STING信号通路在CORM-3减轻肝缺血再灌注大鼠肝细胞焦亡和凋亡中的作用

Role of STING signaling pathway in CORM-3-induced reduction of hepatocyte pyroptosis and apoptosis in a rat model of hepatic ischemia-reperfusion

目的评价干扰素基因刺激蛋白(STING)信号通路在一氧化碳释放分子-3(CORM-3)减轻肝缺血再灌注大鼠肝细胞焦亡和凋亡中的作用。方法清洁级健康雄性SD大鼠48只,9~11周龄,体重320~380 g,采用随机数字表法分为4组(n=12):假手术组(S组)、缺血再灌注组(IR组)、CORM-3组(C组)和STING激动剂ADU-S100组(A组)。IR组、C组和A组采用可逆性结扎肝左中叶肝动脉、门静脉及胆管分支45 min后恢复灌注的方法建立肝缺血再灌注损伤模型。C组于再灌注即刻股静脉注射CORM-34 mg/kg,S组、IR组和A组股静脉注射含二甲基亚砜的等容量生理盐水。股静脉注射后1.5 h,A组腹腔注射ADU-S10010 mg/kg,S组、IR组和C组腹腔注射等量生理盐水。再灌注3 h时,采用速率法检测血清ALT和AST浓度。再灌注12 h时处死,取肝组织,采用比色法检测一氧化碳(CO)含量,进行肝损伤评分,采用Western blot法检测IL-1β、IL-18、Bcl-2、Bax、干扰素调节因子3(IRF3)、磷酸化IRF3(p-IRF3)、STING、NOD样受体蛋白3(NLRP3)、消皮素D(GSDMD)和活化的caspase-1的表达,采用免疫荧光染色法确定肝细胞焦亡率和凋亡率。结果与S组比较,IR组血清ALT和AST浓度、肝损伤评分、CO含量、肝细胞焦亡率和凋亡率升高,IL-1β、IL-18、p-IRF3、STING、NLRP3、GSDMD和活化的caspase-1表达上调,Bcl-2/Bax比值降低(P<0.05);与IR组比较,C组血清ALT和AST浓度、肝损伤评分、肝细胞焦亡率和凋亡率降低,CO含量升高,IL-1β、IL-18、p-IRF3、STING、NLRP3、GSDMD和活化的caspase-1表达下调,Bcl-2/Bax比值升高(P<0.05);与C组比较,A组血清ALT和AST浓度、肝损伤评分、肝细胞焦亡率和凋亡率升高,CO含量降低,IL-1β、IL-18、p-IRF3、STING、NLRP3、GSDMD和活化的caspase-1表达上调,Bcl-2/Bax比值降低(P<0.05)。结论CORM-3减轻肝缺血再灌注大鼠肝细胞焦亡和凋亡的机制可能与抑制STING信号通路激活有关。

Objective To evaluate the role of stimulator of interferon genes(STING)signaling pathway in carbon monoxide(CO)-releasing molecule-3(CORM-3)-induced reduction of hepatocyte pyroptosis and apoptosis in a rat model of hepatic ischemia-reperfusion.Methods Forty-eight clean-grade healthy male Sprague-Dawley rats,aged 9-11 weeks,weighing 320-380 g,were divided into 4 groups(n=12 each)using a random number table method:sham operation group(S group),ischemia-reperfusion group(IR group),CORM-3 group(C group)and STING agonist ADU-S100 group(A group).Hepatic ischemia-reperfusion injury models were developed by reversible ligation of left middle hepatic artery,portal vein and bile duct branches for 45 min,followed by reperfusion in anesthetized animals in IR,C and A groups.In group C,CORM-34 mg/kg was injected into the femoral vein immediately after reperfusion.The equal volume of normal saline containing dimethyl sulfoxide was injected into the femoral vein in S,IR and A groups.At 1.5 h after injection into the femoral vein,ADU-S10010 mg/kg was intraperitoneally injected in A group,and the equal volume of normal saline was given instead in S,IR and C groups.The serum alanine transaminase(ALT)and aspartate transaminase(AST)concentrations were determined at 3 h of reperfusion.The rats were sacrificed at 12 h of reperfusion,and liver tissues were collected for determination of the content of CO(by colorimetry),expression of interleukin-1beta(IL-1β),IL-18,Bcl-2,Bax,interferon regulatory factor 3(IRF3),phosphorylated IRF3(p-IRF3),STING,NOD-like receptor protein 3(NLRP3),aspirin D(GSDMD)and activated caspase-1(by Western blot),and pyroptosis and apoptosis rates of hepatocytes(by immunofluorescence staining).The liver injury was scored.Results Compared with group S,the serum ALT and AST concentrations,liver injury score,CO content,and pyroptosis and apoptosis rates of hepatocytes were significantly increased,and the expression of IL-1β,IL-18,p-IRF3,STING,NLRP3,GSDMD and activated caspase-1 was up-regulated,and the Bcl-2/Bax ratio was decreased in group IR(P<0.05).Compared with group IR,the serum ALT and AST concentrations,liver injury score,and pyroptosis and apoptosis rates of hepatocytes were significantly decreased,the CO content was increased,the expression of IL-1β,IL-18,p-IRF3,STING,NLRP3,GSDMD and activated caspase-1 was down-regulated,and the Bcl-2/Bax ratio was increased in group C(P<0.05).Compared with group C,the serum ALT and AST concentrations,liver injury score,and pyroptosis and apoptosis rates of hepatocytes were significantly increased,the CO content was decreased,the expression of IL-1β,IL-18,p-IRF3,STING,NLRP3,GSDMD and activated caspase-1 was up-regulated,and the Bcl-2/Bax ratio was decreased in group A(P<0.05).Conclusions The mechanism by which CORM-3 attenuates hepatocyte pyroptosis and apoptosis may be related to the inhibition of activation of STING signaling pathway in a rat model of hepatic ischemia-reperfusion.