Protective Effects Of Tetramethylpyrazine On Oxidative Damage Of Retina Through PI3K/AKT-mediated Anti-oxidative Stress

Purpose:To investigate the protective effects of tetramethylpyrazine（TMP）on oxidatively damaged retinal pigment epithelial（RPE）cells and retina.The expression changes of the PI3K/Akt pathway were also examined.To explore whether tetramethylpyrazine can be used as a clinical drug for oxidative damage of the retina.Method1.Oxidative damage model of RPE cells established by hydrogen peroxide damage to RPE cellsThe well-grown log phase RPE cells were cultured in vitro,the cell concentration gradient was established,the number of viable cells was detected by CCK-8 method,and the cell number-absorbance（OD value）curve was established.The well-grown RPE cells cultured in vitro were divided into normal group,100μmmol/L H₂O₂,200μmmol/L H₂O₂,300μmmol/L H₂O₂,400μmmol/L H₂O₂ intervention group and blank group,using CCK-8 method according to the number of cells-absorbance The（OD value）curve calculates the number of viable cells per group.Determine the concentration of hydrogen peroxide used.2.Effects of different concentrations of tetramethylpyrazine on the activity of RPE cells damaged by hydrogen peroxideThe well-grown RPE cells cultured in vitro were divided into normal group and A,B,C,and D treatment groups（100μmmol/L TMP,200μmmol/L TMP,300μmmol/L TMP were added to 200μmmol/L H₂O₂ damage,respectively）.400μmmol/L TMP),the number of viable cells in each group was calculated according to the cell number-absorbance（OD value）curve using the CCK-8 method.The effects of different concentrations of TMP on H₂O₂-induced RPE cells were determined and the concentration of TMP used was determined.3.Effect of tetramethylpyrazine on ROS and PI3K/AKt in RPE cells oxidized by hydrogen peroxide.The well-grown RPE cells cultured in vitro were divided into normal group,200μmmol/L H₂O₂ damage group,200μmmol/L H₂O₂ damage plus 200μmmol/L TMP treatment group,200μmmol/L H₂O₂ damage plus 200μmmol/L TMP plus wortmannin group.After 24 hours of simultaneous culture,the ROS content was detected by fluorescent probe DCFH-DA,the protein expression of PI3K/AKt was detected by western blotting,and the mRNA expression of PI3K/AKt was detected by real-time PCR.4.Effect of tetramethylpyrazine on the retina of SD rats damaged by sodium iodate.Sixty clean SD rats weighing about 150g were randomly divided into normal control group,model group,low concentration tetramethylpyrazine treatment group,medium concentration tetramethylpyrazine treatment group and high concentration tetramethylpyrazine treatment group,12 rats in each group（model group）The retinal oxidative damage model was established by tail vein injection of 50 mg/Kg sodium iodate in the treatment group.The low,medium and high concentration treatment groups were treated with 20 mg/Kg,40 mg/Kg and 80 mg/Kg per day,respectively;Six rats in each group were taken for 1 month.Three eyes were removed and HE staining and tunel detection were performed.Three eyes were removed and the expression of PI3K/AKT was detected by real-time PCR and western blotting.Result:1.The standard curve absorbance（OD value）is linear with the number of cell activities,and the correlation coefficient R2>0.99 indicates a feasible and linear relationship.With the increase of H₂O₂ concentration,the cell survival rate decreased gradually.The cell survival rate of 100μmmol/L H₂O₂,200μmmol/L H₂O₂,300μmmol/L H₂O₂,400μmmol/L H₂O₂ damage group accounted for 90.8%and68.8%of the normal group,respectively.52.3%,15.3%.2.The ratio of 200μmmol/L H₂O₂+200μmmol/L TMP group to normal cells was about 85.9%,which was significantly higher than other TMP groups.Therefore,the concentration of 200μmmol/L TMP was selected as the treatment group.3.The ROS content in the control group was significantly higher than that in the normal group.The ROS content in the treatment group was significantly lower than that in the normal group.The gene expression and protein content of PI3K/Akt in the treatment group were significantly lower than those in the normal group（P<0.05）,which was higher than that in the control group and the treatment plus wortmannin group（P<0.05）.4.The expression of PI3K/AKT gene and protein in the model group and the low,medium and high concentration groups were significantly lower than those in the normal group（P<0.05）.The middle concentration group was compared with the model group and the low concentration group and the high concentration group.The expression of PI3K/AKT gene and protein was significantly increased（P<0.05）.The expression of PI3K/AKT gene and protein was significantly higher in the low concentration group and the high concentration group compared with the model group（P<0.05）.There was no significant difference in the expression of PI3K/AKT gene and protein in the high concentration group.The expression of the gene and protein of tetramethylpyrazine PI3K/AKT in each treatment group was significantly higher than that in the first week of gavage（P<0.05）.HE staining results showed that the tissue structure level of the middle concentration treatment group was significantly closer to normal than the control group and the low concentration and high concentration treatment groups.Tunel results showed that the apoptosis rate of the moderate concentration treatment group was significantly lower than that of the control group and the low concentration and high concentration treatment groups（P<0.05）.Conclusion:Tetramethylpyrazine can increase the survival rate of RPE cells damaged by hydrogen peroxide,reduce the ROS content,and protect the RPE cells damaged by hydrogen peroxide.Ligustrazine protects the retina from oxidative damage caused by sodium iodate.The protective effect of tetramethylpyrazine on hydrogen peroxide-injured RPE cells and sodium iodate oxidatively damaged retina may be mediated through the PI3K/AKT pathway.