紫檀芪调节Keap-1/Nrf2/HO-1信号通路对非酒精性脂肪肝大鼠氧化应激和细胞凋亡的影响

摘要 目的：研究紫檀芪调节Kelch样ECH关联蛋白1（Keap-1）/核因子E2相关因子2（Nrf2）/血红素加氧酶-1（HO-1）信号通路对非酒精性脂肪肝（NAFLD）大鼠氧化应激和细胞凋亡的影响。方法：将60只SD大鼠随机分为对照组、模型组、紫檀芪低剂量组（30 mg/kg）、紫檀芪高剂量组（60 mg/kg）、紫檀芪（60 mg/kg）+N-（4-（2,3-二氢-1-（2''-甲基苯甲酰）-1H-吲哚-5-基）-5-甲基-2-噻唑基）-1,3-苯并二氧唑-5-乙酰胺（ML385）（30 mg/kg）组，每组12只。模型组与药物干预组大鼠以高脂饲料饲养诱导NAFLD模型，对照组大鼠以普通饲料饲养，各组连续喂养12周。以紫檀芪和ML385分组处理14 d后（对照组以等剂量生理盐水处理），检测各组大鼠脂代谢指标三酰甘油（TG）、总胆固醇（TC）及游离脂肪酸（FFA）水平]、肝指数、肝功能指标谷丙转氨酶（ALT）及谷草转氨酶（AST）]水平、血清白细胞介素（IL）-17、IL-6、IL-10、氧化应激指标丙二醛（MDA）、超氧化物歧化酶（SOD）及过氧化氢酶（CAT）]水平；原位末端标记法（TUNEL）染色检测各组大鼠肝细胞凋亡率；蛋白免疫印迹法检测各组大鼠肝组织凋亡相关蛋白及Keap-1/Nrf2/HO-1通路相关蛋白表达。结果：与对照组相比，模型组大鼠血清IL-10、SOD及CAT水平、肝组织Nrf2、HO-1、Bcl-2表达水平显著降低（P＜0.05），TG、TC及FFA水平、肝指数、ALT及AST水平、血清IL-17、IL-6、MDA水平、肝细胞凋亡率、肝组织Keap-1及Bax表达水平显著升高（P＜0.05）。与模型组相比，紫檀芪低、高剂量组大鼠血清IL-10、SOD及CAT水平、肝组织Nrf2、HO-1、Bcl-2表达水平均升高（P＜0.05），TG、TC及FFA水平、肝指数、ALT及AST水平、血清IL-17、IL-6、MDA水平、肝细胞凋亡率、肝组织Keap-1、Bax表达水平均降低（P＜0.05）；与紫檀芪低剂量组相比，紫檀芪高剂量组大鼠血清IL-10、SOD及CAT水平、肝组织Nrf2、HO-1、Bcl-2表达水平升高（P＜0.05），TG、TC及FFA水平、肝指数、ALT及AST水平、血清IL-17、IL-6、MDA水平、肝细胞凋亡率、肝组织Keap-1及Bax表达水平降低（P＜0.05）；与紫檀芪高剂量组相比，紫檀芪+ML385组大鼠血清IL-10、SOD及CAT水平、肝组织Nrf2、HO-1、Bcl-2表达水平降低（P＜0.05），TG、TC及FFA水平、肝指数、ALT及AST水平、血清IL-17、IL-6、MDA水平、肝细胞凋亡率、肝组织Bax表达水平升高（P＜0.05）。结论：紫檀芪可能通过激活Keap-1/Nrf2/HO-1信号通路，改善NAFLD大鼠脂代谢水平，调节炎症反应及氧化应激，减轻肝组织脂肪变性及细胞凋亡。

Effects of Pterostilbene on Oxidative Stress and Apoptosis in Nonalcoholic Fatty Liver Disease Rats by Regulating Keap-1/Nrf2/HO-1 Signaling Pathway

ABSTRACT Objective: To investigate the effects of pterostilbene on oxidative stress and apoptosis in nonalcoholic fatty liver disease (NAFLD) rats by regulating Kelch-like ECH-associated protein 1 (Keap-1)/nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. Methods: 60 SD rats were randomly divided into control group, model group, pterostilbene low-dose group (30 mg/kg), pterostilbene high-dose group (60 mg/kg), pterostilbene (60 mg/kg)+N-(4-(2,3-dihydro-1-(2''-methylbenzoyl)-1H-indole-5-yl)-5-methyl-2-thiazolyl)-1,3-benzodioxazol-5-acetamide (ML385) (30 mg/kg) group, with 12 rats in each group. Rats in model group and drug intervention group were fed with high-fat diet to induce NAFLD model, and rats in control group were fed with normal diet, each group was continuously fed for 12 weeks. 14 days after treatment with pterostilbene and ML385 groups (control group was treated with the same dose of normal saline), the lipid metabolism indexes triglyceride (TG), total cholesterol (TC) and free fatty acid (FFA) levels], liver index, liver function indexes alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] levels, serum interleukin (IL)-17, IL-6, IL-10, oxidative stress indexes malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT)] levels were detected in each group. The apoptosis rate of hepatocytes in each group were detected by in situ end labeling method (TUNEL) staining. The expression of apoptosis-related proteins and Keap-1/Nrf2/HO-1 pathway-related proteins in liver tissue of rats in each group were detected by western blotting. Results: Compared with control group, the levels of serum IL-10, SOD and CAT, the expression levels of Nrf2, HO-1 and Bcl-2 in liver tissue of rats in model group were significantly decreased (P<0.05), and the levels of TG, TC and FFA, liver index, ALT and AST levels, serum IL-17, IL-6, MDA levels, hepatocyte apoptosis rate, liver tissue Keap-1 and Bax expression levels were significantly increased (P<0.05). Compared with model group, the levels of serum IL-10, SOD and CAT, the expression levels of Nrf2, HO-1 and Bcl-2 in liver tissue of rats in pterostilbene low and high dose groups were increased (P<0.05), and the levels of TG, TC and FFA, liver index, ALT and AST, serum IL-17, IL-6, MDA, hepatocyte apoptosis rate, the expression levels of Keap-1 and Bax in liver tissue were decreased (P<0.05). Compared with pterostilbene low-dose group, the levels of serum IL-10, SOD and CAT, and the expression levels of Nrf2, HO-1 and Bcl-2 in liver tissue were increased in pterostilbene high-dose group (P<0.05), and the levels of TG, TC and FFA, liver index, ALT and AST, serum IL-17, IL-6, MDA, hepatocyte apoptosis rate, and the expression levels of Keap-1 and Bax in liver tissue were decreased (P<0.05). Compared with pterostilbene high-dose group, the levels of serum IL-10, SOD and CAT, and the expression levels of Nrf2, HO-1 and Bcl-2 in liver tissue of pterostilbene+ML385 group were decreased (P<0.05), and the levels of TG, TC and FFA, liver index, ALT and AST, serum IL-17, IL-6, MDA, hepatocyte apoptosis rate, and liver tissue Bax expression level were increased (P<0.05). Conclusion: Pterostilbene may improve lipid metabolism, regulate inflammatory response and oxidative stress, and reduce hepatic steatosis and apoptosis in NAFLD rats by activating Keap-1/Nrf2/HO-1 signaling pathway.