槲皮素预处理调节AMPK/mTOR信号通路介导的自噬途径改善大鼠肝缺血再灌注损伤

Quercetin pretreatment modulates the autophagy pathway mediated by the AMPK/m TOR signaling pathway and improves liver ischemia-reperfusion injury in rats

目的 探讨槲皮素预处理通过AMPK/mTOR信号通路介导的自噬对肝缺血再灌注损伤的保护作用。方法 大鼠使用随机数字表法分为对照组、模型组、槲皮素组、氯喹(自噬抑制剂)组和槲皮素+氯喹组。手术前3 d,槲皮素组大鼠灌胃给予0.1 g/kg槲皮素,氯喹组大鼠腹腔注射0.02 g/kg氯喹,槲皮素+氯喹组灌胃给予槲皮素同时腹腔注射氯喹,对照组和模型组给予等体积溶剂,每天1次。构建肝缺血再灌注大鼠模型并于再灌注6 h后处死大鼠。全自动生化分析仪检测大鼠肝功能指标,包括谷氨酸-丙酮酸转氨酶(ALT)和天门冬氨酸酰基转移酶(AST);苏木素-伊红(HE)染色观察各组大鼠肝组织病理学变化;酶联免疫吸附法(ELISA)检测各组大鼠血清中炎性因子含量,包括白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β);单磺尸胺染色法(MDC)观察并计算各组大鼠肝组织细胞自噬率;蛋白免疫印迹法(Western blot)检测各组大鼠肝组织中Beclin1蛋白表达水平及AMPK、mTOR蛋白磷酸化水平。结果 对照组大鼠肝组织结构完整,肝细胞排列整齐,无显著坏死现象。与对照组相比,模型组大鼠肝组织结构紊乱,肝细胞排列疏松,出现坏死,ALT、AST水平、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)含量以及mTOR蛋白磷酸化水平显著升高(P<0.05),肝组织细胞自噬率、Beclin1蛋白表达水平及AMPK蛋白磷酸化水平显著降低(P<0.05)。与模型组相比,槲皮素组大鼠肝组织上述病理损伤显著缓解,ALT、AST水平、IL-6、TNF-α、IL-1β含量以及mTOR蛋白磷酸化水平显著降低(P<0.05),肝组织细胞自噬率、Beclin1蛋白表达水平及AMPK蛋白磷酸化水平显著升高(P<0.05);氯喹组大鼠肝组织上述病理损伤显著加重,ALT、AST水平、IL-6、TNF-α、IL-1β含量以及mTOR蛋白磷酸化水平显著升高(P<0.05),肝组织细胞自噬率、Beclin1蛋白表达水平及AMPK蛋白磷酸化水平显著降低(P<0.05)。与槲皮素组相比,槲皮素+氯喹组大鼠肝组织病理损伤显著加重,ALT、AST水平、IL-6、TNF-α、IL-1β含量以及mTOR蛋白磷酸化水平显著升高(P<0.05),肝组织细胞自噬率、Beclin1蛋白表达水平及AMPK蛋白磷酸化水平显著降低(P<0.05);与氯喹组相比,槲皮素+氯喹组大鼠肝组织病理损伤显著减轻,ALT、AST水平、IL-6、TNF-α、IL-1β含量以及mTOR蛋白磷酸化水平显著降低(P<0.05),肝组织细胞自噬率、Beclin1蛋白表达水平及AMPK蛋白磷酸化水平显著升高(P<0.05)。结论 槲皮素预处理可能通过激活肝缺血再灌注损伤大鼠肝组织中AMPK/mTOR信号通路介导的自噬,从而减少炎症反应,缓解肝损伤。

Objective To investigate the protective effect of quercetin pretreatment on hepatic ischemia-reperfusion injury by autophagy mediated via the AMPK/mTOR signaling pathway. Methods Rats were randomly divided into control, model, quercetin, chloroquine(autophagy inhibitor) and quercetin+chloroquine groups. Three days before the operation, the quercetin group was administered 0.1 g/kg quercetin, the chloroquine group was intraperitoneally injected with 0.02 g/kg chloroquine, the quercetin+chloroquine group was administered quercetin by gavage and chloroquine was injected intraperitoneally at 3 days, and the control and model groups were administered an equal volume of solvent once a day. The rat model of hepatic ischemia-reperfusion was established and rats were sacrificed at 6 hours after reperfusion. Liver function indexes, which included glutamic pyruvate transaminase(ALT) and aspartate acyltransferase(AST), were measured by an automatic biochemical analyzer. Pathological changes of liver tissue were observed by hematoxylin eosin(HE) staining. The serum levels of inflammatory factors, which included interleukin(IL-6), tumor necrosis factor-α(TNF-α) and IL-1β were measured by enzyme-linked immunosorbent assays. The autophagy rate was calculated by monosulfonamide(MDC) staining. Expression of Beclin1 protein and the phosphorylation of AMPK and mTOR proteins in liver tissue were detected by Western blot. Results In the control group, the liver tissue structure was complete, hepatocytes were arranged in order, and there was no significant necrosis. Compared with the control group, the liver tissue structure of the model group was disordered, the arrangement of hepatocytes was loose, and necrosis had appeared, the levels of ALT and AST, contents of IL-6, TNF-α and IL-1β, phosphorylation level of mTOR protein were significantly higher(P<0.05), and the autophagy rate, expression level of Beclin1 protein, and phosphorylation level of AMPK protein were significantly lower(P<0.05). Compared with the model group, the above pathological damage of liver tissue in the quercetin group was alleviated significantly, the levels of ALT and AST, the contents of IL-6, TNF-α, IL-1β and the phosphorylation level of mTOR protein were significantly lower(P<0.05), and the autophagy rate, expression level of Beclin1 protein, and phosphorylation level of AMPK protein were significantly higher(P<0.05). In the chloroquine group, the above pathological damage of liver tissue was aggravated significantly, the levels of ALT and AST, contents of IL-6, TNF-α and IL-1β and phosphorylation level of mTOR protein were significantly higher(P<0.05), and the autophagy rate, expression level of Beclin1 protein, and phosphorylation level of AMPK protein were significantly lower(P<0.05). Compared with the quercetin group, the pathological damage of liver tissue in the quercetin + chloroquine group was more serious, the levels of ALT and AST, contents of IL-6, TNF-α and IL-1β. and phosphorylation level of mTOR protein were significantly higher(P<0.05) and the autophagy rate, expression level of Beclin1 protein, and phosphorylation level of AMPK protein were significantly lower(P<0.05). Compared with the chloroquine group, the pathological damage of liver tissue in the quercetin+chloroquine group was significantly reduced, the levels of ALT and AST, the contents of IL-6, TNF-α and IL-1β, and phosphorylation level of mTOR protein were significantly lower(P<0.05), and the autophagy rate, expression level of Beclin1 protein, and phosphorylation level of AMPK protein were significantly higher(P<0.05). Conclusions Quercetin pretreatment may activate autophagy mediated by the AMPK/mTOR signaling pathway in liver tissue of rats with hepatic ischemia-reperfusion injury, thereby reducing the inflammatory response and alleviating liver injury.