姜黄素对INS-1细胞氧化应激和胰岛素分泌的影响及新机制的初步研究

Pilot study on the mechanism and effection of curcumin on oxidative stress and insulin secretion in INS-1 cells

目的采用H2O2制备INS-1氧化应激模型,观察姜黄素对INS-1细胞氧化应激水平及胰岛素分泌的影响及瓣状核酸内切酶(FEN1)和ERK1/2磷酸化的改变在其中的作用。方法将INS-1细胞分为阴性对照组、模型组(H2O2处理)、干预组(5、7、10μM姜黄素+H2O2处理)。采用CCK-8检测细胞增殖,分光光度计法检测细胞丙二醛(MDA)、还原型谷胱甘肽(GSH)水平,酶标仪检测细胞内活性氧(ROS)水平的变化,放射免疫法检测细胞上清液中胰岛素分泌量,Western blot检测FEN1,p-ERK1/2蛋白表达的改变。结果 (1)模型组MDA、ROS值较对照组明显升高(P<0.05);干预组MDA、ROS较模型组明显降低(P<0.05);模型组GSH较对照组明显降低(P<0.05);干预组GSH较模型组显著升高(P<0.05)。(2)模型组胰岛素分泌水平较对照组明显降低(P<0.05),10μM姜黄素干预组胰岛素分泌水平较模型组明显升高(P<0.05)。(3)与模型组比较,10μM姜黄素干预显著提高INS-1细胞FEN1蛋白的表达和ERK1/2的磷酸化,ERK1/2特异性抑制剂U0126可抑制姜黄素引起INS-1细胞FEN1蛋白表达的改变。结论姜黄素可以通过pERK1/2-FEN1通路改善INS-1细胞的氧化应激状态,以及胰岛素分泌,为姜黄素治疗糖尿病提供新的作用机制。

Objective To observe the effections of curcumin on the oxidative stress and glucose- stimulated insulin secretion （GSIS） and the changes of flap endonuclease 1 （FEN1） and phosphorylation extracellular signal-regulated kinasel/2 （pERK1/2） in this process. Methods INS-1 cells were divided into three groups： the negative control group, the model group （treated with H2O2）, and the intervention group （treated with curcumin 5, 7, 10 μM ＋ H2O2）. Proliferation of INS-1 cells was detected through CCK-8 assay. Malondialdehyde （MDA） and glutathione （GSH） levels were detected by spectrophotometer assay. The level of reactive oxygen species （ROS） was observed by enzyme-labeled instrument and insulin secretion stimulated by glucose in the cell supernate was measured by radioirnmunoassay. The changes of FEN1 and p-ERK1/2 protein expressions were detected by Western blot assay. Results （1） MDA and ROS levels of the model group were significantly higher than those of the control group （P〈0. 01） ; MDA and ROS levels of the intervention group were significantly lower than those of the model group （P〈 0. 05） ; GSH level of model group was significantly lower than that of the control group （P〈0. 05） ; GSH level of intervention group was significantly higher than that of the model group （P〈0. 05）. （2） Insulin secretion of the model group was significantly lower than that of the control group （P〈0. 05）, while the insulin secretion of the 10μM curcumin intervention group was significantly higher than that of the model group （P〈0. 05）. （3） Western blot assay showed that the expressions of Fen1 and pERK1/2 proteins in INS-1 cells were upregulated by 10μM curcumin compared with the model group. U0126, as an ERK1/2 specific inhibitor, could inhibit the expression changes of Fen1 protein which was induced by curcumin in INS-1 cells. Conelmion Curcumin can decrease the oxidative stress damage and has a good effection on insulin secretion through the signal pathway of pERK1/2-FEN1 in INS-1 cells, providing a new mechanism of curcumin treatment on diabetes.