细胞焦亡在肝缺血再灌注大鼠脑损伤中的作用

Role of pyroptosis in brain injury in a rat model of hepatic ischemia-reperfusion

目的评价细胞焦亡在肝缺血再灌注大鼠脑损伤中的作用。方法清洁级健康雄性SD大鼠30只,6~8周龄,体重180~200 g,采用随机数字表法分为3组(n=10):假手术组(S组)、肝缺血再灌注+DMSO组(I/R+DMSO组)和肝缺血再灌注+caspase-1抑制剂Ac-YVAD-cmk组(I/R+YVAD组)。夹闭肝左动脉、门静脉及胆管共干90 min,再灌注6 h,以建立肝缺血再灌注损伤模型。于再灌注前2 h时,I/R+YVAD组腹腔注射Ac-YVAD-cmk 5 mg/kg,I/R+DMSO组腹腔注射等容量DMSO。再灌注结束时取海马及皮质组织,采用DCFH-DA荧光探针检测ROS含量,硫代巴比妥酸法检测MDA含量,黄嘌呤氧化酶法检测SOD活性;采用Western blot法检测NOD样受体蛋白3(NLRP3)、cleaved-caspase-1和凋亡相关微粒蛋白(ASC)表达水平,免疫组织化学染色法观察NLRP3的表达情况;采用ELISA法检测血清IL-1β、IL-18、S-100β蛋白和神经元特异性烯醇化酶(NSE)浓度。结果与S组比较,I/R+DMSO组和I/R+YVAD组海马和皮质组织ROS、MDA含量升高,SOD活性降低,NLRP3、cleaved-caspase-1和ASC表达上调,血清IL-1β、IL-18、S-100β蛋白和NSE浓度升高(P<0.05);与I/R+DMSO组比较,I/R+YVAD组海马和皮质组织ROS、MDA含量降低,SOD活性升高,NLRP3、cleaved-caspase-1和ASC表达下调,血清IL-1β、IL-18、S100-β蛋白和NSE浓度降低(P<0.05)。结论细胞焦亡参与了肝缺血再灌注大鼠脑损伤的病理生理机制。

Objective To evaluate the role of pyroptosis in brain injury in a rat model of hepatic ischemia-reperfusion(I/R).Methods Thirty clean-grade healthy male Sprague-Dawley rats of both sexes,aged 6-8 weeks,weighing 180-200 g,were divided into 3 groups(n=10 each)using a random number table method:sham operation group(group S,n=10),hepatic I/R plus dimethyl sulfoxide(DMSO)group(group I/R+DMSO,n=10),and hepatic I/R plus caspase-1 inhibitor Ac-YVAD-cmk group(group I/R+YVAD,n=10).Hepatic I/R was produced by occluding the left hepatic artery and portal vein for 90 min followed by 6-h reperfusion in anesthetized rats.At 2 h before reperfusion,Ac-YVAD-cmk 5 mg/kg was intraperitoneally injected in group I/R+YVAD,and the equal volume of DMSO was given instead in group I/R+DMSO.Hippocampal and cortical samples were obtained at the end of reperfusion for determination of reactive oxygen species(ROS)content(using DCFH-DA fluorescence probe),malondialdehyde(MDA)content(using thiobarbituric acid method),superoxide dismutase(SOD)activity(by xanthine oxidase method),expression of NLRP3,cleaved-caspase-1 and apoptosis-associated speck-like protein containing a CAR(ASC)(by Western blot),nucleotide-binding domain,leucine rich family(NLR)pyrin domain containing 3(NLRP3)expression(by immunohistochemical staining),and concentrations of IL-1β,IL-18,S100-βprotein and neuron-specific enolase(NSE)in serum(by enzyme-linked immunosorbent assay).Results Compared with group S,the contents of ROS and MDA in hippocampus and cortex were significantly increased,the activity of SOD in hippocampus and cortex was decreased,the expression of NLRP3,cleaved-caspase-1 and ASC was up-regulated,and the concentrations of IL-1β,IL-18,S-100βprotein and NSE in serum were increased in group I/R+DMSO(P<0.05).Compared with group I/R+DMSO,the contents of ROS and MDA in hippocampus and cortex were significantly decreased,the activity of SOD in hippocampus and cortex was increased,the expression of NLRP3,cleaved-caspase-1 and ASC was down-regulated,and the concentrations of IL-1β,IL-18,S-100βprotein and NSE in serum were decreased in group I/R+YVAD(P<0.05).Conclusion Pyroptosis is involved in the pathophysiological mechanism of brain injury induced by hepatic I/R injury in rats.