Study On The Material Basis Of The Active Ingredients Of Huoxue Jiedu Prescription Regulating Endothelial Cell Function Based On Hippo/Notch Signaling Pathwa

ObjectiveThe purpose of this study is to observe the effect of ingredients of Huoxue Jiedu Formula on the retinal vascular endothelial cells and confirm the material foundation of Huoxue Jiedu Formula for preventing diabetic retinopathy.Furthermore the effect of the active ingredients on the Hippo/Notch signaling pathway will be explored.Methods1.Retinal endothelial cells were assigned into control group(5.5 mM glucose),high glucose group(25 mM glucose)and active ingredients groups(25 mM glucose+different ingredients with various doses).After the cells were treated with different medium for three days,CCK-8 assay was used to determine cell vitality.The toxicity and dose range of active ingredients were analyzed.Based on the data of CCK-8 assay,transwell migration assay,tube formation assay and BrdU proliferation assay were used to observe the effect of active ingredients on high glucose-stimulated endothelial cells,and in the end the most active ingredients were determined.2.Real-time PCR method was used to evaluate the mRNA expressions of the Hippo(TEAD1)and Notch(Notchl,Notch2,HES1)signaling pathway in the retinal endothelial cells.Western blot method was used to examine the protein expression of the Hippo(MST1,P-MST1/2,YAP1,P-YAP,TEAD1)and Notch(Notchl,Notch2,DLL1,HES1)signaling pathway in the retinal endothelial cells.Results1.The cell viability results showed that,compared with the control group,the cell viability was increased in high glucose group;compared with the high glucose group,the cell viability was inhibited by T3(20 μg/ml-60 μg/ml),T5(0.001 μg/ml-1 μg/ml),and GinsenosideRb3(100 μg/ml-200 μg/ml).However Tannic acid,Hesperidin,Tetrahydroproline,Gypenosides and T4 were toxic and ineffective.2.The cell migration results showed that,compared with the control group,the number of migrated cells was increased in high glucose group;compared with the high glucose group,the number of migrated cells was decreased in T3,T5 and GinsenosideRb3 group.3.The tube formation results showed that,compared with the control group,the total length was increased in high glucose group;compared with the high glucose group,the total length was decreased in T3 and GinsenosideRb3 group.4.The cell proliferation results showed that,compared with the control group,the percentage of BrdU+cells was increased in high glucose group;compared with the high glucose group,the perceltage of BrdU+ cells was decreased in T3 and GinsenosideRb3 group.5.The Real-time PCR results showed that,in comparison to the control group,the high glucose group showed lower expression of HES1,the high glucose group showed higher expression of TEAD1,Notchl and Notch2;In comparison to the high glucose group,T3 increased the expression of HES1,T3 inhibited the expression of TEAD1,Notch1 and Notch2,and GinsenosideRb3 inhibited the expression of Notchl.6.The Western blot results showed that,in comparison to the control group,the high glucose group showed higher expression of P-MST1/2,MST1,P-YAP1,YAP1,TEAD1,Notch1 and Notch2,the high glucose group showed lower expression of DLL 1 and HES1;In comparison to the high glucose group,T3 increased the expression of HES1,T3 inhibited the expression of P-MST1/2,MST1,P-YAP1,YAP1,TEAD1,Notch1,and Notch2,GinsenosideRb3 inhibited the expression of P-YAP1,YAP 1 and Notch1.Conclusion1.According to the data of cell vitality,cell migration,cell proliferation and tube formation assay,the most active ingredients of Huocue Jiedu Formula are T3 and GinsenosideRb3.It suggests that T3 and GinsenosideRb3 can improve the function of retinal endothelial and they are probably the active ingredients of Huocue Jiedu Formula for preventing diabetic retinopathy.2.T3 and GinsenosideRb3 change the gene and protein levels of core molecules in Hippo/Notch signaling pathway,which suggests the active ingredients of Huocue Jiedu Formula improve endothelial function via regulating the Hippo/Notch signaling pathway.