The Protective Effect And Mechanism Of Qinpi B On Oxidative Damage Of Human Retinal Pigment Epithelial Cells

Purpose:To study the protective effect of esculetin on oxidative damage of human retinal pigment epithelial cell line(ARPE-19)induced by t-BHP.Material and method:Main mterial and reagents: human retinal pigment epithelium(ARPE)、Esculetin、Dimethyl sulfocide、Tert-butyl hydroperoxide、(American Sigma Aldrich),High glucose medium 、 Fetal bovine serum 、 Phosphate buffer saline(Boehringer Ingelheim),Trypsin(Gibico),MTS Cell Proliferation Assay Kit(Promega,USA),Western and IP cell lysates、BCA protein concentration kit、Total SOD activity detection kit、Lipid oxidation detection kit、Catalase detection kit、Glutathione Peroxidase Detection Kit(Shanghai Biyuntian Biotechnology Co.,Ltd.),Microplate reader(Biotek Gen5),Low temperature desktop centrifuge(Siemens,Germany),Inverted phase contrast fluorescence microscope(Nikon Japan),Cytometer 、 Cell Counting Plate(Wuxi Devin Instrument Co.,Ltd.).Methods : To investigate the effect of Esculetin on the viability of ARPE-19 cells after 24 h of treatment.In the anti-oxidation experiment,Qinpiyidin + t-BHP simultaneously acted on the cells for 4 h.The anti-oxidative effect of Qinpiyidin on ARPE-19 cells was detected by MTS method.Fluorescent staining of reactive oxygen species(ROS)was used to detect the effect of genistein on ROS levels in injured cells;kit was used to detect malondialdehyde(MDA)and superoxide dismutase(SOD)in injured cells using kits.Of catalase(CAT)and glutathione peroxidase(GSH-Px)levels.Results:1.t-BHP has an inhibitory effect on ARPE-19 cells,which can reduce the cell survival rate,and the inhibition rate is positively correlated with the concentration.When the t-BHP concentration was 900 μM,ARPE-19 cell survival rate was 50%.2.Esculetin can promote the proliferation of ARPE-19 cells,and has a positive correlation with concentration.3.The results of the simultaneous administration of the model group showed that the four concentrations of Esculetin treatment group 20μM,40μM,80μM,100μM have protectiveeffects on oxidative damage of ARPE-19 cells,and inhibited t-BHP-induced ARPE-19 Cell death.4.The results of the first model after drug addition showed that compared with the model group,the esculetin treatment group at a concentration of 20 μM,40 μM,80 μM had protective effects on oxidatively damaged ARPE-19 cells.5.Modeling for 4 hours and then administration for 4 hours did not protect the t-BHP-induced ARPE-19 cells from oxidative damage.5.ROS fluorescence intensity test results show that: Esculetin 20μM,40μM,80μM,100μM concentration,concentration-dependent inhibition of ROS accumulation in injured ARPE-19 cells.6.The MDA content test results showed that:Esculetin 20μM,40μM,80μM,100μM concentration,in a concentration-dependent manner can significantly inhibit the level of MDA in injured ARPE-19 cells.7.The results of SOD activity test showed that: Esculetin 20μM,40μM,80μM,100μM concentration,in a concentration-dependent manner can significantly promote SOD activity in injured ARPE-19 cells.8.The results of GXH-Px enzyme activity test showed that: Esculetin 20μM,40μM,80μM,100μM concentration,in a concentration-dependent manner can significantly promote the GXH-Px enzyme activity in injured ARPE-19 cells.9.CAT activity test results showed that: Esculetin 20 μM,40 μM,80 μM,100 μM four groups of concentration,concentration-dependent can significantly promote CAT activity in injured ARPE-19 cells.Conclusion:1.Esculetin pre-administration and simultaneous administration have protective effect on oxidative stress injury of ARPE-19 cells cultured in vitro.2.Esculetin inhibits the oxidative stress injury of ARPE-19 cells,and by reducing the levels of ROS and MDA,it up-regulates the antioxidant enzymes SOD,GXH-Px and CAT to exert antioxidant effects.