保健食品中非法添加物二氢吡啶类化合物诱导的肝细胞毒性与作用机制研究

Hepatocyte toxicity and mechanisms induced by illegal dihydropyridine additives in health foods

中文关键词:  二氢吡啶类化合物  尼莫地平  丝裂原活化蛋白激酶  自噬

英文关键词:dihydropyridines  nimodipine  mitogen-activated protein kinase  autophagy

中文摘要:

      目的 探究尼莫地平所致肝细胞毒性及分子机制并研究保健食品中非法添加物二氢吡啶类化合物对自噬通路的影响。方法 以不同浓度(0、3、10、30、100 μmol/L)的尼莫地平作用于人肝癌细胞HepG2 24 h, 测定肝损伤指标谷丙转氨酶(alanine amino transferase, ALT)和谷草转氨酶(aspartate amino transferase, AST)活力, 并利用Western blot检测细胞中丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)、氧化应激通路、凋亡蛋白、自噬相关通路蛋白的表达; 以不同浓度(0、3、10、30、100 μmol/L)的氯维地平、硝苯地平、尼群地平、西尼地平、阿雷地平及马尼地平作用HepG2细胞24 h, 利用Western blot检测细胞中自噬相关通路蛋白的表达。结果 100 μmol/L的尼莫地平可上调ALT和AST活力、激活MAPK通路蛋白的表达、抑制核因子E2相关因子2 (nuclear factor erythroid 2-related factor 2, Nrf2)及下游解毒蛋白血红素加氧酶1 (heme oxygenase-1, HO-1)的表达、增加微管相关蛋白1轻链3 (microtubule-associated protein 1 light chain 3, LC3)Ⅱ/LC3Ⅰ、降低选择性自噬接头蛋白(sequestosome 1, SQSTM1)表达; 氯维地平在3 μmol/L时引起细胞自噬, 其他6种二氢吡啶类化合物均在100 μmol/L时使细胞发生自噬。结论 尼莫地平所致的肝细胞毒性机制中自噬的发生可能是由MAPK通路介导的, 其中细胞外信号调节激酶(extracellular signal-regulated kinase, ERK)通路促进HepG2细胞自噬的作用相对最强。此外, 其他6种二氢吡啶类化合物氯维地平、硝苯地平、尼群地平、西尼地平、阿雷地平及马尼地平均可引起自噬。

英文摘要:

      Objective To explore the hepatotoxicity and molecular mechanism caused by nimodipine, and study the effect of illegal dihydropyridine additives in health foods on the autophagic pathway. Methods Nimodipine was treated with different concentrations (0, 3, 10, 30 and 100 μmol/L) on human hepatoma cells HepG2 for 24 h. The liver injury indexes alanine amino transferase (ALT) and aspartate amino transferase (AST) viability were measured, and the expressions of proteins related with mitogen-activated protein kinase (MAPK), oxidative stress pathway, apoptosis protein and autophagy-related pathway were analyzed by Western blot. Clevidipine, nifedipine, nifedipine, cilnidipine, aranidipine and manidipine at different concentrations (0, 3, 10, 30 and 100 μmol/L) were used to treat HepG2 for 24 h. The expressions of autophagy-related pathway proteins were analyzed by Western blot. Results Nimodipine at 100 μmol/L up-regulated ALT and AST activity, activated the expressions of MAPK pathway protein, inhibited the expressions of nuclear factor E2-related factor 2 (Nrf2) and downstream detoxification protein heme oxygenase 1 (HO-1), increased microtubule-associated protein 1 light chain 3 (LC3)II/LC3Ⅰ, and decreased the expressions of sequestosome 1 (SQSTM1); clevidipine caused autophagy at 3 μmol/L, and the other 6 kinds of dihydropyridines caused autophagy at 100 μmol/L. Conclusion The onset of autophagy in the mechanism of nimodipine-induced hepatocytotoxicity might be mediated by the MAPK pathway, with the extracellular signal-regulated kinase (ERK) pathway promoting autophagy in HepG2 cells relatively most strongly. In addition, other 6 kinds of dihydropyridines, clevidipine, nifedipine, nifedipine, cilnidipine, aranidipine and manidipine, can cause autophagy.