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目的探讨褪黑素(MT)对亚油酸(LA)引起Hep G2细胞胰岛素信号缺陷的修复作用及其机制。方法将培养的Hep G2细胞分为对照组(BSA组)、亚油酸组(LA组)、亚油酸+褪黑素组(LA+MT组)、亚油酸+维生素C组(LA+Vit C组)。BSA组予以10%牛血清白蛋白处理24 h;LA组予以0.5 mmol·L-1亚油酸处理24 h,LA+MT组予以10μmol·L-1褪黑素和0.5 mmol·L-1亚油酸处理24 h;LA+Vit C组予以1 mmol·L-1维生素C和0.5 mmol·L-1亚油酸处理24 h。用荧光比色法测定各组细胞胞内氧自由基水平,用Western印迹法检测胰岛素刺激后胞内胰岛素信号蛋白磷酸化水平,包括蛋白激酶B、叉头转录因子以及c-Jun氨基末端激酶。结果 LA组的氧自由基含量明显高于BSA组(P<0.05);LA+MT组的氧自由基含量明显低于LA组(P<0.05);LA+MT组与BSA组相比,氧自由基含量差异无统计学意义(P>0.05)。LA+Vit C组与LA组相比,氧自由基的含量减少,但仍高于LA+MT组,差异有统计学意义(P<0.05)。LA组与BSA组相比,蛋白激酶B、叉头转录因子磷酸化水平减少,而c-Jun氨基末端激酶磷酸化水平升高;LA+MT组与LA组相比,蛋白激酶B、叉头转录因子磷酸化水平显著升高,c-Jun氨基末端激酶磷酸化水平明显降低,组间比较差异有统计学意义(P<0.05)。结论亚油酸可导致细胞内氧自由基增加,褪黑素能够清除亚油酸产生的过量氧自由基,减少c-Jun氨基末端激酶的活化,修复胰岛素下游信号损伤。
Objective To investigate the effect and mechanism of melatonin (MT) on the insulin signal defect in Hep G2 cells induced by linoleic acid (LA). Methods Hep G2 cells were divided into control group (BSA group), linoleic acid group (LA group), linoleic acid + melatonin group (LA + MT group), linoleic acid + vitamin C group (LA + Vit C group). The BSA group was treated with 10% bovine serum albumin for 24 h, the LA group was treated with 0.5 mmol·L-1 linoleic acid for 24 h, the LA + MT group was treated with 10 μmol·L -1 melatonin and 0.5 mmol·L -1 subunit Oleic acid for 24 h. LA + Vit C group was treated with 1 mmol·L-1 vitamin C and 0.5 mmol·L-1 linoleic acid for 24 h. The level of intracellular oxygen free radicals in each group was measured by fluorescent colorimetric assay. The level of intracellular insulin signaling protein phosphorylation was detected by Western blotting, including protein kinase B, forkhead transcription factor and c-Jun N-terminal kinase. Results The content of oxygen free radicals in LA group was significantly higher than that in BSA group (P <0.05). The content of oxygen free radical in LA + MT group was significantly lower than that in LA group (P <0.05) Free radical content difference was not statistically significant (P> 0.05). The content of oxygen free radicals in LA + Vit C group was lower than that in LA group, but still higher than that in LA + MT group (P <0.05). LA group compared with BSA group, protein kinase B, forkhead transcription factor phosphorylation decreased, while the c-Jun amino terminal kinase phosphorylation level increased; LA + MT group compared with the LA group, protein kinase B, fork Phosphorylation of transcription factor was significantly increased, phosphorylation of c-Jun N-terminal kinase was significantly decreased, there was significant difference between the two groups (P <0.05). CONCLUSION: Linoleic acid induces an increase in intracellular free radicals. Melatonin clears excessive oxygen free radicals produced by linoleic acid, decreases activation of c-Jun N-terminal kinase, and restores signal damage downstream of insulin.