富氢液对脓毒症相关性脑病小鼠海马线粒体生物合成和动力学的影响

Effect of hydrogen-rich saline on mitochondrial biogenesis and dynamics in hippocampus of mice with sepsis-associated encephalopathy

目的评价富氢液对脓毒症相关性脑病(SAE)小鼠海马线粒体生物合成和动力学的影响。方法雄性C57BL/6J小鼠128只,6~8周龄,体重20~25 g,采用随机数字表法分为4组(n=32):假手术组(Sham组)、假手术+富氢液组(Sham+HRS组)、SAE组和SAE+富氢液组(SAE+HRS组)。采用盲肠结扎穿孔法建立小鼠SAE模型。Sham+HRS组和SAE+HRS组分别于造模后1和6 h时腹腔注射富氢液10 ml/kg。每组随机取20只小鼠,记录术后7 d生存情况。于造模后3、5和7 d进行Y迷宫迷路分辨测试。于造模后24 h时采用ELISA法测定海马组织TNF-α、IL-6和高迁移率族蛋白B1(HMGB1)的含量;采用分光光度法测定超氧化物歧化酶(SOD)和过氧化氢酶(CAT)的活性;采用Western blot法测定海马过氧化物酶体增殖活化受体γ共激活因子-1α(PGC-1α)、核呼吸因子2(NRF2)、线粒体转录因子A(Tfam)、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组术后7 d生存率降低,新异臂停留时间缩短,海马组织TNF-α、IL-6和HMGB1含量升高,SOD和CAT活性降低,PGC-1α、NRF2、Tfam和Drp1表达上调,Mfn2表达下调(P<0.05);与SAE组比较,SAE+HRS组术后7 d生存率升高,新异臂停留时间延长,海马组织TNF-α、IL-6和HMGB1含量降低,SOD和CAT活性升高,PGC-1α、NRF2和Tfam表达上调,Drp1表达下调,Mfn2表达上调(P<0.05)。结论富氢液减轻小鼠SAE的机制可能与促进线粒体生物合成,调节线粒体动力学,减轻海马氧化应激有关。

Objective To evaluate the effect of hydrogen-rich saline(HRS)on mitochondrial biogenesis and dynamics in hippocampus of mice with sepsis-associated encephalopathy(SAE).Methods One hundred and twenty-eight male C57BL/6J mice,aged 6-8 weeks,weighing 20-25 g,were divided into 4 groups(n=32 each)using a random number table method:sham operation group(Sham group),sham operation plus HRS group(Sham+HRS group),SAE group and SAE plus HRS group.Sepsis was developed by cecal ligation and puncture(CLP)in anesthetized mice.HRS 10 ml/kg was intraperitoneally injected at 1 and 6 h after CLP in Sham+HRS and SAE+HRS groups.Twenty mice were randomly selected from each group to record the 7-day survival after operation.The working memory of the mice was observed by Y-maze test on days 3,5 and 7 after CLP.The hippocampal tissues were obtained at 24 h after CLP for determination of the content of tumor necrosis factor-alpha(TNF-α),interleukin-6(IL-6)and high-mobility group box 1 protein(HMGB1)(by enzyme-linked immunosorbent assay),activities of superoxide dismutase(SOD)and catalase(CAT)(by spectrophotometry),and expression of peroxisome proliferator-activated receptor gamma coactivator 1α(PGC-1α),nuclear respiratory factor 2(NRF2),mitochondrial transcription factor A(Tfam),dynamin-related protein 1(Drp1)and mitochondrial fusion protein mitofusin 2(Mfn2)(by Western blot).Results Compared with group Sham,the postoperative 7-day survival rate was significantly decreased,the time spent in novel arm was shortened,the contents of TNF-α,IL-6 and HMGB1 were increased,the activities of SOD and CAT were decreased,the expression of PGC-1α,NRF2 and Tfam was up-regulated,the expression of Drp1 was up-regulated,and the expression of Mfn2 was down-regulated in group SAE(P<0.05).Compared with group SAE,the postoperative 7-day survival rate was significantly increased,the time spent in novel arm was prolonged,the contents of TNF-α,IL-6 and HMGB1 were decreased,the activities of SOD and CAT were increased,the expression of PGC-1α,NRF2 and Tfam was up-regulated,the expression of Drp1 was down-regulated,and the expression of Mfn2 was up-regulated in group SAE+HRS(P<0.05).Conclusions The mechanism by which HRS alleviates SAE may be related to promotion of mitochondrial biogenesis,regulation of dynamics,and reduction of oxidative stress in hippocampus of mice.