+H_(2).Conclusion The mechanism by which hydrogen improves mitochondrial function is probably associated with promoting mitochondrial fusion and inhibiting mitochondrial fission in hippocampus of mice with SAE.