大黄素对肠缺血再灌注小鼠肝损伤的影响及HO-1介导的自噬在其中的作用

Effect of emodin on liver injury in a mouse model of intestinal ischemia-reperfusion and role of heme oxygenase-1-mediated autophagy

目的评价大黄素对肠缺血再灌注小鼠肝损伤的影响及血红素加氧酶-1(HO-1)介导的自噬在其中的作用。方法SPF级健康雄性C57BL/6小鼠24只,6~8周龄,体质量18~22 g,采用随机数字表法分为4组(n=6):假手术组(Sham组)、肠缺血再灌注组(I/R组)、大黄素组(E组)和大黄素+HO-1抑制剂锡原卟啉Ⅸ组(ES组)。采用夹闭肠系膜上动脉45 min再灌注120 min的方法制备小鼠肠缺血再灌注损伤模型。E组缺血前5 d每天灌胃给予大黄素40 mg/kg;ES组缺血前5 d每天灌胃给予大黄素40 mg/kg,缺血前12 h尾静脉注射锡原卟啉Ⅸ7.5 mg/kg。于再灌注120 min时采集眶静脉血样,采用比色法检测血清ALT和AST浓度,随后处死小鼠取肝组织,观察肝组织病理学变化,分别采用WST-1法和TBA法检测SOD活性和MDA含量,荧光定量PCR法测定IL-6 mRNA和TNF-αmRNA的表达,Western blot法检测HO-1、自噬蛋白-苄氯素1(Beclin1)和微管相关蛋白1轻链3(LC3)的表达,计算LC3-Ⅱ/Ⅰ比值。结果与Sham组比较,I/R组肝组织SOD活性降低,MDA含量、血清ALT和AST浓度升高,IL-6 mRNA、TNF-αmRNA和HO-1表达上调,Beclin1表达下调,LC3-Ⅱ/Ⅰ比值降低(P<0.05),出现明显肝组织病理学损伤;与I/R组比较,E组肝组织SOD活性升高,MDA含量、血清ALT和AST浓度降低,IL-6 mRNA和TNF-αmRNA表达下调,HO-1和Beclin1表达上调,LC3-Ⅱ/Ⅰ比值升高(P<0.05),肝组织病理学损伤减轻;与E组比较,ES组肝组织SOD活性降低,MDA含量、血清ALT和AST浓度升高,IL-6 mRNA和TNF-αmRNA表达上调,HO-1和Beclin1表达下调,LC3-Ⅱ/Ⅰ比值降低(P<0.05),肝组织病理学损伤加重。结论大黄素可减轻小鼠肠缺血再灌注诱导的肝损伤,其机制可能与激活HO-1介导的自噬有关。

Objective To evaluate the effect of emodin on liver injury in a mouse model of intestinal ischemia-reperfusion(I/R)and the role of heme oxygenase-1-mediated autophagy.Methods Twenty-four SPF-grade healthy male C57BL/6 mice,aged 6-8 weeks,weighing 18-22 g,were divided into 4 groups(n=6 each)using a random number table method:sham operation group(Sham group),I/R group,emodin group(E group)and emodin plus HO-1 inhibitor Zinc ProtoporphyrinⅨ(ZnPP)group(ES group).The intestinal I/R injury model was established by clamping the superior mesenteric artery for 45 min followed by 120 min of reperfusion.Emodin 40 mg/kg dissolved in 5%methylcellulose sodium was given by gastric gavage once a day for 5 days before ischemia in E group.Emodin 40 mg/kg dissolved in 5%methylcellulose sodium was given by gastric gavage once a day for 5 days before intestinal I/R,and ZnPP 7.5 mg/kg was injected via the tail vein at 12 h before ischemia in ES group.Orbital venous blood samples were collected at the end of reperfusion for determination of serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)concentrations.Then the mice were sacrificed,and liver tissues were obtained for microscopic examination of the pathological changes(after HE staining)and for determination of the activity of superoxide dismutase(SOD),content of malondialdehyde(MDA),expression of interleukin-6(IL-6)and tumor necrosis factor-alpha(TNF-α)mRNA(by fluorescent quantitative polymerase chain reaction),the expression of HO-1,autophagy-related protein Beclin1 and microtubule-associated protein 1 light chain 3(LC3)(by Western blot).The LC3-Ⅱ/Ⅰratio was calculated.Results Compared with Sham group,the activity of SOD was significantly decreased,the content of MDA and serum ALT and AST concentrations were increased,the expression of IL-6 and TNF-αmRNA and HO-1 was up-regulated,the expression of Beclin1 was down-regulated,the LC3-Ⅱ/Ⅰratio was decreased(P<0.05),and the pathological changes of liver tissues were found in I/R group.Compared with I/R group,the activity of SOD was significantly increased,the content of MDA and serum ALT and AST concentrations were decreased,the expression of IL-6 and TNF-αmRNA was down-regulated,the expression of HO-1 and Beclin1 was up-regulated,the LC3-Ⅱ/Ⅰratio was increased(P<0.05),and the pathological changes of liver tissues were significantly attenuated in E group(P<0.05).Compared with E group,the activity of SOD was significantly decreased,the content of MDA and serum ALT and AST concentrations were increased,the expression of IL-6 and TNF-αmRNA was up-regulated,the expression of HO-1 and Beclin1 was down-regulated,the LC3-Ⅱ/Ⅰratio was decreased(P<0.05),and the pathological changes of liver tissues were aggravated in ES group.Conclusions Emodin can alleviate liver injury induced by intestinal I/R in mice,and the mechanism may be related to the activation of HO-1-mediated autophagy.