Astragalosides Ⅳ Inhibits Proliferation, Migration Of Vascular Smooth Muscle Cells Induced By PDGF-BB Via Activating The MTOR

Objective: The development of percutaneous coronary intervention(PCI)is a major historical breakthrough and is widely used in the treatment of coronary heart disease.However,the occurrence of instent restenosis(IRS)hinders the long-term benefits of patients.At present,studies have found that the occurrence of IRS is mainly due to the formation of neointima,and blood vessels are obstructed.Current studies suggest that neointima may be related to endothelial injury,inflammation,activation of vascular smooth muscle cells(VSMCs),and increased synthesis of extracellular mechanisms.Astragalosides Ⅳ(ASIV)is an effective substance extracted from traditional Chinese medicine Astragalus membranaceus,which has a variety of pharmacological effects including anti-inflammatory,antioxidant,anti-apoptosis,anti-fibrosis,etc.Experimental studies show that ASIV can protect endothelial cell function,inhibit proliferation and migration of vascular smooth muscle cells,and inhibit inflammatory response.Therefore,ASIV has important potential role in inhibiting neointimal hyperplasia.This study is based on the proliferation of VSMCs induced by platelet-derived growth factor-BB(PDGF-BB)and is designed to clarify the effect and underlying mechanisms of ASIV on the proliferation and migration of VSMCs induced by PDGF-BB.Methods:(1)VSMCs were isolated from SD rat thoracic aorta by tissue block attachment method,then were identified and subcultured.Cell morphology was observed under optical microscope and was stained by α-SMA by underfluorescent inverted microscope.VSMCs of 3-8 generations were taken for following experiments,and the PDGF-BB and ASIV of different concentrations were used to interfere with VSMCs.Cell counting kit 8(CCK8)assay was used to detect VSMCs proliferation ability,transwell assay and wound healing assay experiment was used to detect migration ability and to determine the best intervention concentration of PDGF-BB.(2)The experiment was divided into 5 groups:the control group,the PDGF-BB group,the PDGF-BB+ASIV 25μ mol/L group,the PDGF-BB+ASIV 50μ mol/L group,and the PDGF-BB+ASIV 100μmol/L group.CCK8 assay was used to detect the proliferation of VSMCs and transwell assay and wound healing assay were used to evaluate the migration of VSMCs.Western blot(WB)was used to detect the protein expression of α-SMA,PCNA,P62,LC3,mTOR and p-mTOR.Results:(1)VSMCs were successfully isolated and 3-8 generations of VSMCs were used for experiments.(2)PDGF-BB significantly stimulated the proliferation and migration of primary VSMCs as compared with control group(P<0.05),and the best intervention concentration was 40ng/ml.ASIV had no significant difference on proliferation and migration of primary VSMCs(P<0.05).(3)ASIV can inhibit the proliferation and migration of primary VSMCs induced by PDGF-BB in a concentration-dependent manner(P<0.05).The concentration gradient of 25,50,100μmol/L were selected.(4)The expression of PCNA of PDGF-BB group was significantly higher than the control group(P<0.05),and the expression of α-SMA protein was significantly lower than that in the control group(P<0.05).ASIV decreased the expression of PCNA induced by PDGF-BB,while ASIV increased the expression of α-SMA induced by PDGF-BB in a concentration-dependent manner(P<0.05).(5)The expression of P62 protein was significantly lower than that of control group(P<0.05),and the expression of LC3 Ⅱ/LC3 I protein was significantly higher than that of control group(P<0.05).AS-Ⅳ increased the expression of P62 protein and decreased the expression of LC3 Ⅱ/LC3 I protein induced by PDGF-BB in a concentration-dependent manner(P<0.05).The expression of p-mTOR /mTOR protein in PDGF-BB group were decreased significantly compare with the control group(P<0.05).ASIV exposure up-regulated the expression level of p-mTOR/mTOR protein induced by PDGF-BB in a concentration-dependent manner(P<0.05).Conclusion:(1)PDGF-BB induces proliferation and migration of primary VSMCs and upregulates autophagy.(2)ASIV inhibits proliferation,migration and phenotypic transformation of primary VSMCs induced by PDGF-BB.(3)ASIV downregulates PDGF-BB induced primary VSMCs autophagy via activating the mTOR.