吸入高浓度氢气对脓毒症小鼠急性肾损伤及线粒体动力学的影响

Effects of inhalation of high-concentration hydrogen on acute kidney injury and mitochondrial dynamics in septic mice

目的评价吸入高浓度氢气对脓毒症小鼠急性肾损伤(AKI)及线粒体动力学的影响。方法雄性C57BL/6J小鼠128只,6~8周龄,体质量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+氢气组(Sham+H组)、脓毒症AKI组(S-AKI)和脓毒症AKI+氢气组(S-AKI+H组)。采用盲肠结扎穿孔法建立小鼠脓毒症模型。Sham+H组和S-AKI+H组于假手术或造模后1和6 h时分别吸入67%氢气+33%氧气1 h。取20只小鼠观察造模后7 d的生存情况。于造模后24 h时,取血标本,采用比色法测定血清BUN和Cr浓度;取肾组织,HE染色后进行肾小管损伤评分,采用ELISA法测定肾组织TNF-α、IL-1β和高迁移率族蛋白B1(HMGB1)的含量,采用分光光度法测定SOD和过氧化氢酶(CAT)的活性,采用Western blot法测定动力相关蛋白1(Drp1)和线粒体融合蛋白2(Mfn2)的表达水平。结果与Sham组比较,S-AKI组生存率降低,血清BUN和Cr浓度、肾小管损伤评分、肾组织TNF-α、IL-1β和HMGB1含量升高,SOD和CAT活性降低,Drp1表达上调,Mfn2表达下调(P<0.05),Sham+H组上述指标差异无统计学意义(P>0.05);与S-AKI组比较,S-AKI+H组生存率升高,血清BUN和Cr浓度、肾小管损伤评分、肾组织TNF-α、IL-1β和HMGB1含量降低,SOD和CAT活性升高,Drp1表达下调,Mfn2表达上调(P<0.05)。结论吸入高浓度氢气可减轻脓毒症小鼠AKI,机制可能与抑制肾线粒体分裂,促进线粒体融合有关。

Objective To evaluate the effects of inhalation of high-concentration hydrogen on acute kidney injury(AKI)and mitochondrial dynamics in septic mice.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(group Sham),sham operation+hydrogen group(group Sham+H),sepsis AKI group,and sepsis AKI+hydrogen group(group S-AKI+H).A mouse model of sepsis-induced AKI was developed by cecal ligation and puncture in anesthetized animals.In Sham+H and S-AKI+H groups,67%H2+33%O_(2) was inhaled for 1 h starting from 1 and 6 h after sham operation or developing the model,respectively.Twenty mice were selected to observe the survival at 7 days after developing the model.At 24 h after developing the model,blood samples were collected for determination of serum BUN and Cr concentrations(by colorimetric analysis),and renal tissues were obtained for determination of the contents of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and high mobility group protein B1(HMGB1)(by enzyme-linked immunosorbent assay),activities of superoxide dismutase(SOD)and catalase(CAT)(by spectrophotometry)and expression of dynamin-related protein 1(Drp1)and mitofusin 2(Mfn2)(by Western blot).The damage to the renal tubules was scored after HE staining.Results Compared with Sham group,the survival rate was significantly decreased,the serum BUN and Cr concentrations,renal tubular damage score and contents of TNF-α,IL-1βand HMGB1 were increased,the activities of SOD and CAT were decreased,the expression of Drp1 was up-regulated,and the expression of Mfn2 was down-regulated in S-AKI group(P<0.05),and no significant change was found in the parameters mentioned above in Sham+H group(P>0.05).Compared with S-AKI group,the survival rate was significantly increased,the serum BUN and Cr concentrations,renal tubular injury score and contents of TNF-α,IL-1βand HMGB1 were decreased,the activities of SOD and CAT were increased,the expression of Drp1 was down-regulated,and the expression of Mfn2 was up-regulated in S-AKI+H group(P<0.05).Conclusions Inhalation of high-concentration hydrogen can alleviate AKI in septic mice,and the mechanism may be related to inhibition of renal mitochondrial fission and promotion of mitochondrial fusion.