Intervention Effect Of Yougui Pill On MTOR/HIF-1?/VEGF Signal Pathway In Rat Retinal Microvascular Endothelialcells Under High Glucose And Hypoxia Conditions

Objective:Aim at yang deficiency mechanism of proliferative diabetic retinopathy?PDR?,our study starts with the mTOR/HIF-1?/VEGF signal pathway which is related to retinal neovascularization to ex plore the effect way,specific target and molecular mechanism of Yougui Pill in the treatment of PDR.It could provide a new thinking and scientific basis for the prevention and treatment of diabetic retinopathy by traditional Chinese medicine.Methods:Rat retinal microvascular endothelial cells were selected for the experiment.33mmol/L glucose and 100umol/L CoCl ₂ were added to the culture medium,then cultured cells for 96h.The cell model of hyperglycemia and hypoxia in vitro was established to simulate the pathological state of excessive proliferation of PDR.According to different culture conditions,endothelial cells were divided into normal control grou p,high glucose model group and different concentrations of Yougui pill drug serum?high,middle and low dose?.The proliferation and migration of model cells were observed by methyl thiazolyl tetrazolium?MTT?method and cell scratch test res pectively.Flow cytometry was used to detect the effect of Yougui pill drug serum on model cell cycle and apoptosis.The expression of mTORmRNA,4EBP1mRNA,HIF-1?mRNA and VEGFmRNA in cells of each group were detected by a quantitative real-time RT-PCR assay.The protein expression levels of p-mTOR,p-4EBP1,HIF-1?and VEGF in each group were detected by Western blot.Results:1.Adding 33mmol/L glucose and 100umol/L CoCl₂ to the culture medium of rat retinal microvascular endothelial cells for 96h,the pathological state of excessive proliferation of PDR endothelial cells could be successfully simulated,which was consistent with the previous results.2.The results of cell proliferation assay showed that at 24 hours,the OD value of the model grou p was lower than that of the blank group,the difference was statistically significant?p<0.003?,and the OD value of the high and middle dose grou ps of Yougui pill serum was higher than that of the model grou p,and the difference was statistically significant?p<0.003?.At 48 hours,there was no significant difference among blank group,model group,high,middle and low dose groups?p>0.003?.At 96 h,the OD value in the model group was significantly higher than that in the blank group?p<0.003?,and the OD value in the high and middle dose grou ps of Yougui pill was significantly lower than that in the model grou p?p<0.003?.The results of cell migration assay showed that at 48 hours,the migration rate of the model group was significantly lower than that of the blank grou p?p<0.003?,and the migration rate of the high dose grou p of Yougui pill was higher than that of the model group,and the difference was statistically significant?p<0.003?.At 96hours,the mobility in the model grou p was significantly higher than that in the blank group?p<0.003?,while that in the high and middle dose grou ps of Yougui pills was significantly lower than that in the model group?p<0.003?.3.RT-PCR assay showed that compared with the blank grou p,the gene expressions of mTOR,4EBP1,HIF-1?and VEGFmRNA in the model grou p were significantly increased?p<0.003?.Compared with the model group,the gene expression of mTOR,4EBP1,HIF-1?and VEGFmRNA in each concentration grou p of Yougui pill decreased in varying degrees?p<0.003?,and the expression level of each gene decreased with the increase of Yougui pill concentration.Western blot assay showed that compared with the blank group,the protein expression levels of p-mTOR,p-4EBP1,HIF-1?and VEGF in the model grou p were significantly higher than those in the blank grou p?p<0.003?.Compared with the model group,the protein expression of p-mTOR,p-4EBP1,HIF-1?and VEGF in each concentration group of Yougui pill decreased in varying degrees?p<0.003?,and the expression level of each protein decreased with the increase of Yougui pill concentration.Conclusion:The drug serum of Yougui Pill can inhibit the proliferation and migration of rat retinal microvascular endothelial cells caused by high glucose and hypoxia,thereby inhibiting the retinal neovascularization generate.The mechanism may be to inhibit the ex pression of related genes and proteins in the mTOR/HIF-1?/VEGF signaling pathway.