Effect And Mechanism Of Diosgenin On Proliferation And Migration Of Vascular Smooth Muscle Cells

Background:Arteriosclerotic obliterans mainly occur in the elderly,so intravascular interventional therapy has become the main treatment method with its minimally invasive advantages.However,target vessel restenosis seriously affects the medium and long-term patency rate,which is the bottleneck of interventional therapy.How to overcome this difficulty has become a research hotspot.Arteriosclerosis obliterans is characterized by decreased vessel wall compliance,thickening of atherosclerotic plaques,and calcification of the middle layer,resulting in limb artery stenosis and reduced blood flow.Intimal hyperplasia has been the main factor of stent restenosis,and the abnormal proliferation and migration of vascular smooth muscle cells(VSMC)are the important pathological basis of intimal hyperplasia.Therefore,the key to prevent stent restenosis is to find drugs that can effectively inhibit the abnormal proliferation and migration of VSMC.Diosgenin(Dgn),as a naturally phytosteroidal saponin,has high biological activity.Dioscin,the derivative of diosgenin,has been reported to inhibit the proliferation and migration of VSMC.However,the mechanisms remain unclear.It has been reported that Dgn inhibits hepatocellular carcinoma by inhibiting intracellular Signal transducers and activators of transcription 3(STAT3)signaling pathway.Moreover,STAT3 activation has been reported to contribute to the proliferation and migration of vascular smooth muscle cells,which is an important pathway triggering intimal hyperplasia.Therefore,this study hypothesized that Dgn could inhibit proliferation and migration of VSMC,possibly through regulating and inhibiting the STAT3 signaling pathway,which was further verified by in vitro and in vivo experiments.Purpose:This study aims to verify whether Dgn has an inhibitory effect on VSMC proliferation and migration,and explore whether Dgn can inhibit intimal hyperplasia by regulating and inhibiting STAT3 signaling pathway,so as to provide new ideas for the clinical solution of target vessel restenosis.Methods:(1)To study the effect of Dgn on balloon injury-induced carotid intimal hyperplasia in rats.The intimal hyperplasia model was established by carotid artery balloon injury in rats,and the rats were randomly divided into control group,low-dose Dgn intervention group,and high-dose diosgenin intervention group.The common carotid arteries in each group were collected and stained with hematoxylin and eosin to study the vascular morphological.(2)To study the expression of STAT3 in atherosclerotic tissues.The GSE20129 chip dataset was downloaded from GEO database,and the expression profiles of 119 peripheral blood samples(including 48 atherosclerotic patients and 71non-atherosclerotic patients)were extracted.The STAT3 gene expression and pathway score were studied using difference analysis and correlation test,respectively.(3)To study the effect and molecular mechanism of Dgn on proliferation of VSMC.The in vitro model of intimal hyperplasia was established by Platelet-derived growth factor(PDGF)or 15% Foetal Bovine Serum(FBS)induced proliferation of A10 cells.Western blotting was used to detect Retinoblastoma protein(p Rb),cyclindependent kinase,Expression of proliferation-related proteins such as(Cyclindependent kinase,CDK)2,CDK4 and Proliferating cell Nuclear antigen(PCNA).The changes of PCNA were detected by immunocytochemical staining.Plateletderived growth factor receptor(PDGFR),Extracellular signal-regulated kinases(PDGFR),Phosphorylation levels of ERK 1/2,Serine-Threonine kinase(Akt)and STAT3 proteins were detected by western blotting.The Sarcoma gene(Src)and Janus kinase(JAK)small-molecule inhibitors were used to block the Src and JAK pathways,respectively,to observe the phosphorylation level of STAT3 protein.The expression of Cyclin D1 and Survivin in STAT3 target gene was detected by real-time fluorescence quantitative Polymerase chain reaction(PCR).(4)To study the effect and molecular mechanism of Dgn on migration of VSMC.The in vitro model of intimal hyperplasia was established by PDGF or15%FBS-induced migration of A10 cells.Cell migration was detected by cell scratch assay and Transwell cell migration assay.F-actin were detected by rhodaminephalloidin staining.The ratio of two forms of actin in cells was detected with G-actin/FActin detection kit.The phosphorylation level of Focal adhesion kinase(FAK)protein was determined by western blot.(5)To study the toxicity of Dgn.Cell viability was detected by Methyl thiazolyl Tetrazolium(MTT)assay.The Live/Dead cell Viability Assay kit detects living and dead cells.TUNEL cell apoptosis detection kit was used to detect cell apoptosis.Western blot detection of B-cell lymphoma-2(Bcl-2),Bcl-2 Associated X protein(Bcl-2)Bax,Cleaved caspase-3 and other apoptosis-related proteins;Caspase 3activity assay kit was used to detect changes in enzyme activity.Results:(1)When Dgn concentration reached 80 mg/kg by gavage,the area of new intima induced by carotid artery injury induced by balloon was significantly decreased(37844±3588μm~2)compared with that of control group(116748±20754μm~2)(P<0.05).(2)GEO database analysis indicated that STAT3 was significantly upregulated in peripheral blood of patients with atherosclerosis,and showed a significant positive correlation with the formation of atherosclerosis.(3)Dgn inhibited DNA synthesis and expression of CDK2,CDK4,PCNA and p Rb in a concentration dependent manner(P<0.05).Dgn inhibited tyrosine phosphorylation of PDGFR in A10 cells(P<0.05),and had no significant effect on phosphorylation of Akt and ERK1/2 protein downstream of PDGFR.Dgn significantly inhibited STAT3 phosphorylation at Tyrosine(Tyr)705(P<0.05),but had no effect on phosphorylation of Serine(Ser)727.Application of JAK2 small molecule inhibitor AG490 and Src small molecule inhibitor SU6656 showed that Dgn inhibited STAT3 protein activation by inhibiting Src signal(P<0.05),this process is independent of JAK2 signal.STAT3 downstream target genes Cyclin D1 and Survivin were involved in Dgn inhibition of vascular smooth muscle cell proliferation(<0.05).(4)Scratch test and Boyden chamber test showed that Dgn significantly inhibited the migration of A10 cells induced by PDGF-BB or 15%FBS(P<0.05).In addition,Dgn inhibited the expression of FAK protein in A10 cells induced by PDGF-BB,decreased the ratio of fibroactin to globular actin,prevented the aggregation of fibroactin,and then inhibited the migration of A10 cells(P<0.05).(5)Diosgenin had no significant effect on the viability of A10 cells at the concentration of 25 μM.Meanwhile,diosgenin had no significant effect on apoptosis of A10 cells and protein expression of Bax,Bcl-2 and Cleaved caspase-3 at 25 μM.Conclusions: Diosgenin alleviates balloon injury-induced neointimal hyperplasia of carotid artery in rats and inhibits the proliferation and migration of vascular smooth muscle cells without affecting its cell ability.Diosgenin inhibits the proliferation of vascular smooth muscle cells through Src/STAT3/cyclin D1 signaling pathway.Diosgenin inhibits the migration of vascular smooth muscle cells through FAK signaling pathway.