Copper Nanoparticles Induced Oxidative Stress In Ovarian Granulosa Cells And The Protective Mechanism Of HO-1

Part 1 Toxic effects of copper nanoparticles on ovarian granulosa cells[Purpose] Phenotypic analysis and verification of granulosa cells before and after exposure to copper nanoparticles(CuNPs)to prove the oxidative stress damage.[Methods] According to our preliminary research results,granulosa cells(COV434)were cultured in vitro,and treated with different concentrations of CuNPs or without any treatment(the control group).Cell samples were obtained at the corresponding time points for subsequent treatment and detection.Cell viability was detected by CCK-8 assay,apoptosis and mitochondrial membrane potential were detected by flow cytometry,and oxidative stress makers were detected by automatic microplate reader.[Results] Compared with the control group,the proliferation activity of COV434 cells decreased after exposure to CuNPs,and decreased with the increase of CuNPs concentration and treatment time.After CuNPs co-incubation with COV434 cells for 12 h,the 50% growth inhibition concentration of CuNPs on COV434 cells was 150 μg/ml;The apoptosis rate increased and mitochondrial membrane potential decreased.Compared with the control group,the oxidation products,such as reactive oxygen species(ROS)and lipid peroxidation products(MDA),were significantly increased in the CuNPs group;The activities of superoxide dismutase(SOD),catalase(CAT),and the content of glutathione peroxidase(GSH)were decreased.[Conclusions] Oxidative stress was involved in the toxic damage of CuNPs on granulosa cells,and the oxidative stress damage induced by CuNPs may be related to mitochondria-dependent apoptosis.Part 2 The potential protective effect of HO-1 on oxidative stress damage induced by copper nanoparticles[Purpose] Based on the results of Part 1,we found that CuNPs induced oxidative stress damage in COV434 cells.The purpose of this study was to preliminarily explore the expression of HO-1 related protein pathway before and after CuNPs exposure,and verify the protective effect of HO-1 on granulosa cells by activating the antioxidant defense system in the CuNPs-induced damage model.[Methods] The CuNPs exposure model of COV434 cells was established,treated with CuNPs or without any treatment(the control group),and protein was extracted from cell samples at corresponding time points.The protein expressions of MAPK14,phosphorylated MAPK14(p-MAPK14),Nrf2,Keap1,HO-1 and NQO1 were detected by Western blot.HO-1 intervention model was established by pretreating COV434 cells with heme(hemin)and inhibiting HO-1 activity with zinc protoporphyrin(Znpp).Oxidation-stress related markers before and after exposure to CuNPs in these model were detected.[Results] Compared with the control group,HO-1 protein expression was significantly increased,Nrf2 protein expression was increased,and Keap1 protein expression was significantly decreased after CuNPs treatment;There was no significant difference in the expression of NQO1 protein before and after CuNPs treatment.HO-1 protein expression showed a dynamic change.HO-1 protein expression began to increase significantly at 12 h,continued to increase within 24 h,and decreased sharply at 30 h.Compared with the control group,the expression of p-MAPK14 protein increased after CuNPs treatment,which began to increase at 6h and continued to increase at 9h;There was no significant difference in the expression of MAPK14 protein.In cells exposed to CuNPs,ROS production was significantly decreased,GSH level was significantly increased and SOD2 protein expression was upregulated in hemin pretreated cells compared with unpretreated cells.Similarly,in cells exposed to CuNPs,ROS production was significantly increased in Znpp pretreated cells compared with unpretreated cells.[Conclusions] After COV434 cells were exposed to CuNPs,HO-1 protein expression was increased and MAPK14/Nrf2 signaling pathway was activated.These results showed that the high expression of HO-1 protein may be mediated by the activation of MAPK14/Nrf2 signaling pathway.HO-1 intervention experiments in vitro showed that HO-1 high expression plays an important role in the anti-oxidative damage in the CuNPs-induced damage of COV434 cells.