Study On The Protective Mechanism Of Benzomorpholine Compound Q2 On Oxidative Damaged PC12 Cells

Background:Oxidative stress is a phenomenon caused by imbalance in the production and elimination of ROS.As we known,reactive oxygen species and oxidative stress are very harmful to human health.A large number of studies have shown that reactive oxygen species are closely related to the occurrence and development of a variety of diseases,such as neurodegenerative diseases,heart system diseases,and cancer.ROS regulates the survival and death of cells at multiple levels.Apoptosis,oxidative stress and autophagy are triggered mainly through the oxidation of intracellular substances.Nrf2-ARE signaling pathway has been widely studied as the main anti-oxidation pathway in cells.Nrf2 is a key transcription factor of intracellular antioxidant and phase II detoxifying enzymes,and plays an important role in maintaining redox homeostasis of cells.Therefore,the development of Nrf2 activators is particularly important for protecting cells from oxidative damage.Objective:In this study,the oxidative damage of PC12 cells induced by H₂O₂ was used as an oxidative stress model to study the effect of benzomorpholine compound Q2 on oxidative damaged cells and to explore the potential mechanism of its antioxidant effect.Methods:The survival rate of H₂O₂ on PC12 cells was measured by MTT method,and the appropriate concentration was selected to establish the oxidative damage model.MTT assay was used to measure the cytotoxicity of Q2 on PC12 cells,and then the cell viability effect of different concentrations of Q2 on oxidative damaged cells was examined.LDH cytotoxicity assay kit was used to detect the effect of Q2 on the release of LDH from oxidative damaged cells.The morphological change of the oxidative damaged cell nucleus treated by Q2 were observed by inverted fluorescence microscope after the staining of hoechst33342.The effects of different concentrations of Q2 on apoptosis,autophagy,mitochondrial membrane potential and ROS in oxidative injured cells were detected by flow cytometry?FCM?with FITC annexin V and PI,MDC,JC-1,DHE fluorescence staining,respectively.The effect of 3-MA on the survival rate of oxidized cells was measured by MTT method.The effects of Q2 on antioxidant related protein?HO-1,NQO1,Nrf2,p-Nrf2?S40??were detected in PC12 cells by western blot assay.The effects of Q2 on apoptosis related protein?Casepase-3,Casepase-8,Casepase-9,Bax,Cytochrome C,Bcl-2?,autophagy associated protein?Beclin-1,LC3,p62?,antioxidant related protein?HO-1,NQO1,Nrf2,p-Nrf2?S40?,Keap1?were detected by western blot assay,and the nucleoprotein of Nrf2,p-Nrf2?S40?was examined by the extraction of nucleoprotein in oxidative damaged PC12 cells treated by H₂O₂.Real time-PCR was used to detect the effect of Q2 on the expression of Nrf2 mRNA in PC12 cells.Total SOD activity assay kit,lipid oxidation?MDA?kit and GSH and GSSH kit were used to detect the effects of Q2on SOD enzyme activity,MDA and GSH content in oxidative damaged cells.Results:The survival rate of PC12 cells treated by H₂O₂ found that 400?M H₂O₂ was the best concentration for establishing oxidative damage model.The results of MTT revealed that Q2 could promote the proliferation of PC12 cells,being non-toxic to the cells,and increase the survival rate of PC12cells after oxidative injury.Besides,Q2 could reduce the amount of LDH released by oxidative damaged cells,and the release of LDH from 0.001 and 0.01?M Q2 group was the lowest.The results of nuclear staining indicated that Q2 could protect the nucleus from oxidative damage.The results of flow cytometry showed that Q2 could decrease the apoptosis rate of oxidative damaged cells and inhibit the decrease of mitochondrial membrane potential.Among them,0.001,0.01,0.1?M Q2groups had the best inhibitory effect on cell apoptosis and significantly inhibited the decrease of mitochondrial membrane potential.Western blot assay sugested that Q2 could inhibition of Casepase-3,Casepase-8,Casepase-9 activation and up-regulate the expression of anti-apoptotic gene Bcl-2and inhibit the expression of pro-apoptotic gene Bax,Cytochrome C.MDC staining results showed that Q2 could inhibit the autophagy induced by H₂O₂ in PC12 cells. 3-MA could increase the survival rate of cells treated with 200?M and 400?M H₂O₂.This meaned H₂O₂-induced autophagy promoted cell death,while Q2 reduced the cell death by inhibiting this autophagy and the inhibitory effect at low concentration is better than that at high concentration..The results of western blot showed that Q2 could inhibit the expression of autophagy protein beclin1,LC3-II and up-regulate the expression of autophagy inhibitor protein p62.Q2 could inhibit the increase of intracellular ROS content induced by H₂O₂,enhance the enzyme activity of SOD,augment the content of intracellular GSH,decrease the release of MDA,and reduce the oxidative damage of cells.At protein level,the expression levels of antioxidant proteins HO-1,NQO1,Nrf2,p-Nrf2?S40?in PC12 cells were up-regulated by Q2.After oxidative damage induced by H₂O₂,Q2 could also increase the expression of these proteins and depress the expression of Keap1.The detection of nucleoprotein showed that Q2 could promote the translocation of Nrf2,p-Nrf2?S40?protein into the nucleus.Q2 could up-regulate the expression of Nrf2 gene mRNA.Conclusions:Benzomorpholine compound Q2 can significantly protect PC12 cells from oxidative damage.The mechanism for the protection of Q2 may be mainly by reducing ROS content,stabilizing mitochondrial membrane potential and then inhibiting apoptosis,inhibit autophagy,thus increasing the cell survival rate.At the same time,Q2 activates the antioxidant protein Nrf2 and then play an anti-oxidative role by activating the transcription of antioxidant enzymes and phase II detoxifying enzymes in cells and ultimately maintain the redox homeostasis.