Effects And Mechanisms Of GSC Extracts Against Vascular Endothelial Senescence Via Regulating Mitophagy

Diabetes mellitusis a chronic metabolic disease characterized by abnormal glucose and lipid metabolism.Cardiovascular disease is the main complication of diabetes,and about 60%of diabetic patients die from cardiovascular complications.There is increasing evidence that vascular endothelial cell senescence caused by disorders of glycolipid metabolism is the key to endothelial dysfunction and cardiovascular disease.After acquiring a senescence-related secretory phenotype,senescence endothelial cells secrete a large number of inflammatory cytokines,promote the vascular chronic inflammation proliferation,mononuclear cell accumulation and thrombosis formation,and accelerate the vascular endothelial dysfunction and the formation of cardiovascular disease in diabetes.Therefore,exploring the pathological mechanism of vascular endothelial senescence in high glucose and high fat environment,is of great significance for the research of diabetes and cardiovascular complications.Environmental factors and genetic factors together lead to cellular senescence,of which oxidative stress is one of the main mechanisms.Hyperglycemia and hyperlipidemia in diabetic patients damage mitochondria in vascular endothelial cells and accelerate the production of reactive oxygen species(ROS).ROS could result in accumulation of protein and DNA damage;decrease telomere length and telomerase activity;or directly regulate p16/Ras or p53/p21 signaling pathways as signaling molecules affecting cell growth cycle;all of which are responsible for accelerating cell senescence.Mitophagy is a form of selective autophagy that automatically recognizes and degrades it,thereby improving the integrity of mitochondrial structure and function,and preventing excessive accumulation of mitochondria-derived ROS,which plays a protective role in delaying cellular senescence.So,mitophagy as a potential target might be an important role for the prevention and treatment of vascular endothelial cells senescence.Chinese medicine ginseng,Sanqi and Chuanxiong have been widely used in the treatment of cardiovascular and cerebrovascular diseases.Pharmacological studies have shown that the active ingredients of the above three traditional Chinese medicines have good anti-aging effects.Our previous study found that ginseng,sanqi and chuanxiong extracts can effectively reduce the level of intracellular ROS to delay cell senescence in replicative and Angll-induced senescence of vascular endothelial cells.However,whether ginseng,sanqi and chuanxiong extracts can inhibit cell senescence in hyperglycemia and hyperlipidemia-induced vascular endothelial cell senescence and the specific mechanism involved has not yet been elucidated.Therefore,based on the network pharmacology analysis technology,this study first explored the possible targets and mechanisms of ginseng,sanqi and chuanxiong extracts on inhibitin vascular aging.Then,based on diabetic mice and hyperglycemia and hyperlipidemia-induced endothelial senescence,this study has explored the relationship between mitophagy,AMPK/mTOR pathway and senescence,at the same time,the target and mechanism of ginseng,sanqi and chuanxiong extracts to delay the endothelial senescence were discussed.Part 1 Network pharmacology study of ginseng,sanqi and chuanxiong on delaying vascular agingObjective:To analyze the possible targets and mechanisms of Network pharmacology study of ginseng,sanqi and chuanxiong on delaying vascular aging based on network pharmacology.Methods:The active ingredients of ginseng,sanqi and chuanxiong were screened by oral bioavailability(OB)and drug-like(DL)in the TCMSP database and the target was predicted.Construction of a pharmaceutical active ingredient-target network,a component target interaction network,and a vascular aging-related target protein interaction network using Cytoscape 3.7.1.The BisoGenet plugin was used to map the interaction between the target of the active ingredient and the target of human vascular aging-related diseases,and to extract the key targets of ginseng,sanqi and chuanxiong to delay vascular aging.Finally,the key targets obtained by the above screening were subjected to KEGG signal pathway enrichment analysis using the ClueGO plugin.Results:In the TCMSP database,33 active components were collected from ginseng,sanqi and chuanxiong with OB?30%and DL?0.18 as parameters.The 130 predicted targets were extracted using the relevant target prediction technology.146 human vascular aging related genes were obtained from NCBI,AgeFactDB and HAGR databases.The protein interaction network of regulating vascular aging was constructed,and 253 key targets were obtained by topological attribute analysis.ClueGO enrichment analysis showed that the mechanism of ginseng,sanqi and chuanxiong on delaying the vascular aging involved in cell cycle,longevity regulatory pathway,FoxO signaling pathway,neurotrophin signaling pathway,mitophagy and ubiquitin-mediated proteolysis,and so on.Conclusion:Ginseng,sanqi and chuanxiong delay the vascular aging with multi-component,multi-pathway and multi-target regulation.Mitophagy is likely to be one of the key points.Part 2 Effects of ginseng,sanqi and chuanxiong(GSC)extracts on endothelial senescence induced by hyperglycemia and hyperlipidemiaObjective:To observe the mechanism of GSC extracts on endothelial senescence induced by hyperglycemia and hyperlipidemia in vivo and in vitro.Methods:A diabetic mouse model was established by intraperitoneal injection of streptozotocin(STZ)and a high-fat diet.After 28 weeks of modeling,drug intervention was lasted for 12 weeks.The experiment was divided into control group,model group,GSC extracts low(0.819 g/kg)and high(1.638 g/kg)group,and metformin group(150 mg/kg).The biochemical analysis of blood glucose and lipid changes in mice,?-galactosidase(SA-?-gal)staining method to calculate the proportion of blue stained tissue,immunohistochemistry and Western blot to detect the expression of p16 and p21 protein.In vitro experiments,40 mM glucose(HG)and 100 ?M palmitic acid(PA)for 48 h induced human aortic endothelial cell(HAEC)senescence model.The experiment was divided into control group,model group,GSC extracts different dose groups and Metformin group.Cell viability was detected by CCK-8,cellular senescence degree was detected by SA-P-gal staining,the expression of p16 and p21 was detected by Western blot,the expression of p-H2A.X(Ser139),mitochondrial ROS(mtROS)and mitochondrial membrane potential(MMP)were detected by immunofluorescence.Results:Compared with control group,in model group the levels of random blood glucose(GLU),cholesterol(CHO),triglyceride(TG)and low-density lipoprotein(LDL-C)were significantly increased and the level of high-density lipoprotein(HDL-C)decreased,the proportion of SA-?-gal blue staining in aortic tissue increased,and the expression level of p16 and p21 protein increased.After GSC extracts intervention,the blood glucose level of mice was decreased,and the influence of various blood lipid indexes was not obvious.The proportion of SA-(3-gal blue staining was significantly decreased,and the expression levels of p16 and p21 proteins were decreased.In vitro experiment,compared with control group,HAEC cell viability and MMP levels were decreased,the number of SA-?-gal blue-stained cells,mtROS production,and p16,p21,p-H2A.X(Serl39)protein expression increased in model group.Compared with model group,the GSC extracts can increase the cell viability and MMP levels,reduce the number of SA-p-gal blue-stained cells and mtROS production,and reduce the expression level of p16,p21 and p-H2A.X(Serl39).Conclusion:Long-term hyperglycemia and hyperlipidemia stimulation can accelerate the aging process of mouse aorta and HAEC cells,while GSC extracts can delay the senescence of vascular tissue and HAEC cells,and significantly interfere with HAEC mitochondrial function and mtROS production,which might be the mechanism of the GSC extracts on vascular endothelial senescence.Part 3 Effects of GSC extracts on inhibiting vascular endothelial senescence by regulating mitophagyObjective:To observe the effect of GSC extracts on mitophagy in senescent vascular endothelial cells,and to further explore the possible mechanism of GSC extracts on inhibiting cellular senescence after specifically inhibit mitophagy Methods:40 mM glucose(HG)and 100 ?M palmitic acid(PA)for 48 h induced HAEC senescence model.The experiment was divided into control group,model group,GSC extracts group and metformin group.The mitochondrial autophagosome formation was assessed by GFP-LC3,Parkin and mitotracker co-localization.Western blot was used to detect the expression levels of mitochondrial autophagy-related proteins PINK1,Parkin,LC3B-II,p62 and senescence-associated proteins p16 and p21.Cell viability was detected by CCK-8.Cell senescence was detected by SA-?-gal staining.p-H2A.X(Ser139)expression,mtROS production and MMP level were detected by immunofluorescence.Results:(1)Compared with control group,the co-localization of GFP-LC3 and mitotracker decreased,the expression and translocating of Parkin and BNIP3L/NIX protein was down-regulated,and the expression of p62 was increased in HAEC cells of model group.Compared with model group,GSC extracts can effectively increase the colocalization of GFP-LC3,Parkin and mitotracker,up-regulate the expression levels of Parkin,and LC3B-II,and down-regulate the expression of p62.Metformin can increase the colocalization of GFP-LC3 and mitotracker,and improve Parkin and LC3B-? protein expression.(2)Mdivi-1 was administered to inhibit mitophagy.Compared with model group,the co-localization of GFP-LC3 and mitotracker and the expression and translocatioin of Parkin were unchanged in the GSC extracts group;the expression level of LC3B-? and p62 were up-regulated;there was no significant difference in mtROS production,MMP level,cell viability,number of SA-?-gal blue-stained cells and p16,p21 and p-H2A.X(Ser139)protein expression.Conclusion:Hyperglycemia and hyperlipidemia stimulation can inhibit mitophagy in HAEC cells,and GSC extracts can restore the ability of mitophagy,which may be an important mechanism to improve mitochondrial function,reduce mtROS production and delay cell senescence.Part 4 The GSC extracts exerts the regulation of mitophagy through the AMPK/mTOR pathwayObjective:To observe the effect of GSC extracts on AMPK/mTOR signaling pathway in senescent HAEC.By specifically interfering with the activity and expression of AMPK,the mechanism of GSC extracts on regulating mitophagy and delaying cellular senescence was further explored.Methods:40 mM glucose(HG)and 100 ?M palmitic acid(PA)for 48 h induced HAEC senescence model.The experiment was divided into control group,model group,GSC extracts group and metformin group.Western blot was used to detect the expression of p-AMPK,AMPK,p-mTOR,mTOR,p-p70S6K and p70S6K proteins.Compound C and AMPKa 1 shRNA lentiviral particles were used to interfere with AMPK activity and expression.On the basis of this,the effects of GSC extracts on the regulation of AMPK/mTOR signaling pathway,mitophagy and cellular senescence were studied.Results:(1)Compared with control group,the levels of AMPK protein and phosphorylation(Thr172)were down-regulated,and the phosphorylation levels of mTOR and p70S6K protein were up-regulated in model group.After drug intervention,AMPK protein expression and phosphorylation were up-regulated,and the level of mTOR and p70S6K phosphorylation was down-regulated with statistically significant.(2)Compound C was applied to inhibit AMPK activity.Compared with model group,there was no difference in phosphorylation levels of AMPK,mTOR and p70S6K after drug intervention;the degree of binding of LC3 and Parkin to mitochondria in HAEC cells was not increased;there was no significant difference in the expression of PINK1,Parkin,p62 and LC3B-?;there was no difference in HAEC cell viability,number of blue staining of SA-?-gal cells,mtROS production,and expression of p16,p21 and Y-H2AX proteins.(3)RNAi technology specifically interfered with AMPKa 1 protein expression.Compared with the negative control group(SCR shRNA),AMPKa 1 shRNA transfected cells significantly down-regulated AMPK protein expression and phosphorylation,and mTOR and p70S6K phosphorylation levels were up-regulated;the expression level of Parkin protein was significantly down-regulated;the expression level of p62 protein was up-regulated;the cell viability level was decreased,but the number of blue staining of SA-?-gal cells,mtROS production and the expression of p16,p21 and Y-H2AX proteins were increased.(4)In AMPKa 1 shRNA interference cells,compared with model group,there was no significant difference in AMPK protein expression and phosphorylation in GSC extracts group,but mTOR and p70S6K phosphorylation levels were down-regulated with but lower degree;the expression levels of PINK1,Parkin and LC3B-II were not significantly different,and the level of p62 protein was significantly down-regulated;there was no difference in cell viability,number of blue staining of SA-?-gal cells,mtROS production and p21 protein expression,but the levels of p16 and y-H2AX proteins were significantly down-regulated.Conclusion:The AMPK/mTOR signaling pathway was inhibited in senescent HAEC cells.This signaling pathway may be one of the important mechanism of GSC extracts to activate mitophagy and antagonize cell senescence.In summary,this study used a diabetic mouse model and a senescent HAEC model induced by a combination of HG/PA demonstrating that the GSC extracts can delay vascular endothelial cell aging in a dose-dependent manner,which has not been reported in the past.Based on "AMPK/mTOR signaling pathway-mitophagy-mitochondrial dysfunction" axis,we have clarified the mechanism of GSC extracts in delaying vascular endothelial cellular senescence in hyperglycemia and hyperlipidemia environment,providing new ideas for the prevention and treatment of diabetes and cardiovascular diseases by Chinese medicine.