Study On The Mechanism Of Shuangdan Mingmu Capsule’s Regulation Of Biological Function Of Endothelial Progenitor Cells And VEGF/KDR Loop

Objective : To investigate the inhibition of neovascularization of diabetic retinopathy(DR)rats and the effect on the endothelial progenitor cells(EPCs)by Chinese herbal formula Shuangdan Mingmu capsule from both the whole animal experiment and the in vitro cell experiment.Inhibitory effect of proliferation,taking the VEGF/KDR loop as an entry point,clarifying the specific mechanism of Shuangdan Mingmu capsules’ therapeutic effect,providing experimental basis for the study of the pathogenesis of DR and prevention and treatment of traditional Chinese medicine.Methods:1.EPCs were isolated from human umbilical cord blood and identified using their specific antigen CD34.After subculture digestion,they were divided into 8 groups: control group,blank serum group,Shuangdan Mingmu capsule drug-containing serum group(5 groups in total: 11.2 mg/ml,2.24 mg/ml,1.12 mg/ml,0.56 mg/ml,and 0.28 mg/ml)and Guizhi Ming-containing serum were treated with drugs for 24 h and 48 h respectively.MTT assay was used to determine the cell proliferation inhibition rate.The optimal administration concentration and intervention time of Shuangdang Mingmu Capsules;After determining the concentration and duration of the intervention,the EPCs were divided into 5 groups: blank serum group,Shuangdan Mingmu capsule drug-containing serum group,and diazepam-containing serum group.In serum group,VEGF/KDR blocker group,and Shuangdan Mingmu capsule drug-containing serum + VEGF/KDR blocker group,the VEGF content in the cell supernatant was detected by Elisa method,and detected by Western-blotting and RT-PCR,respectively.Expressions of VEGF/KDR loops and its downstream factors Ras,Raf-1,MEK,ERK proteins and genes.2.68 SD rats were randomly divided into 2 groups,one group of 10 rats was a normal control group,and the other group of 58 was a model group.Diabetic models were induced by a single intraperitoneal injection of STZ(50 mg/kg).Divided into model group,guided Shengming group,Shuangdan Mingmu capsule high,middle and low dose groups,11 in each group.One week after modeling,intragastric administration was continued for 12 weeks.At the end of the last administration,the fasting blood glucose level was measured and fundus fluorescein angiography was performed.Four items of blood lipids and hemorheology indexes were detected by kit method.The degree of retinopathy was detected by HE staining.The level of VEGF in plasma was detected by Elisa method.The number of VEGF-positive cells in the retina was observed by immunofluorescence double labeling method.Western blotting was used to detect retinal VEGF/ KDR and its upstream factors Spred-1,HIF-1α,NF-κB protein expression.Results:1.The CD133、CD34、KDR marker was identified as EPCs.Compared with the blank serum group,Shuangdan Mingmu capsule drug-containing serum significantly inhibited the proliferation of EPCs,and showed a certain degree of dose-dependent,but there was no significant difference between the intervention of 48 hours and the intervention of 24 hours.Comprehensive analysis of the concentration and time of intervention with the serum containing 1.12 mg/ml and 24 h was the best.2.Compared with the blank serum group,the VEGF content in the cell supernatant of the Shuangdan Mingmu capsule serum group was significantly decreased,and the expression of VEGF and KDR was down-regulated.The downstream regulatory factors Ras,Raf-1,MEK,ERK protein and gene expression were significant.After down-regulation,after the combination of a specific blocker of VEGF/KDR loop,the expression of the above indicators was further decreased on the basis of the intervention of TCM serum alone,showing a good synergistic effect.3.Compared with the control group,the blood glucose level of the DR model group increased abnormally,blood lipids and blood rheology were disordered,retinal microvessels increased,capillary blood vessels expanded,reticular structure was loose,interstitial edema,plasma VEGF content increased,and VEGF-positive cells in the retina significantly proliferated,VEGF,KDR protein expression was upregulated,and its upstream angiogenesis-related factors HIF-1α,NF-κB expression increased,while inhibition of angiogenesis-related factor Spred-1 expression decreased.4.After intervention with Shuangdan Mingmu Capsule,the hyperglycemia of DR model rats was controlled to some extent,and the disorder of blood lipid and blood rheology was improved.At the same time,the retinopathy was alleviated,the number of microvessels was reduced,and the structure of the retina was clear,and the edema reduced.Plasma VEGF content decreased significantly,the number of VEGF-positive cells in the retina decreased,and its proliferation was inhibited.The expression of VEGF,KDR,HIF-1α,and NF-κB proteins was significantly downregulated compared with the model group,while Spred-1 expression was up-regulated.Conclusions: In summary,the present study found that Shuangdang Mingmu capsule has good effects in reducing blood glucose and lipids,inhibiting microangiogenesis,relieving the damage of the retina,inhibiting the proliferation of endothelial progenitor cells,and the like through a comprehensive study of animal experiments and cell experiments.This is achieved by reducing VEGF synthesis by inhibiting the VEGF/KDR loop and regulating the upstream and downstream factors of the loop.