目的 探究川陈皮素防治肝缺血再灌注损伤（hepatic ischemia reperfusion injury，HIRI）的作用及机制。方法 SD大鼠随机分为对照组、模型组和川陈皮素（10 mg/kg）组，采用ELISA法检测各组大鼠血清中丙氨酸转氨酶（alanine aminotransferase，ALT）和天冬氨酸转氨酶（aspartate aminotransferase，AST）活性；采用苏木素-伊红（HE）染色法考察各组大鼠肝组织病理变化；采用透射电子显微镜（TEM）观察各组大鼠肝细胞线粒体结构；采用Western blotting和qRT-PCR考察各组大鼠肝组织自噬相关蛋白Beclin1、微管相关蛋白1轻链3-II（microtubule associated protein 1 light chain 3-II，LC3-II）、自噬相关基因-7（autophagy-related gene-7，ATG-7）、p62蛋白和mRNA表达情况。采用H₂O₂诱导大鼠肝细胞BRL建立氧化损伤细胞模型，设置对照组、模型组和川陈皮素（10 μmol/L）组，采用Western blotting和qRT-PCR考察各组细胞Beclin1、LC3-II、ATG-7、p62蛋白和mRNA表达情况；采用MitoTracker染色和免疫荧光法考察各组细胞自噬蛋白与线粒体的共定位情况；考察自噬抑制剂和诱导剂对川陈皮素细胞保护作用的影响。结果 与对照组比较，模型组大鼠血清ALT和AST活性均显著升高（P<0.05），肝组织病变，肝细胞线粒体数目增加，结构错乱，受损的线粒体被吞噬，肝组织中LC3-II、Beclin1、ATG-7蛋白和mRNA表达水平显著升高（P<0.05），p62蛋白和mRNA表达水平显著降低（P<0.05）；与模型组比较，川陈皮素组大鼠血清ALT和AST活性明显降低（P<0.05），肝组织病变程度减轻，线粒体形态变化得到明显改善，肝组织中LC3-II、Beclin1、ATG-7蛋白和mRNA表达水平显著降低（P<0.05），p62蛋白和mRNA表达水平显著升高（P<0.05）。氧化损伤细胞模型中BRL细胞LC3-II、Beclin1、ATG-7蛋白和mRNA表达水平显著升高（P<0.05），p62蛋白和mRNA表达水平显著降低（P<0.05），Beclin1和MitoTracker的共定位数目增加；与模型组比较，川陈皮素组BRL细胞中LC3-II、Beclin1、ATG-7蛋白和mRNA表达水平显著降低（P<0.05），p62蛋白和mRNA表达水平显著升高（P<0.05），Beclin1和MitoTracker的共定位数目减少；自噬抑制剂3-MA（1、5 mmol/L）显著增强川陈皮素对BRL细胞的保护作用（P<0.05），自噬诱导剂雷帕霉素显著抑制川陈皮素对BRL细胞Beclin1蛋白和mRNA表达水平的下调作用（P<0.05）。结论 川陈皮素对HIRI具有较好的防治作用，其机制可能与抑制线粒体自噬有关。

Objective To explore the effect and mechanism of nobiletin on preventing and treating hepatic ischemia reperfusion injury (HIRI). Methods SD rats were randomly divided into control group, model group, and nobiletin (10 mg/kg) group. Activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum of rats in each group were detected by ELISA; Hematoxylin-eosin (HE) staining method was used to observe the pathological changes of liver tissue in rats; Transmission electron microscope (TEM) was used to observe the mitochondrial structure of hepatocytes in rats; Western blotting and qRT-PCR were used to investigate protein and mRNA expressions of autophagy-related protein Beclin1, microtubule associated protein 1 light chain 3-II (LC3-II) and autophagy-related gene-7 (ATG-7) and p62. H₂O₂ was used to induce BRL cells to establish oxidative damage cell models. Control group, model group and nobiletin (10 μmol/L) group were set up, Western blotting and qRT-PCR were used to investigate the protein and mRNA expressions of Beclin1, LC3-II, ATG-7 and p62 in BRL cells; MitoTracker staining and immunofluorescence were used to investigate the co-localization of autophagy proteins and mitochondria in BRL cells of each group; Effects of autophagy inhibitors and inducers on protective effect of cells of nobiletin was investigated. Results Compared with control group, activities of ALT and AST in serum of rats in model group were significantly increased (P < 0.05), liver tissue was diseased, number of hepatocyte mitochondria was increased and structure was disordered, the damaged mitochondria were phagocytosed, protein and mRNA expressions of LC3-II, Beclin1, ATG-7 were significantly increased (P < 0.05), p62 protein and mRNA expressions were significantly reduced (P < 0.05); Compared with model group, activities of ALT and AST in serum of rats in nobiletin group was significantly reduced (P < 0.05), liver tissue lesions were reduced, mitochondrial morphological changes were improved, protein and mRNA expressions of LC3-II, Beclin1, ATG-7 in liver tissue were significantly reduced (P < 0.05), p62 protein and mRNA expressions were significantly increased (P < 0.05). In oxidative damage cells model, protein and mRNA expressions of LC3-II, Beclin1, ATG-7 in BRL cells were significantly increased (P < 0.05), p62 protein and mRNA expressions were significantly reduced (P < 0.05), Co-localization numbers of Beclin1 and MitoTracker were increased; Compared with model group, protein and mRNA expressions of LC3-II, Beclin1, ATG-7 in BRL cells of nobiletin group were significantly reduced (P < 0.05), p62 protein and mRNA expressions were significantly increased (P < 0.05), co-localizations numbers of Beclin1 and MitoTracker was reduced; Autophagy inhibitor 3-MA (1, 5 mmol/L) significantly enhanced the protective effect of nobiletin on BRL cells (P < 0.05), autophagy inducer rapamycin significantly inhibited the down-regulation of nobiletin on protein and mRNA expressions of Beclin1 in BRL cells (P < 0.05). Conclusion Nobiletin has a good preventive effect on HIRI, and the mechanism may be related to the inhibition of mitochondrial autophagy.

R285.5

国家自然科学基金资助项目（82003755）