| ObjectiveTo investigate the intervention effect of six flavonoids monomers from chrysanthemum on H2O2-induced retinal ARPE-19 cell injury and the mechanism of the reduction of ARPE-19 cell injury induced by H2O2-induced by acacetin.and to verify the effect of acacetin on the reduction of retinal injury induced by sodium iodate in rats.MethodsIn vitro cultured rat Retina ARPE-19 cells,MTT experiments to detect the proliferation of six flavonoids(acacetin,quercetin,apigenin,luteolin,diosmetin,baicalein)to ARPE-19 cells,MTT experiment to detect the effects of six flavonoids on H2O2 induced ARPE-19 apoptosis,FRAP method to detect the total antioxidant capacity of six flavonoids,Flow cytometry was used to detect the effects of acacetin on SOD.ROS and apoptosis of arpe-19 cells induced by H2O2.QPCR detected the expression of ARPE-19 cell oxidative stress and apoptosis pathway induced by H2O2,and Western Bot detected the expression of ARPE-19 cell apoptosis and oxidative stress-related protein induced by H2O2.Rat model of acute retinal injury induced by sodium iodate was established in vivo.SD rats were randomly divided into 5 groups:normal group,saline group,model group,0.2um/ml acacetin group and 0.4um/ml acacetin group.Three days before the modeling,the right eye was injected with the corresponding concentration of acacetin and 0.9%normal saline via vitreous cavity puncture.Three days later,the rats in the saline group,model group.0.2um/ml acacetin group and 0.4um/ml acacetin group were injected with 40mg/ml sodium iodate(NaIO3)1ml via sublingual vein.After 7 days,the rats were killed and the right eye tissue samples were collected.Retinal tissue changes were detected by RPE staining,HE staining and TUNEL staining.ResultsMTT test showed that:six flavonoids monomers can promote the proliferation of human ARPE-19 cells;acacetin,quercetin and luteolin had anti-apoptotic effects on H2O2-induced ARPE-19 cells,and that the combination of quercetin and acacetin could enhance the anti-apoptotic effects.The total antioxidant capacity of quercetin.baicalin and luteolin was higher than that of the control group,and the difference was statistically significant.SOD activity in the monomer groups was higher than that in the control group(P<0.05),and the difference was statistically significant.The activity of SOD decreased with the dose increasing.The ROS production of quercetin,baicalein,luteolin,acacetin and apigenin groups was higher than that of the control group(P<0.05),and the difference was statistically significant.The ROS production of different concentrations of acacetin decreased with the increase of dose.Flow cytometry was used to detect the apoptosis of ARPE-19 cells induced by H2O2 after treatment with acacetin,and the apoptosis was minimal at 1uM concentration.QPCR detection showed that the expressions of cleaved caspase-3,PEDF,and VEGF decreased after treatment with acacetin,Nrf2 expression increased in a dose-dependent manner.Western Blot analysis showed that BAX expression decreased after pretreatment with acacetin,and bcl-2,bcl-2/BAX ratio,caspase 9 and Nrf2 increased in a dose-dependent manner.Nuclear apoptosis and fusion were observed in the model group,the saline group and the acacetin group,with structural disorder,increased intercellular space and smaller nuclear dissolution in the acacetin group.HE staining showed that the structure of model group,the saline group and the acacetin group was disordered,and the arrangement of cell nuclei in the outer nuclear layer and the inner nuclear layer was irregular.Compared with the model group,the changes were obvious,and the arrangement of cell nuclei in the acacetin group was orderly.After TUNEL staining,it was found that the number of apoptotic cells increased significantly in the saline group and the model group,and further decreased in the acacetin group compared with the model group.Conclusion1.Acacetin,quercetin,apigenin,luteolin,diosmetin,and baicalein have antioxidant effects.Acacetin,quercetin and luteolin have anti-apoptotic effects.These six monomers can protect the oxidative damage of arpe-19 cells induced by H2O2,with the strongest effect of acacetin.2.Acacetin can reduce the oxidative stress response induced by H2O2 stimulation of ARPE-19 cells,repair the damaged ARPE-19 cells under oxidative stress,reduce the apoptosis induced by H202 stimulation of ARPE-19 cells,inhibit the expression of ARPE-19 cell apoptosis pathway related factors,and maintain cell homeostasis.Acacetin can protect the oxidative damage of ARPE-19 cells induced by H2O2 stimulation by anti-oxidation and anti-apoptosis.3.As an antioxidant,acacetin can inhibit the secretion of vascular growth factor.4.Acacetin can reduce the necrosis of RPE cells induced by NaIO3,reduce the apoptosis of RPE,and significantly protect the morphological changes after acute retinal injury. |
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