Xiao Xin, Xu Wen-Hua, Zhang Xiao-Qing, Ding Jun-Feng, Jiang Yue, Tu Jun
Research Center for Differentiation and Development of Chinese Medicine Basic Theory & Jiangxi Province Key Laboratory of Chinese Medicine Etiopathogenisis, Jiangxi University of Chinese Medicine Nanchang 330004, China.
Research Center for Differentiation and Development of Chinese Medicine Basic Theory & Jiangxi Province Key Laboratory of Chinese Medicine Etiopathogenisis, Jiangxi University of Chinese Medicine Nanchang 330004, China Key Laboratory of Traditional Chinese Medicine Pharmacology of Jiangxi Province Nanchang 330004, China.
Zhongguo Zhong Yao Za Zhi. 2022 Aug;47(16):4403-4410. doi: 10.19540/j.cnki.cjcmm.20220119.402.
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.
本研究探讨了梓醇对H₂O₂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H₂O₂刺激不同时间诱导并优化INS-1细胞的氧化损伤模型。采用CCK-8法检测梓醇干预（1、5、10、20、40、80和160 μmol·L⁻¹）24 h后细胞活力。分别用DCFH-DA荧光探针、WST-1和TBA检测细胞内活性氧（ROS）、超氧化物歧化酶（SOD）和脂质过氧化物丙二醛（MDA）。此外，通过AO-EB和Annexin V-FITC/PI染色检测凋亡效应。另外，采用Western blot检测蛋白表达水平，用ELISA检测细胞内胰岛素浓度。结果表明，50 μmol·L⁻¹ H₂O₂处理2 h可稳定诱导INS-1细胞氧化损伤模型，1-80 μmol·L⁻¹梓醇对INS-1细胞活力无影响。与模型组相比，1、5和10 μmol·L⁻¹梓醇干预2 h可保护INS-1细胞免受氧化损伤（P<0.001），降低ROS和MDA水平，提高SOD水平，抑制细胞过度凋亡。此外，1、5和10 μmol·L⁻¹梓醇还可上调核转录因子NF-E2相关因子的磷酸化水平，负向调节Kelch样ECH相关蛋白1（Keap1）、细胞外信号调节激酶（ERK）的磷酸化水平以及血红素加氧酶1（HO-1），并促进胰腺十二指肠同源盒因子-1（PDX-1）和葡萄糖转运蛋白2（GLUT2）的蛋白表达。另外，1、5和10 μmol·L⁻¹梓醇可增加高糖培养基中氧化损伤状态下INS-1细胞的胰岛素分泌，提示胰腺β细胞功能恢复。PDX-1是胰腺β细胞功能的关键核转录因子，直接调节GLUT2和胰岛素合成，影响葡萄糖稳态。综上所述，梓醇可减轻INS-1细胞的氧化损伤和凋亡，激活抗氧化途径，保护胰腺β细胞功能，改善胰岛素合成与分泌。
