Study on the Effect and Mechanism of Huayu Mingmu Recipe-Containing Serum on Angiogenesis in High Glucose-Induced Functional Disorder Model of HRMECs / 中药新药与临床药理

ABSTRACT

Objective To explore the effect and potential mechanism of Huayu Mingmu Recipe(HMR)-containing serum on high glucose-induced angiogenesis of human retinal microvascular endothelial cells(HRMECs).Methods CCK-8 method is used to screen the optimal sugar concentrations,as well as volume fraction of drug-containing serum.A model of high glucose-induced HRMECs dysfunction was established.The HRMECs were divided into 6 groups such as normal control group which was treated with normal ECM culture medium(containing 5.5 mmol·L-1 glucose)and 10%blank serum,the mannitol control group and the high glucose model were given 19.5 mmol·L-1 mannitol and 19.5 mmol·L-1 D-glucose on the basis of the treatment of normal control group,respectively.The low-,medium-,and high-dose groups of HMR were given 10%low-,medium-,and high-dose medicated serum(without blank serum)on the basis of the model group.The colony experiment was used to detect the number of cell colonies.Transwell experiment was used to test the number of cell migration,and tube formation experiment was used to determine the forming of tubes.Immunofluorescence,Western Blot and RT-PCR were used to detect levels of protein and mRNA expression of FactorⅧ,platelet endothelial cell adhesion molecule-1(CD31),cell differentiation factor(CD34),vascular endothelial growth factor A(VEGFA),vascular endothelial growth factor receptor 2(VEGFR2).Results Compared with the normal control group,the model group could promote colony formation of HRMECs(P＜0.01),cell migration(P＜0.01),and lumen formation(P＜0.01).The levels of protein and mRNA expressions of Factor VIII,CD31,CD34,VEGFA,VEGFR2 were significantly increased(P＜0.01)in model group.Compared with the model group,low-,medium-and high-dose HMR-containing serum groups could inhibit colony formation of HRMECs(P＜0.05,P＜0.01),cell migration(P＜0.01),and lumen formation(P＜0.05,P＜0.01).The levels of protein and mRNA expressions of Factor VIII,CD31,CD34,VEGFA,VEGFR2 were significantly reduced(P＜0.05,P＜0.01)in HMR-containing serum groups.There was no statistically significant difference in results of various tests between the normal control group and the mannitol control group(P＞0.05).Conclusion HMR-containing serum can inhibit the proliferation,migration,and tube formation of HRMECs induced by high glucose,and then prevent or reduce angiogenesis.The mechanism may be related to the regulation of VEGFA/VEGFR2 signaling pathway.