HMGB1/Caspase-1/GSDMD信号轴介导肝细胞焦亡在肝脏缺血-再灌注损伤中的作用

Effect of HMGB1/Caspase-1/GSDMD signaling axis-mediated hepatocyte pyroptosis on liver ischemia-reperfusion injury

    摘要
  • 摘要:
      目的  探讨高迁移率族蛋白1（HMGB1）/半胱氨酸天冬氨酸蛋白酶（Caspase）-1/Gasdermin D（GSDMD）信号轴介导肝细胞焦亡在肝脏缺血-再灌注损伤（IRI）中的作用。
      方法  将C57BL/6小鼠随机分为假手术组（Sham组）、IRI 2 h组、IRI 6 h组、IRI 12 h组、甘草酸（GA）+Sham组和GA+IRI 12 h组（每组8只）；将AML12细胞大致均匀地分为Sham组、IRI 12 h组、GA+Sham组和GA+IRI 12 h组。采用酶联免疫吸附试验（ELISA）检测各组小鼠血清丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）、白细胞介素（IL）-1β和IL-6的水平；采用逆转录聚合酶链反应（RT-PCR）检测肝组织中IL-1β和IL-6信使核糖核酸（mRNA）水平；比较各组小鼠肝脏缺血病理学评分和细胞凋亡情况；采用免疫组织化学（免疫组化）法检测各组小鼠肝组织中HMGB1的表达情况；采用蛋白质印迹法检测小鼠肝组织和AML12细胞中HMGB1、Caspase-1、GSDMD蛋白的表达水平。
      结果  与Sham组比较，IRI后各组小鼠血清中ALT、AST、IL-1β、IL-6水平，以及小鼠肝组织中的IL-1β、IL-6 mRNA相对表达量均升高（均为P < 0.05），且随着再灌注时间的延长呈明显的时间依赖性。与Sham组比较，IRI后各组小鼠肝脏缺血病理学评分和肝细胞凋亡率均升高（均为P < 0.05）。免疫组化结果显示，IRI后HMGB1在肝组织中的表达明显增多，且在2~12 h内随着时间延长而增多。蛋白质印迹法结果显示，在体内和在体外，与Sham组比较，IRI 12 h组HMGB1、Caspase-1和GSDMD蛋白相对表达量均升高；与IRI 12 h组比较，GA+IRI 12 h组HMGB1蛋白相对表达量升高，Caspase-1和GSDMD蛋白相对表达量均降低，差异均有统计学意义（均为P < 0.05）。
      结论  肝细胞可能通过释放HMGB1激活Caspase-1/GSDMD通路，从而触发肝细胞发生焦亡，导致肝脏IRI，而通过GA抑制HMGB1的胞外释放可减轻肝脏IRI。

     

    Abstract:
      Objective  To evaluate the effect of high mobility group box 1 (HMGB1)/ cysteinyl aspartate specific proteinase (Caspase)-1/Gasdermin D (GSDMD) signaling axis-mediated hepatocyte pyroptosis on liver ischemia-reperfusion injury (IRI).
      Methods  C57BL/6 mice were randomly divided into the sham operation group (Sham group), IRI 2 h group, IRI 6 h group, IRI 12 h group, glycyrrhizic acid (GA)+Sham group and GA+IRI 12 h group (n=8 in each group). AML12 cells were evenly divided into the Sham group, IRI 12 h group, GA+Sham group and GA+IRI 12 h group. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), interleukin (IL)-1β and IL-6 in each group were detected by enzyme-linked immune absorbent assay(ELISA). The messenger ribonucleic acid (mRNA) levels of IL-1β and IL-6 were detected by reverse transcription polymerase chain reaction(RT-PCR). The pathological score of liver ischemia and cell apoptosis were compared among all groups. The expression level of HMGB1 in the liver tissues of each group was determined by immunohistochemistry. The expression levels of HMGB1, Caspase-1 and GSDMD proteins in the mouse liver tissues and AML12 cells were measured by Western blot.
      Results  Compared with the Sham group, the serum levels of ALT, AST, IL-1β and IL-6 and the relative expression levels of IL-1β and IL-6 mRNA in the liver tissues were all significantly up-regulated after IRI in each group (all P < 0.05), and showed significant time-dependent pattern along with the prolongation of reperfusion time. Compared with the Sham group, the pathological score of hepatic ischemia and the apoptosis rate of hepatocytes were significantly increased after IRI in each group (all P < 0.05). Immunohistochemical results showed that the expression level of HMGB1 in the liver tissues was significantly up-regulated after IRI, which showed an increasing trend along with the prolongation within the period of 2-12 h. Western blot showed that compared with the Sham group, the relative expression levels of HMGB1, Caspase-1 and GSDMD proteins in vivo and in vitro were up-regulated in the IRI 12 h group. The relative expression level of HMGB1 protein was significantly up-regulated, whereas those of Caspase-1 and GSDMD proteins were significantly down-regulated in the GA+IRI 12 h group compared with those in the IRI 12 h group (all P < 0.05).
      Conclusions  Hepatocytes probably activate the Caspase-1/GSDMD signaling pathway by releasing HMGB1, thereby triggering hepatocyte pyroptosis and leading to liver IRI. Inhibition of extracellular release of HMGB1 by GA may mitigate liver IRI.

     

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