雷帕霉素对肝脏缺血再灌注损伤大鼠的影响及相关机制

Effect of rapamycin on hepatic ischemia reperfusion injury in rats and its possible mechanism

目的分析不同剂量雷帕霉素对肝脏缺血再灌注损伤(HIRI)大鼠的影响及可能机制。方法48只Sprague-Dawley大鼠随机分为四组,各12只,低剂量组(术前注射低剂量雷帕霉素+缺血),高剂量组(术前注射高剂量雷帕霉素+缺血),模型组(术前注射0.9%氯化钠溶液+缺血),假手术组(术前注射0.9%氯化钠溶液,仅解剖出第一肝门)。术后24、72 h留取血清,检测ALT、AST、TNF-α、IL-6浓度,Western印迹以及PCR检测肝组织自噬相关蛋白表达,肝组织行HE染色。结果术后24 h,假手术组肝组织基本正常,模型组肝小叶结构消失,肝细胞大量水肿,空泡样变性、坏死,低剂量组和高剂量组损伤减轻。术后24 h,模型组、低剂量组、高剂量组ALT、AST、TNF-α、IL-6水平呈下降趋势,差异有统计学意义(P<0.05)。术后24 h,假手术组、模型组、低剂量组和高剂量组ULK1(1.00±0比4.76±2.62比8.26±3.46比12.95±6.45)、微管相关蛋白1轻链3(LC3,1.00±0比2.88±0.59比4.66±1.22比7.10±0.85) mRNA相对表达量呈升高趋势,而哺乳动物雷帕霉素靶蛋白(mTOR,1.00±0比0.31±0.09比0.18±0.04比0.02±0.01)、P70核糖体蛋白激酶(S6K1,1.00±0比0.57±0.34比0.27±0.14比0.03±0.01)mRNA相对表达量呈降低趋势,各组间差异有统计学意义(P<0.05)。术后24 h,假手术组、模型组、低剂量组和高剂量组ULK1、LC3蛋白相对表达呈升高趋势,而磷酸化mTOR、磷酸化S6K1及磷酸化ULK1呈降低趋势,差异有统计学意义(P<0.05)。术后72 h,各数据结果与术后24 h基本一致。结论雷帕霉素通过mTORC1-ULK1信号通路激活细胞自噬减轻HIRI,且高剂量保护作用优于低剂量。

Objective To analyze the effects of different doses of rapamycin on hepatic ischemia reperfusion injury in rats and the possible mechanism. Methods 48 Sprague-Dawley rats were randomly divided into four groups(n=12), low dose group (preoperative injection of low dose of rapamycin+ ischemia), high dose group (preoperative high dose of rapamycin injection + ischemia), model group (preoperative injection of 0.9% sodium chloride solution + ischemia), sham group (preoperative injection of 0.9% sodium chloride solution, only dissected the first hepatic portal). Serum was collected 24 and 72 hours after surgery, ALT, AST, TNF-α and IL-6 concentrations were detected, Western blotting and PCR were performed to detect the expression of autophagy proteins, and HE staining was performed. Results 24 h after the operation, the liver tissue of the sham group was almost normal, the hepatic lobule structure of the model group disappeared, the liver cells were edema, vacuolar degeneration and necrosis, and the damage was reduced in the low dose and high dose group. 24 h after surgery, levels of ALT, AST, TNF-α and IL-6 was on a declining curve in all groups, with statistically significant differences (P<0.05). 24 h after surgery, the relative expression levels of ULK1 (1.00±0 vs. 4.76±2.62 vs. 8.26±3.46 vs. 12.95±6.45), microtubule-associated protein 1 light chain 3(LC3, 1.00±0 vs. 2.88±0.59 vs. 4.66±1.22 vs. 7.10±0.85) mRNA in sham group, model group, low dose group and high dose group were increased. While the relative mRNA expression levels of mammalian target of rapamycin (mTOR, 1.00±0 vs. 0.31±0.09 vs. 0.18±0.04 vs. 0.02±0.01), P70 ribosomal protein kinase (S6K1, 1.00±0 vs. 0.57±0.34 vs. 0.27±0.14 vs. 0.03±0.01) showed a decreasing trend, and the differences were statistically significant (P<0.05). 24 h after surgery, the relative expressions of ULK1 and LC3 proteins in sham group, model group, low dose group and high dose group increased, while the phosphorylation of mTOR, S6K1 and ULK1 decreased, with statistically significant differences (P<0.05). 72 h after the operation, the results were agreed with those at 24 h after the operation. Conclusion Rapamycin activates autophagy through mTORC1-ULK1 signaling pathway to reduce HIRI, and the protective effect of high dose is better than that of low dose.