| Objective:To explore the role of BYHWD glycosides in repairing damaged vascular endothelium and clarify its mechanism from the perspective of integrin/extracellular matrix.Methods:(1)Establish a rat AS model.Rats were fed high-fat feed for 14 days and underwent balloon injury surgery to establish an endothelial injury model.(2)EPCs were extracted by density gradient centrifugation.Immunofluorescence assay was used to identify whether CD34,CD133,Dil-ac-LDL,and FITC-UEA-1 were positive.After 72 hours of transfection with lentivirus,observe the transfected results under a fluorescence microscope.(3)SD rats were randomly divided into sham-operated group,model group,BYHWD group,glycosides group,EPCs group,BYHWD+EPCs group,glycosides+EPCs group,and atorvastatin group,with 6 rats in each group.After sampling,the following tests were performed:HE staining was used to observe the thickening changes of the vascular intima;Western blot detected the levels of eNOS,ITGβ3,ITGβ6,FAK,P-FAK,ERK1/2,P-ERK1/2;Elisa detected the levels of TC,TG,HDL-C,LDL-C,Col-1,MMP-9,and TIMP-1;Frozen sections were used to detect the homing status of EPCs in vivo.Results:(1)Compared with the sham-operated group,the model group had significant hyperplasia,lower eNOS levels,higher TG,TC,LDL-C levels,and lower HDL-C levels,demonstrating successful modeling.(2)After identification,EPCs showed positive results for CD34,CD133,Dil-ac-LDL,and FITC-UEA-1,confirming their identification as EPCs.The cell transfection rate is about 90%,and the cell transfection and labeling are successful.(3)①Compared with the shamoperated group,the model group had significantly higher intima/media area ratio(P<0.01);Compared with the model group,intima/media area ratio of each group significantly decreased(P<0.01);Compared with the EPCs group,the glycosides+EPCs group showed a significant decrease in intimal/media area ratio(P<0.05);Compared with the BYHWD group,the glycosides group showed no differences in intimal/media area ratio.② Compared with the sham-operated group,the eNOS level in the model group was significantly lower(P<0.01);Compared with the model group,the level of eNOS in BYHWD group,glycosides+EPCs group,atorvastatin group significantly increased(P<0.01),other groups increased(P<0.05);Compared with the EPCs group,the eNOS levels in the glycosides+EPCs group increased(P<0.05);Compared with the BYHWD group,there was no difference in eNOS levels among the glycosides group.③ Compared with the sham-operated group,the levels of TG,TC and LDL-C in the model group were significantly higher(P<0.01),and the levels of HDL-C were significantly lower(P<0.01);Compared with the model group,the levels of TG,TC,and LDL-C in all groups except for the EPCs group reduced significantly(P<0.01),HDL-C level increased significantly(P<0.01),HDL-C level in the atorvastatin group decreased(P<0.05),and there was no difference in TG,TC,LDL-C and HDLC levels in the EPCs group.④Compared with the sham-operated group,the fluorescence brightness of EPCs group,BYHWD+EPCs group and glycosides+EPCs group were significantly enhanced(P<0.01);Compared with the BYHWD+EPCs group,the fluorescence brightness of glycosides+EPCs was significantly enhanced(P<0.01).⑤Compared with the sham-operated group,the levels of ITGβ3 and ITGβ6 in the model group significantly increased(P<0.01);Compared with the model group,the levels of ITGβ3 in BYHWD group,glycosides group and EPCs group decreased(P<0.05),the levels of ITGβ3 in other groups significantly decreased(P<0.01),the levels of ITGβ6 in glycosides group,BYHWD+EPCs group,glycosides+EPCs group and atorvastatin group decreased(P<0.05);Compared with the EPCs group,the levels of ITGβ6 in the glycosides+EPCs group decreased(P<0.05);Compared with the BYHWD group,there was no difference in the levels of ITGβ3 and ITGβ6 in the glycosides group.⑥Compared with the sham-operated group,the levels of MMP-9 and col-1 in the model group were significantly increased,while the levels of TIMP-1 were significantly reduced(P<0.01);Compared with the model group,the col-1 level in each group decreased significantly,and the TIMP-1 level increased significantly(P<0.01);The levels of MMP-9 in EPCs group decreased(P<0.05),while MMP-9 levels of the other groups significantly decreased(P<0.01);Compared with the EPCs group,glycosides+EPCs group showed a decrease in col-1 and MMP-9 levels,while the TIMP-1 level increased(P<0.05);Compared with the BYHWD group,there was no difference in the levels of MMP-9,col-1,and TIMP-1 in the glycosides group.⑦ Compared with the sham-operated group,the levels of P-FAK and P-ERK in the model group were significantly higher(P<0.01);Compared with the model group,the levels of P-FAK in each group were significantly lower(P<0.01),the levels of P-ERK in BYHWD group,BYHWD+EPCs group and atorvastatin group were significantly lower(P<0.05),the levels of P-ERK in glycosides group and glycosides+EPCs group were significantly lower(P<0.01);Compared with the BYHWD group,there was no difference in the levels of in P-FAK and P-ERK in glycosides group.Conclusion:Glycosides can promote EPCs to repair damaged vascular endothelium,and their effects are similar to BYHWD,indicating that they are the main pharmacological substances of BYHWD;the mechanism of glycosides protecting vascular endothelium may be related to their inhibition of integrin/extracellular matrix and FAK/ERK pathway protein expression. |
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