本研究探讨了catalpol对H_2O_2诱导的胰腺β细胞(INS-1细胞)的抗氧化和抗凋亡作用及其分子机制。通过不同浓度的H_2O_2刺激不同时间,诱导并优化了INS-1细胞的氧化损伤模型。采用CCK-8法检测catalpol干预(1、5、10、20、40、80和160μmol·L~(-1))24h后的细胞活力。超氧化物歧化酶(SOD),用DCFH-DA荧光探针检测脂质过氧化丙二醛(MDA),分别为WST-1和TBA。此外,AO-EB和AnnexinV-FITC/PI染色检测到凋亡效应。此外,通过Wes-tern印迹检测蛋白质表达水平,通过ELISA测量细胞内胰岛素浓度。结果表明,50μmol·L~(-1)H_2O_2处理2h稳定诱导INS-1细胞氧化损伤模型,1-80μmol·L〜(-1)的catalpol不影响INS-1细胞的细胞活力。与模型组的条件相比,1、5、10μmol·L~(-1)catalpol干预2h可保护INS-1细胞免受氧化损伤(P<0.001),减少ROS和MDA,增加SOD,抑制细胞过度凋亡。此外,1,5,10μmol·L~(-1)catalpol也能上调核转录因子NF-E2相关因子的磷酸化,负调节Kelch样ECH相关蛋白1(Keap1),细胞外信号调节激酶(ERK)的磷酸化,和血红素氧合酶1(HO-1),并促进胰十二指肠同源异型盒因子-1(PDX-1)和葡萄糖转运蛋白2(GLUT2)的蛋白表达。此外,1,5,10μmol·L~(-1)catalpol在高糖培养基中增加氧化损伤下INS-1细胞的胰岛素分泌,提示胰岛β细胞功能恢复。PDX-1是胰岛β细胞功能的关键核转录因子,直接调节GLUT2和胰岛素合成,并影响葡萄糖稳态。总之,catalpol可以减轻INS-1细胞的氧化损伤和凋亡,激活抗氧化途径,保护胰腺β细胞的功能,改善胰岛素的合成和分泌。
The present study investigated the anti-oxidative and anti-apoptotic effects and molecular mechanisms of catalpol on the H_2O_2-induced pancreatic β-cells(INS-1 cells).The oxidative damage model of INS-1 cells was induced and optimized by the stimulation of H_2O_2 of different concentrations for different time.CCK-8 assay was used to detect cell viability after catalpol intervention(1, 5, 10, 20, 40, 80, and 160 μmol·L~(-1)) for 24 h.Intracellular reactive oxygen species(ROS), superoxide dismutase(SOD), and lipid peroxide malondialdehyde(MDA) were measured by DCFH-DA fluorescent probe, WST-1, and TBA respectively.Moreover, the apo-ptotic effect was detected by AO-EB and Annexin V-FITC/PI staining.In addition, the protein expression levels were detected by Wes-tern blot, and intracellular insulin concentration was measured by ELISA.The results showed that the oxidative damage model of INS-1 cells was stably induced by 50 μmol·L~(-1) H_2O_2 treatment for 2 h, and catalpol at 1-80 μmol·L~(-1) did not affect cell viability of INS-1 cells.Compared with the conditions in the model group, 1, 5, and 10 μmol·L~(-1) catalpol intervention for 2 h could protect INS-1 cells from oxidative damage(P<0.001), reduce ROS and MDA, increase SOD, and inhibit excessive cell apoptosis.Moreover, 1, 5, and 10 μmol·L~(-1) catalpol could also up-regulate the phosphorylation of nuclear transcription factor NF-E2 related factors, negatively regulate Kelch-like ECH-associated protein 1(Keap1), phosphorylation of extracellular signal-regulated kinase(ERK), and heme oxyge-nase 1(HO-1), and promote the protein expression of pancreatic-duodenal homeobox factor-1(PDX-1) and glucose transporter 2(GLUT2).In addition, 1, 5, and 10 μmol·L~(-1) catalpol increased insulin secretion of INS-1 cells under oxidative damage in the high-glucose culture medium, indicating function recovery of pancreatic β cells.PDX-1 is a key nuclear transcription factor of pancreatic β cell function that directly regulates GLUT2 and insulin synthesis, and affects glucose homeostasis.In conclusion, catalpol can reduce the oxidative damage and apoptosis of INS-1 cells, activate antioxidant pathway, protect the function of pancreatic β cells, and improve insulin synthesis and secretion.