Research About Protective Effect Of NBP On Oxidative Stress And Apoptosis Of Retina Induced By Hydrogen Peroxide

ObjectiveMany ophthalmic diseases involve oxidative stress injury,such as diabetic retinopathy,glaucoma,central retinal artery occlusion,etc.The diagnosis and treatment of these diseases are complex,so the exploration of the disease mechanism is of great importance,and it is also to find the target of treatment for early intervention and treatment.Butyl phthalide（DL-3-n-butylphthalide,NBP）is a drug discoveried by our own country,most used for ischemic stroke caused by injury,many studies suggest that the NBP protective cells in ischemic anoxia,cell apoptosis,we introduce the NBP in ophthalmology,explore the protective effect of the NBP on retinal Müller cells induced by hydrogen peroxide oxidative stress damage,which provides a new thought for our future diagnosis and treatment.Methods1.A total of female SD rats were divided into three groups:control group,NBP group and PBS group.One week before modeling,NBP group was intraperitoneally injected（concentration dose:80mg/kg）,and PBS group was intraperitoneally injected（dose:80mg/kg）.All groups were subjected to anterior chamber irrigation and pressurization to build an acute ocular hypertension mouse model,and paraffin sections were prepared after modeling.Nrf2+GFAP+DAPI and HO-1+GFAP+DAPI were stained with HE and immunofluorescence to observe the expressions of Nrf2and HO-1 in the retina and retinal cells of the experimental animals.2.In this study,human Müller cell line（mio-m1）was selected,and the experimental concentrations of H₂O₂ and butylphthalide were determined after experimental exploration,and were divided into the following three groups:normal control group（group C）,H₂O₂ model stimulation group（200 mol/L,M group）,and NBP treatment group（H₂O₂ 200 mol/L+NBP 1 g/ml,N group）.3.Müller cell lines were identified by GFAP and GS cell immunofluorescence staining.Cell morphology was observed by HE staining.The activity of Müller cells in each group was detected by MTT assay.Cell viability was determined by Calcein AM staining.The expression levels of reactive oxygen species（ROS）in ER cells were observed by ER-Tracker Red staining with DCFDA+ER.The cell mitochondrial membrane potential was detected with jc-1 kit,and the Müller cell membrane potential of each group was observed.Hoechst33258 staining was used to observe the changes of early apoptosis in each group.The fluorescence expression of Nrf2 and HO-1 cells was observed by immunofluorescence staining,and the expression levels of Nrf2 and HO-1 proteins in each group were recorded by Western Blot.4.Univariate anova and Dunnett statistical method were used for data analysis.Results1.The eye and retinal conditions of the rats were observed by HE staining,and no retinal detachment was observed.Compared with the control group and PBS group,the expression of Nrf2 and HO-1 in the AOH animal model and Müller cells in NBP group were up-regulate,and the results were statistically significant.2.MTT method was used to detect the growth activity and viability of Müller cells.According to the data analysis at 24h,48h and 72h,H₂O₂ combined with NBP could effectively improve the growth activity and viability of Müller cells.Calcein AM staining showed that NBP could effectively improve the survival and growth force of Müller cells under the action of H₂O₂.Compared with N group,P=0.0092 in M group,and the difference was statistically significant.In this study,it was believed that the addition of NBP,when stimulated by oxidative stress,could effectively reduce the damage and apoptosis of Müller cells and further increase the ability of cells to resist oxidative stress.3.Results of Hoechst33258 staining of Müller cells:Compared with group M,P₁=0.0015 in group C,and the difference was statistically significant.Compared with N group,P₂=0.0359 in M group,and the difference was statistically significant. Compared with N group,P<0.0001 in group C,the difference was statistically significant.Oxidative stress stimulation with H₂O₂ and NBP can effectively improve the ability of Müller cells to resist oxidative stress,reduce apoptosis,and further improve the growth activity and growth force of Müller cells.4.JC-1 staining suggested that,according to the analysis of changes in mitochondrial membrane potential fluorescence intensity,the oxidative stress stimulation with H₂O₂ and the administration of NBP could effectively improve the ability of Müller cells to resist oxidative stress.Aggregation result:Compared with group M,P₁=0.0015 in group C,and the difference was statistically significant.Compared with group N,P₂=0.0103 in group M,and the difference was statistically significant.Compared with group N,P=0.0308 in group C,the difference was statistically significant.Monomer result:Compared with group M,P₁=0.0002 in group C,and the difference was statistically significant.Compared with N group,P₂=0.0296 in M group,and the difference was statistically significant.Compared with group N,P=0.0014 in group C,the difference was statistically significant.5.DCFDA+ER-tracker Red staining confirmed that H₂O₂ stimulation could lead to the increase of ROS expression level in Müller cells,and the results were statistically significant.The effect of NBP could reduce the expression of ROS.Results:Compared with group M,P₁=0.0020 in group C,and the difference was statistically significant.Compared with N group,P₂=0.0272 in M group,and the difference was statistically significant.Compared with N group,P<0.0049 in group C,the difference was statistically significant.Oxidative stress stimulation with H₂O₂ combined with NBP can reduce ROS accumulation in Müller cells and reduce the damage caused by ROS accumulation.6.Cell immunofluorescence staining showed that the fluorescence staining intensity of Nrf2 in the NBP group was significantly enhanced compared with that in the control group,suggesting that NBP could significantly enhance the expression of Nrf2.Results:Compared with group M,P₁=0.0117 in group C,and the difference was statistically significant.Compared with group N,P₂=0.0491 in group M,and the difference was statistically significant.Compared with N group,P=0.1607,and the difference was not statistically significant.Oxidative stress stimulation with H₂O₂ combined with NBP increased Nrf2 expression in Müller cell nucleus,indicating nuclear translocation of Nrf2.7.Cell immunofluorescence staining showed that the fluorescence staining intensity of HO-1 in the NBP group was significantly enhanced compared with that in the control group,suggesting that NBP could significantly enhance the expression of HO-1.Results:Compared with group M,P₁=0.0026 in group C,and the difference was statistically significant.Compared with N group,P₂=0.0022 in M group,and the difference was statistically significant.Compared with group N,P=0.0652 in group C,the difference was not statistically significant.The expression of HO-1 in Müller cells was increased by oxidative stress stimulation with H₂O₂ and NBP.8.The results of Western blotting showed that the expression of HO-1 in the NBP group was higher than that in the H₂O₂ stimulation group,and showed a time-dependent change.The longer the drug was used,the more significant the increase of HO-1 expression.Translocation of Nrf2 in the nucleus could be observed after 2h of H₂O₂ treatment,and increased expression of Nrf2 in the nucleus could be observed after 48h.Conclusion1.NBP can effectively improve the biological behavior of Müller cells caused by H₂O₂.2.NBP can increase the nuclear translocation of Nrf2 in the retina of rats with acute ocular hypertension.3.The protective effect of NBP on Müller cells induced by H₂O₂ may be related to Nrf2 pathway.