自噬调控Sprague-Dawley大鼠梗阻性黄疸肝损伤机制研究

The underlying mechanism of autophagy regulating liver injury with obstructive jaundice in Sprague-Dawley rats

目的探讨自噬对Sprague-Dawley(SD)大鼠梗阻性黄疸肝损伤的影响及其机制。方法健康雄性SD大鼠35只,SPF级,6~8周龄,200~300 g,分为5组:假手术组(单纯游离胆总管,不结扎,腹腔内注射生理盐水)、胆总管结扎(OJ)组(游离胆总管,并双重丝线结扎,腹腔内注射生理盐水)、OJ+3-甲基腺嘌呤(3-MA)组、OJ+雷帕霉素(Rapamycin)组和OJ+3-MA+VX-765组,每组7只。HE染色观察肝脏组织学变化。免疫组化染色检测自噬相关蛋白Atg5表达水平。全自动生化仪检测肝功能。酶联免疫吸附试剂盒检测血清白细胞介素(IL)-18水平。蛋白质印迹法检测自噬相关蛋白水平及内质网应激相关凋亡通路。结果通过免疫组化染色,OJ组自噬相关蛋白Atg5相对表达量较假手术组增加[(5.0±1.0)比(2.8±1.3)],差异有统计学意义(t=-3.00,P<0.05)。与假手术组比较,OJ组自噬活性增加,半胱氨酸天冬氨酸蛋白酶(Caspase)-1/p-65及炎症因子IL-18水平升高,同时内质网发生应激并诱导其相关性凋亡。应用自噬抑制剂3-MA后,与OJ组比较,OJ+3-MA组中Caspase-1/p-65蛋白水平及炎症因子IL-18水平增加,同时肝损伤加重;而应用自噬激动剂Rapamycin后,与OJ组比较,OJ+Rapamycin组中Caspase-1/p-65蛋白水平及炎症因子IL-18水平下降,同时肝损伤减轻。应用Caspase-1特异性阻断剂VX-765后,与OJ+3-MA组比较,OJ+3-MA+VX-765组中Caspase-1/p-65蛋白水平及炎症因子IL-18水平下降,同时肝损伤减轻。结论胆总管结扎后,诱导内质网应激相关性凋亡及激活自噬。活化的自噬减轻SD大鼠梗阻性黄疸肝损伤,可能与抑制Caspase-1/p-65炎症通路相关。

Objective To investigate the effect of autophagy on liver injury with obstructive jaundice in Sprague-Dawley(SD)rats and its underlying mechanism.Methods Thirty-five healthy male SD rats,SPF grade,aged 6-8 weeks,weighting 200-300 g,were divided into 5 groups with 7 rats in each group,including sham group(simple free common bile duct,without ligation,intraperitoneal injection of normal saline),obstructive jaundice(OJ)group(established by common bile duct ligation,intraperitoneal injection of normal saline),OJ group with 3-MA,OJ group with Rapamycin,and OJ group with 3-MA and VX-765.Morphological changes in liver tissues were analyzed with HE staining.Expression of autophagy-related protein Atg5 was detected by immunohistochemistry staining.Liver function was analyzed by automatic biochemical instrument and the level of serum interleukin(IL)-18 was detected using ELISA assay.Protein levels of autophagy related-proteins and endoplasmic reticulum stressed(ERs)-related apoptosis proteins were detected by Western Blot.Results The relative expression of autophagy related protein Atg5 in OJ group was significantly higher than that in sham group[(5.0±1.0)vs.(2.8±1.3),t=-3.00,P<0.05].Compared with sham group,the activity of autophagy was enhanced and the protein levels of Caspase-1/p-65 and IL-18 were significantly increased in OJ group.At the same time,apoptosis was induced by activating ERs.In OJ group,the autophagy inducer 3-MA improved the expression levels of Caspase-1/p-65 and IL-18,and aggravate liver injury.While after applying the autophagy agonist Rapamycin in OJ rat models,the expression of Caspase-1/p-65 and IL-18 was repressed and liver damage was also reduced.In addition,in rat OJ groups with 3-MA,inhibition of Caspase-1 by VX-765 could down regulate the expression of Caspase-1/p-65 and IL-18,and protect against liver injury.Conclusions Both ERs related apoptosis and autophagy were activated after ligation of common bile duct.Besides,activation of autophagy could reduce OJ-induced liver injury in SD rats by inhibiting the Caspase-1/p-65 inflammatory pathway.