目的 基于p62-Kelch样ECH相关蛋白1（Kelch-like ECH-associated protein 1，Keap1）-核因子E2相关因子（nuclear factor erythroid 2-related factor 2，Nrf2）信号通路探讨荆防颗粒对酒精性肝损伤（alcoholic liver injury，ALI）小鼠氧化应激的保护作用及机制。方法 SPF级KM小鼠按体质量随机分为对照组、模型组、荆防颗粒高、低剂量（15.0、10.5 g/kg）组及N-乙酰半胱氨酸（N-acetylcysteine，NAC，0.3 g/kg）组，除对照组外，其余各组小鼠连续ig给药15 d并ig白酒构建ALI模型，末次ig白酒12 h后处死小鼠，收集血清及肝组织。采用苏木素-伊红（HE）染色、油红O染色和透射电镜观察肝组织病理形态变化；采用全自动生化仪检测小鼠血清中天冬氨酸氨基转移酶（aspartate aminotransferase，AST）和丙氨酸氨基转移酶（alanine aminotransferase，ALT）活性；采用ELISA法和流式细胞术检测肝组织炎症因子和氧化应激水平；采用免疫组化法检测小鼠肝组织细胞色素P450 2E1（cytochrome P450 family 2 subfamily E member 1，CYP2E1）表达；采用Western blotting检测小鼠肝组织CYP2E1、Nrf2、血红素加氧酶-1（heme oxygenase 1，HO-1）、超氧化物歧化酶1（superoxide dismutase 1，SOD1）、还原型烟酰胺腺嘌呤二核苷酸磷酸醌脱氢酶1（nicotinamide adenine dinucleotide phosphate quinone oxidoreductase 1，NQO1）、p62、Keap1、微管相关蛋白1轻链3B-II/Ⅰ（microtubule-associated protein 1 light chain 3B-II/Ⅰ，LC3B-Ⅱ/Ⅰ）、乙醇脱氢酶1（alcohol dehydrogenase 1，ADH1）、乙醛脱氢酶2（aldehyde dehydrogenase 2，ALDH2）蛋白表达。结果 与模型组相比，荆防颗粒高剂量组小鼠肝脏指数、血清AST及ALT活性显著降低（P＜0.05、0.01）；肝脏中超氧化物歧化酶（superoxide dismutase，SOD）、过氧化氢酶（catalase，CAT）、谷胱甘肽（glutathione，GSH）水平显著升高（P＜0.05、0.01）；肝组织中活性氧（reactive oxygen species，ROS）降低，脂质过氧化产物丙二醛（malondialdehyde，MDA）水平和CYP2E1蛋白表达明显降低（P＜0.05、0.01），肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）水平明显降低（P＜0.05）；肝脏脂肪堆积、肝组织炎性细胞浸润得到明显改善（P＜0.05、0.01），肝细胞形态结构异常变化也得到缓解。荆防颗粒能显著上调ALI小鼠肝脏ADH1、ALDH2、Nrf2、HO-1、NQO1、SOD1和LC3B-Ⅱ/Ⅰ蛋白表达（P＜0.05、0.01），并下调NOX4、p62和Keap1蛋白表达（P＜0.05、0.01、0.001）。结论 荆防颗粒可通过激活p62-Keap1-Nrf2信号通路，提高机体抗氧化能力并激活自噬来发挥抗ALI作用。

Objective To explore the protect effects of Jingfang Granules (荆防颗粒, JFGR) on oxidative stress in mice with alcoholic liver injury (ALI) and its mechanism related to p62- Kelch-like ECH-associated protein 1 (Keap1) - nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. Methods According to body weight, KM mice were randomly divided into five groups of control group, model group, JFGR high-, low-dose (15.0, 10.5 g/kg) groups and N-acetylcysteine (NAC, 0.3 g/kg) group. Except control group, mice of other groups were intragastric administrated the corresponding drug and white wine every day for 15 d to establish the ALI model. The pathological changes of liver tissue were observed by hematoxylin-eosin (HE) staining, oil red O staining and transmission electron microscopy. The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum of mice were detected by automatic biochemical analyzer. The levels of liver inflammation and oxidative stress were detected by ELISA and flow cytometry. The expression of cytochrome P450 family 2 subfamily E member 1 (CYP2E1) in liver was detected by immunohistochemistry. The protein expressions of CYP2E1, Nrf2, heme oxygenase 1 (HO-1), superoxide dismutase 1 (SOD1), nicotinamide adenine dinucleotide phosphate quinone oxidoreductase 1 (NQO1), p62, Keap1, microtubule-associated protein 1 light chain 3B-II/Ⅰ (LC3B-II/Ⅰ), alcohol dehydrogenase 1 (ADH1) and aldehyde dehydrogenase 2 (ALDH2) in liver were determined by Western blotting. Results Compared with model group, JFGR (15 g/kg) significantly decreased the liver index and the activities of AST, ALT in serum (P < 0.05, 0.01), significantly increased the levels of superoxide dismutase (SOD), Catalase (CAT) and glutathione (GSH) in liver (P < 0.05, 0.01), reduced the level of reactive oxygen species (ROS), and significantly decreased malondialdehyde (MDA) level. CYP2E1 expression and the content of tumor necrosis factor-α (TNF-α) in liver (P < 0.05). In addition, JFGR significantly decreased the liver fat accumulation and the hepatocyte inflammatory infiltration (P < 0.05, 0.01), and ameliorated the morphological and structural changes. JFGR significantly up-regulated the expression levels of ADH1, ALDH2, Nrf2, HO-1, NQO1, SOD1, LC3B-II/LC3B-Ⅰ in liver (P < 0.05, 0.01), and inhibited the expressions of NOX4, p62, Keap1 proteins (P < 0.05, 0.01). Conclusion JFGR could alleviate the alcohol-induced liver injury in mice, which the mechanism maybe related to activate p62-Keap1-Nrf2 signaling pathway so as to improve the antioxidant level and activate autophagy.

R285.5

国家自然科学基金资助项目(82074094);2022年度“天府青城计划”天府名师项目(川青城第1206号)