The Protective Effect And Mechanism Of Nrf2 Pathway On Oxidative Stress Injury Induced By H 2 O 2 Were Regulated By Keqi Capsule

Objective:Through in vitro experiments to investigate whether Kelong capsule has protective effect on oxidative stress injury induced by hydrogen peroxide,and to elucidate the role and mechanism of Nrf2 signaling pathway in this process.Method:Cultured PC-3 cells in vitro,Oxidative stress injury model induced by H2O2 in PC-3 cells.In the preliminary experiment,we used the MTT assay to determine the activity of cells and the time of drug action in each group.PC-3 cells were cultured in 37 ℃.50-%CO2 and saturated humidity cell incubator.When the cell density is about 80%cells were seeded into 4 cell culture flasks for cell grouping,Divided into normal control group(A group),oxidative stress model group(100 μmol·L-1 H2O2 intervention 2h.B group),tBHQ group(50 μmol·L-1 tBHQ intervention 24h+ 100μmol·L-1 H2O2 intervention 2h,C group),KLC group(KLC 100μg/ml intervention 24h+ 100μmol·L-1 H2O2 intervention 2h,D group).According to the above grouping,the cells were collected for the next experiment.Using DCFH-DA as a fluorescent probe,detection the content of intracellular ROS by flow cytometry,to evaluate the level of intracellular oxidative stress.Glutathione(GSH)were measured by chemical coloration method.The cells were collected and the total protein was extracted,Nrf2 signaling pathway related proteins,such as Nrf2,COX-2,HO-1,NQO1 was detected by Western blot analysis.The total RNA was extracted from the cells of each group,detected the expression of Nrf2,COX-2,HO-1 and NQO1 mRNA of each group by RT-PCR assay.Results:KLC pretreatment significantly decreased ROS levels and increased intracellular GSH content.Additionally,KLC pretreatment obviously increased the expression of Nrf2 signaling pathway related protein and mRNA such as Nrf2,COX-2,HO-1 and NQO1.Conclusion:KLC can effectively improve the environment of oxidative stress in PC-3 cells,activation of Nrf2 signaling pathway,and increase the expression of Nrf2 related antioxidant genes HO-1,NQO1,so as to play a role in antioxidant stress.This study proposes the antioxidant mechanism of Kelong capsule from Nrf2 signal pathway perspective,provides a new theoretical basis for the clinical application of Kelong capsule.