异鼠李素对人晶状体上皮细胞(HLE-B3)氧化损伤的保护作用及其机制OA

Objective To explore the protective effect of isorhamnetin(ISO)on oxidative damage in human lens ep-ithelial cells(HLE-B3)and its underlying mechanism.Methods HLE-B3 cells were randomly divided into 6 groups:the control group(received no treatment);the H2O2 group(treated with 300 μmol·L-1 H2O2 alone for 24 hours);the ISO-L,ISO-M,and ISO-H groups(pretreated with 25.0,50.0,and 100.0 μmol·L-1 ISO for 24 hours,respectively,and then cultured in the medium containing 300 μmol·L-1 H2O2 for 24 hours);the ISO-H+ML385 group(pretreated with 100.0 μmol·L-1 ISO together with 2.0 μmol·L-1 nuclear factor E2-related factor 2(Nrf2)inhibitor ML385 for 24 hours and then cultured in the medium containing 300 μmol·L-1 H2O2 for 24 hours.The morphology of cells in each group was observed.The MTT as-say and TUNEL assay were used to detect cell proliferation and apoptosis,respectively.The enzyme-linked immunosorbent assay was used to detect glutathione peroxidase(GSH-Px),superoxide dismutase(SOD),and catalase(CAT)levels.The DCFH-DA probe method was used to detect the level of reactive oxygen species(ROS).Western blot was used to detect the relative protein expression of Nrf2,Keap 1,heme oxygenase-1(HO-1),caspase-3,apoptosis-related B cell lymphoma 2(Bcl-2),and Bcl-2-associated X protein(Bax).Results Microscopic observations showed that H2O2 induced typical morpho-logical damage in HLE-B3 cells,characterized by a reduced cell number,cell shrinkage,and widened intercellular spaces.In contrast,pretreatment with different concentrations of ISO ameliorated the cell morphology in a concentration-dependent manner,increasing cell density and promoting a normal appearance.At the molecular level,compared with the control group,the apoptosis rate and the expression of the pro-apoptotic proteins Bax and Caspase-3 significantly increased in the H2O2 group,while the expression of the anti-apoptotic protein Bcl-2 decreased(all P＜0.05).However,ISO pretreatment reversed these apoptosis-related indicators in a concentration-dependent manner.In addition,the activities of SOD,GSH-Px,and CAT significantly decreased,and the ROS level increased in the H2O2 group(all P＜0.05),whereas ISO treatment re-stored antioxidant enzyme activities and reduced ROS levels in a concentration-dependent manner.Further mechanism stud-ies indicated that H2O2 inhibited the activity of the Nrf2/HO-1 pathway,as evidenced by the downregulation of Nrf2 and HO-1 expression and the upregulation of Keap1 expression(all P＜0.05),while ISO activated this pathway in a concentration-dependent manner.Furthermore,after the addition of the Nrf2 inhibitor ML385,the activation effect of ISO on the Nrf2/HO-1 pathway and its cytoprotective effect were significantly reversed(all P＜0.05).Conclusion ISO activates the Nrf2/HO-1 pathway,upregulates the protein expression of Nrf2 and HO-1 in HLE-B3 cells,and downregulates the expression of its negative regulator Keap1,thereby inhibiting oxidative stress and mitigating H2O2-induced HLE-B3 cell injury.