Study On The Mechanism Of Ginsenoside Rg1 In Promoting Vascular Repair Based On Exosome Mediated Information Transmission Of ECs-VSMCs

Objective: To investigate the effects of Ginsenoside Rg1(GS-Rg1)on inhibiting the phenotypic transformation and proliferation of vascular smooth muscle cells(VSMCs)via affects the information cross talk between vascular endothelial cells(ECs)and VSMCs mediated by exosomes,and to explore the mechanism of exosomes of vascular ECs intervened by GS-Rg1 inhibiting the phenotypic transformation and proliferation of VSMCs and promoting vascular repair.Methods:(1)Culture and identification of VSMCs and human umbilical vein ECs(HUVECs): Cellular morphologies were observed.Proteins of α-Smooth muscle agonist protein(α-SMA)and v WF,with the surface markers of VSMCs and HUVECs,respectively,were detected by immunofluorescence.(2)Effects of different concentrations of GS-Rg1 on the viability and secretory function of HUVECs cells.Cells were divided into four groups: Normal control group,GS-Rg1(1,5,10)μM groups.After GS-Rg1 acted on HUVECs for 48 hours,cells viability were detected by CCK8,and the contents of related cytokines including endothelin-1(ET-1),nitric oxide(NO)and angiotensin II(Ang II)in cell supernatant were detected by enzymelinked immunosorbent assay(ELISA).(3)HUVEC exosomes(HUVEC-exos)was extracted by ultrafiltration and exosomes extraction kit.The morphology of HUVECexos was observed by transmission electron microscope,the particle size of HUVECexos was detected by nanoparticle tracking analysis,and the specific marker proteins CD9,CD63,CD81 and TSG101 of HUVEC-exos were detected by Western blot(WB).(4)CCK8 method was used to explore the concentration platelet-derived growth factor(PDGF-BB)inducing abnormal proliferation of VSMCs.(5)Effect of HUVEC-exos on phenotype transformation and proliferation of VSMCs.Cells were divided into 7groups: Control group,PDGF-BB group,PDGF-BB + HUVEC-exos control group,GS-Rg1(1,5,10 μM)intervents HUVEC-exos group,PDGF-BB + GS-Rg1(10 μM)group.The concentration of PDGF-BB was 20 ng/m L.CCK8 was used to detect cell viability,flow cytometry(FCM)was used to detect the proliferation index of VSMCs,and WB was used to detect the expression of phenotype specific proteins of VSMCs,including synthetic marker osteopontin(OPN),Non muscle myosin heavy chain isoform-b(Smemb)and cellular retinol binding protein-1(CRBP-1),contractile markers α-SMA,smooth muscle myosin heavy chain(SM-MHC)and Smoothelin-B.HUVEC-exos was labeled by PKH67 and the endocytosis of HUVEC-exos by VSMCs was observed by laser confocal microscope.(6)Next Generation Sequencing(NGS)and biological information analysis were performed on HUVEC-exos,and verified by q PCR;And then to explore the differentially expressed mi RNA of HUVEC-exos intervented by GS-Rg1,and to predict the target genes and verify the function by dual luciferase reporter gene assay.Result:(1)The marker proteins α-SMA and v WF of VSMCs and HUVECs were positive,and the cells purity were 100%.(2)There were no significant effect of GSRg1 on the viability of HUVECs,and no significant effects of GS-Rg1 on the ET-1,NO and Ang II secreted by HUVECs.(3)Under transmission electron microscope,the extracted HUVEC-exos showed a concave tea-tray like structure,the particle size ranged was from 30 to 200 nm,and the expression of exosomes marker proteins CD9,CD63,CD81 and TSG101 were positive.(4)20 ng/m L PDGF-BB induced abnormal proliferation of VSMCs successfully.(5)GS-Rg1(1,5,10 μM)intervented HUVECexos decreased the cell viability of VSMCs induced by PDGF-BB(P<0.05)and decreased the proliferation index of VSMCs(P<0.01)significantly.It was found that HUVEC-exos could be internalized by VSMCs and existed around the nucleus.(6)The results of NGS of HUVEC-exos showed that there were differences in the mi RNA expression after GS-Rg1 intervention.Wayne diagram showed 97 differentially expressed mi RNAs.It was found that 10 mi RNAs differences were statistically significant,of which 3 increased and 7 decreased.5 mi RNAs expressed in mouse(mmu-)and 2 mi RNAs expressed in unknown species(PC-).has-mir-7977,has-mir-10400 and has-mir-142 expressed in 3 human sources(has-),and it was found that only the PCR verification results of has-mir-7977 were consistent with the sequencing results.The expression of has-mir-7977 was not only significantly increased in HUVEC-exos after GS-Rg1 intervention,but also highly expressed in VSMCs treated by HUVEC-exos after GS-Rg1 intervention.(7)The results of CCK8 showed that mir-7977 mimics could reduce the proliferation of VSMCs significantly induced by PDGF-BB(P<0.01).The target gene MAPK13 was obtained by target gene prediction and combined with the literature,and the results of dual luciferase reporter gene assay showed that transfection of mi R-7977 could target inhibit the expression of MAPK13 of VSMCs.Conclusion: HUVEC-exos after GS-Rg1 intervention can reduce the abnormal proliferation and phenotypic transformation of VSMCs induced by PDGF-BB.The mechanism is that the increased mi RNA of has-mi R-7977 delivered by HUVEC-exos intervened by GS-Rg1 is internalized by VSMCs and targets MAPK13 gene to inhibit the proliferation and phenotypic transformation of VSMCs,but this mechanism needs to be further verified.