Study On The Effect And Mechanism Of Ginseng-Sanqi-Chuanxiong Extracts Inhibiting Endothelial Progenitor Cell Senescence And Delaying Vascular Aging

The trend of population aging is deepening,which has become a serious public health problem.According to epidemiological studies,by the end of 2018,the number of Chinese citizens aged 65 and above was nearly 167 million,accounting for 11.9 percent of Chinese total population.With the increase of aging population,age-related diseases(such as cardiovascular and cerebrovascular diseases)are also gradually threatening people’s health.Vascular senescence(also known as vascular aging)is the main risk factor leading to aging and age-related cerebrovascular diseases.Vascular structural changes and endothelial dysfunction are the main manifestations of vascular aging.Therefore,it is of great significance to explore methods or drugs to improve vascular structure and endothelial function for the prevention and treatment of vascular aging.Endothelial Progenitor Cells(EPCs)are a type of bone marrow stem cells which could promote the formation and repair of blood vessels through differentiation and secretion.A growing number of studies have demonstrated that EPCs therapy is an important endogenous vascular repair strategy for treating disorders related to endothelial integrity and dysfunction.Autologous EPCs transplantation and drug mobilization of EPCs contribute to vascular repair and neovascularization.In addition,it has been shown in animal and human studies that the number and/or functional activity of EPCs is impaired by aging.Therefore,it is of great significance to explore and improve the aging of autologous endothelial progenitor cells.Mitochondrial dysfunction has an important effect on the aging of the body.Mitochondria,as an important organelle,is crucial to maintain its internal structural integrity and homeostasis.Mitochondria constantly change in number and shape through fusion and fission.This "mitochondrial dynamics" contributes to maintaining the level of reactive oxygen species(ROS)in cells through the efficient transport and distribution of mitochondrial content,which plays an important role in the process of cell aging.AMP-Activated protein kinase(AMPK)is a central metabolic sensor that can be activated by a variety of mitochondrial injuries,including mitochondrial fission.Activation of AMPK can inhibit mitochondrial ectopic mitoprotein,reduce mitochondrial fragmentation and production of ROS,and improve age-related dysfunction.The level and activity of AMPK also decreased during aging.The theory of traditional Chinese medicine believes that qi and blood constitute the basic substances of the body,qi and blood deficiency and failure,and blood blockage are the risk factors of aging.The preliminary study of our research team has proved that Ginseng-Sanqi-Chuanxiong extracts(GSC)has a good effect on improving endothelial cell function and delaying vascular aging.However,studies on whether GSC can inhibit vascular aging by improving EPCs aging have not been carried out yet.Therefore,in this study,d-gal-induced vascular aging mouse model was constructed to observe the protective effect of GSC on mouse aorta and the function and aging of EPCs;in vitro experiments,an EPCs aging model induced by D-gal was constructed to study the effect of GSC on mitochondrial function and mitochondrial fusion and fission-related indicators of EPCs.AMPK inhibitor was used to interfere with the expression of AMPK in EPCs,and then to explore the protective mechanism of GSC on EPCs aging by regulating the AMPK pathway.Based on the results of in vitro experiments,from the perspective of autologous transplantation,in vitro GSC intervenes in EPCs of D-gal-induced vascular aging mice,and transplants them into vascular aging mice to observe the effect of GSC on aortic aging in mice by intervening EPCs and mechanism.Experiment 1:Effect of GSC on D-gal induced aortic aging and EPCs senescence in miceObjective:To explore the effect of GSC on mouse aortic aging and EPCs.Methods:Forty-eight C57 BL/6N mice were randomly divided into Control,Model,GSC-L,GSC-M,GSC-H and Met groups.Mouse model of vascular aging was induced by D-gal dorsal injection for 11 weeks.At the third week of modeling,pure water,Met and GSC were administered by gavage.The aging of mouse aorta was evaluated by HE staining,Elisa detection and immunohistochemistry.Furthermore,the mouse bone marrow cells were induced and cultured.The identification and functions of EPCs were evaluated by cell morphology,immunofluorescence,CCK-8,transwell,adhesion assay and Elisa.Cell senescence was evaluated by SA-β-gal staining and Western blot detection of senescence proteins.Results:Continuous intervention of D-gal can cause structural disorder of the aorta and increase of IMT in mice(P<0.001).The level of oxidative stress in vivo was increased,resulting in a decrease in SOD and an increase in MDA(P<0.05).The endothelial function of mice was damaged,resulting in the decrease of NO and the increase of ET-1(P<0.05).In addition,the aorta of mice appeared sclerosis and senescence protein accumulation,which increased the expression of AGE,MMP-2 and P53(P<0.05).GSC-L,GSC-M and GSC-H groups can improve the aortic structure of mouse aortic vessels to different degrees,reduce IMT(P<0.01),increase SOD and NO content(P<0.05),and reduce MDA and ET-1 level(P<0.05),decreased the expression of aortic AGE,MMP-2 and P53(P<0.05).At the same time,the improvement trend of GSC-H group was more obvious.After the cell morphology,FITC-UEA-I and Dil-ac-LDL double fluorescent staining was positive,and the expressions of CD34,CD 133 and CD309 were positive.It was proved that the cells induced by extraction were EPCs.Compared with the Model group,GSC-L,GSC-M and GSC-H could improve the function of EPCs in different degrees,increase the cell proliferation rate,the number of migrating cells,the number of adherent cells,the contents of NO,VEGF and SDF-1(P<0.05).And alleviated the senescence of EPCs,decreased the staining rate of SA-β-gal(P<0.01),and down-regulated the protein expressions of P53,P21 and P16(P<0.05).Compared with GSC-L and GSC-M,the improvement trend of GSC-H was more obvious.Conclusion:GSC can improve the aortic pathological changes,oxidative stress level,endothelial function,vascular sclerosis and senescence protein accumulation caused by D-gal-induced aortic aging in mice.And GSC can improve the cell senescence and proliferation,adhesion,migration and secretion of NO,VEGF and SDF-1 of EPCs in vascular aging mice.It is suggested that GSC may improve the biological function of EPCs by inhibiting the aging of EPCs in mice,thereby delaying D-gal-induced aging of mouse aortic vessels.Experiment 2:Effect of GSC on the dynamic balance of mitochondrial fusion and fission in D-gal induced aging EPCsObjective:To explore the mechanism of GSC delaying the senescence of EPCs by studying the effect of GSC on mitochondrial fusion and fission in aging EPCs.Methods:Referring to the experiment 1,EPCs were extracted and induced,and the optimal in vitro modeling and intervention concentrations of D-gal,GSC and Met were screened by CCK-8 and SA-β-gal.The cells were divided into Control group,Model group,GSC group and Met group.Western blot was used to detect the expression of senescence proteins in each group.The differences in mitochondrial function and morphology were evaluated by detecting the content of mtROS,mitochondrial membrane potential,ATP content and mitotracker staining in each group.The expression of mitochondrial fusion and fission-related mRNA and protein was detected by qPCR and Western blot,and the effect of GSC on mitochondrial fusion and fission protein and gene expression was explored.Results:20g/L,200mg/L and 8mM were finally determined as the optimal intervention concentrations of D-gal,GSC and Met.In vitro experiments verified that GSC and Met could improve the decrease of cell viability caused by D-gal-induced senescence of EPCs(P<0.05),the increase in the staining rate of SA-β-gal and the expression of senescence proteins P53,P21,and P16 proteins(P<0.05).Compared with the Model group,GSC and Met could significantly reduce mtROS level(P<0.01),increase mitochondrial membrane potential(P<0.05)and ATP content(P<0.01).In terms of mitochondrial morphology,the mitochondrial morphology of the Model group was cloudy and granular,and the control,GSC and Met groups showed obvious mitochondrial stick-like structure,suggesting that D-gal-induced EPCs senescence can cause excessive mitochondrial fission,while GSC and Met can inhibit mitochondrial fission.The results of mitochondrial morphology were verified by Western blot and qPCR.It was found that compared with the Model group,GSC and Met could significantly reduce the expression of mitochondrial fission-related proteins and mRNAs Drp1,Mff and Fis1(P<0.05).Conclusion:GSC can ameliorate the decrease of cell viability,increase of SA-β-gal,P53,P21 and P16 proteins,excessive mitochondrial fission and mitochondrial dysfunction caused by D-gal-induced senescence of mouse bone marrow-derived EPCs.It is suggested that GSC may be able to delay D-gal-induced senescence of EPCs by inhibiting Drpl-mediated mitochondrial fission.Experiment 3:To explore the mechanism of GSC delaying D-gal-induced senescence in EPCs based on the regulation of mitochondrial fission by AMPK pathwayObjective:To observe the effect of GSC on AMPK pathway proteins in senescent EPCs.By interfering the expression of AMPK with AMPK inhibitor(Compound C),we further explored the mechanism of GSC regulating mitochondrial fission to delay the senescence of EPCs.Methods:On the basis of Experiment 2,Compound C group was divided into Control+C.C group,Model+C.C group,GSC+C.C group and Met+C.C group,and the rest of the groups were the same as Experiment 2.The expressions of AMPK and p-AMPK were detected by Western blot.Cell and mitochondrial functions were evaluated by CCK-8,transwell,adhesion assay,SA-β-gal staining,ROS,mitochondrial membrane potential,and ATP.The protein expression of P53,P21,P16,Drpl and Fis1 was detected by Western blot to evaluate cell senescence and mitochondrial fission.Results:Compared with the Model group,the ratio of p-AMPK/AMPK in the GSC and Met groups was increased(P<0.05).After Compound C interfered with the expression of AMPK in each group,there was no significant difference in the cell proliferation rate,cell migration number,cell adhesion number,mtROS activity,mitochondrial membrane potential level,ATP content,mitochondrial morphology,SA-β-gal staining,P53,P21,P16,Drp1,Fis1,AMPK,p-AMPK protein expression compared with Model+C.C(P>0.05).Conclusion:GSC can improve D-gal-induced senescence of EPCs by inhibiting Drpl-mediated mitochondrial fission through activating the AMPK pathway.Experiment 4:Preliminary study on the efficacy of GSC intervening in senescent EPCs in vitro to improve D-gal-induced aortic senescence in miceObjective:To observe the interventional effect of GSC on autologous EPCs transplantation in improving aortic aging in mice.Methods:Before modeling,the mice were randomly divided into Control group and Model group.With the same modeling method in experiment 1,the Model group mice were randomly divided into transplantation group,model blank group(Model),and model transplantation group(Model+M),GSC transplantation group(Model+G),and metformin transplantation group(Model+Met).After modeling,the EPCs of the mice in the transplantation group were injected with 1×106 cells in the tail vein after in vitro drug intervention.After 14 days of transplantation,the general condition of the mice was recorded,and the expression of VEGF in the aorta of the mice was detected by immunohistochemistry.Western blot was used to detect the expression of P53 and P21 proteins in the aorta.The specific indicators were detected with the detection method in Experiment 1.Results:In the end,10 rats in the Model group were successfully injected into the tail vein,8 rats in the Model+M group,9 rats in the Model+G group,and 10 rats in the Model+Met group.Mice in each group did not experience mental disorders and death.Compared with the Model group,there was no significant difference in the general condition of the Model+M group,the Model+G group and the Model+Met group were more active and agile;the Model+M group,Model+G group and Model+Met group could be different improved aortic structural disorder,increased SOD and NO content,decreased MDA and ET-1 content,down-regulated the expression of AGE,MMP-2,P53 and P21(P<0.05).Moreover,the improvement trend of the appeal indicators in the Model+G and Model+Met groups is more obvious than that in the Model+M group.However,there was no significant difference in the expression of VEGF in Model+M group,Model+G group and Model+Met group compared with Model group(P>0.05).Conclusion:GSC in vitro intervention can significantly improve the pathological changes,endothelial function and senescence protein expression of D-gal-induced vascular aging by EPCs transplantation,but it has certain limitations,and its transplantation scheme and mechanism need to be further optimized and improved.In summary,the D-gal-induced vascular aging mouse model and the in vitro-induced EPCs aging model prove that GSCs can improve the aging,function and mitochondrial fusion-fission homeostasis of EPCs while delaying mouse aortic aging.Furthermore,through the interference of AMPK inhibitors,it was further revealed that GSCs can inhibit the senescence of EPCs by inhibiting Drpl-mediated mitochondrial fission through regulating the AMPK pathway.From the study of GSC interfering with autologous EPCs in vitro,it was found that EPCs autologous transplantation can improve vascular aging,while in vitro intervention of GSC can promote the efficacy of transplantation.This study is the first to explore the protective effect and mechanism of GSC on D-gal-induced vascular aging and EPCs aging in mice from the perspective of AMPK-mediated mitochondrial fission,and apply it to the transplantation therapy of vascular aging.It also provides a new idea for traditional Chinese medicine to promote stem cell therapy and prevent vascular aging.