Protective Effect And Mechanism Of Ginsenoside Rg2 On Atherosclerosis

Objective: Atherosclerosis is a chronic progressive inflammatory disease,which is the basis of a variety of cardiovascular and cerebrovascular diseases.The dysfunction of endothelial cells(ECs)and vascular smooth muscle cells(VSMC)is the main cause of its pathogenesis.During the occurrence and development of atherosclerosis,it is very important to study the functional changes of these two cells for the formation and stability of plaque.Ginseng,which has been used in China for thousands of years,has been widely used to treat a variety of diseases,and ginsenosides have been proved to be its main active substance.Ginsenoside Rg2(Rg2)is one of the most active substances with high content of ginseng.It has a variety of pharmacological activities and has been reported to have good therapeutic effects in diseases.However,there is no relevant report on the role of Rg2 in atherosclerosis.Methods: VSMC and Human umbilical vein endothelial cells(HUVEC)were studied in this research.In endothelial cell experiments,1.Cell activity of HUVEC was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.HUVEC were treated with different concentrations of Rg2(0,10,20,30,40 μM)and the effect of Rg2 on HUVEC was determined with MTT.2.Cell counting kit-8(CCK8)and Transwell assay were used to detect cell proliferation and migration.After pretreatment with different concentrations of Rg2 for 12 h,and then treated with platelet derived growth factor BB(PDGF-BB)for 12 h.The effect of Rg2 on cell proliferation was detected by CCK8.The effect of Rg2 on cell migration was detected by Transwell assay.3.Real-time fluorescence quantitative PCR(RT-q PCR).We detected the expression of inflammatory factors stimulated by lipopolysaccharide(LPS)in HUVEC.4.Western blot was used to detect the inflammatory signal pathway.In vascular smooth muscle cells experiments,1.Cell activity of VSMC was detected by MTT assay.VSMC were treated with different concentrations of Rg2(0,10,20,30,40 μM)and the effect of Rg2 on VSMC was determined with MTT.Then,VSMC were pretreated with different concentrations of Rg2(10,20 μM)for 12 h,and then stimulated with 20 ng/m L PDGF-BB for 12 h.Follow-up experiments were carried out under this experimental condition.2.The effect of Rg2 on VSMC proliferation was detected by CCK8.3.The effect of Rg2 on cell migration was detected by Transwell assay and scratch experiments.4.RT-q PCR.Detection of expression at the m RNA level of PDGFBB-induced cell phenotype transformation markers in VSMC.5.Western blot was used to detect the expression of cell phenotype transformation markers at the protein level.In animal experiments,1.the model of carotid artery balloon injury in rats was established.Male Sprague-Dawley rats aged 8 weeks were purchased to establish a carotid balloon injury model.Rats were randomly divided into normal group,balloon injury group,low concentration(8 mg/kg/day)Rg2 treatment group after balloon injury,high concentration(40 mg/kg/day)Rg2 treatment group after balloon injury,and atorvastatin(Ator)(4mg/kg/day)positive control treatment group after balloon injury.After the rats were injured,the rats in the treatment group were continuously intragastric administration for 14 days.Then we anesthetized the rats in each group and took the carotid artery tissue.2.The proliferation of carotid artery intima in rats was observed by frozen section and HE staining.3.RT-q PCR.Vascular tissues were used for RNA extraction to detect the effect of Rg2 on phenotype transformation markers in vivo.4.Western blot.Vascular tissues were used for protein extraction to detect the regulation of phenotype transformation markers and the effect on inflammatory pathways in vivo.Results: In endothelial cell experiments,follow-up experiments are performed with10 μM and 20 μM concentrations of Rg2 after MTT detection.The result of CCK8 showed that Rg2 could inhibit the proliferation of HUVEC induced by PDGF-BB.Transwell experiments indicated that Rg2 could inhibit the migration of HUVEC induced by PDGFBB.In addition,Rg2 decreased the m RNA level of LPS-induced inflammatory factors in HUVEC.Rg2 can down regulate NF-κB signaling pathway and phosphorylation of ERK1/2 reduce inflammatory response.In vascular smooth muscle cell experiments,follow-up experiments are carried out with 10 μM and 20 μM Rg2 after MTT assay.CCK-8 assay indicated that Rg2 can inhibit PDGF-BB-induced VSMC over proliferation.Scratch experiments and transwell experiments showed the ability of Rg2 to inhibit the overmigration of VSMC.In addition,the extraction of cell RNA and protein were performed for RT-q PCR and western blot experiments,showing that Rg2 can improve the expression of contractile markers of VSMC effectively.In animal experiments,rats with balloon injured carotid artery,HE staining showed that Rg2 could reduce the proliferation of intima after injury.Through the extraction of tissue RNA and protein,RT-q PCR and western blot were performed.It was found that Rg2 could increase the expression of contractile markers of VSMC in vivo.In addition,NF-κB signaling pathway and phosphorylation of ERK1/2can also be down regulated in vivo to reduce inflammatory response.Conclusion: Rg2 can inhibit the over proliferation and migration of HUVEC.Rg2 can down regulate NF-κB signaling pathway and phosphorylation of ERK1/2 to reduce inflammatory response in HUVEC.Rg2 can inhibit the over proliferation and migration of VSMC induced by PDGF-BB.Rg2 can inhibit the conversion of VSMC from contractile to synthetic phenotype.In animal experiments,Rg2 can reduce intimal hyperplasia after carotid artery injury in rats and can inhibit phenotypic transformation as well as vascular inflammation in vivo.The above results show that Rg2 can play an anti-atherosclerotic role.This study provides a theoretical basis and scientific basis for the development of ginseng as a functional food or drug to regulate cardiovascular diseases.