| According to the International Diabetes Federation data,in 2019,about 11.3%of the global deaths among adults aged 20-79 years will be caused by diabetes.The prevalence of diabetes in the 65-99 age group accounts for about 19.3%(135.6 million).It is estimated that by 2030,the number of diabetic patients over 65 years(65-99 years)will reach 195.2 million,and about 276.2 million by 2045.These data show that diabetes is a chronic disease burden in the aging society of the global population,and the prevention of diabetes and its complications is of great significance to the study of delaying aging and age-related diseases and coping with population aging.Epidemiological investigations show that diabetic vasculopathy is the main common complication of diabetic patients,which is closely related to the blood glucose fluctuations of diabetes,and is the pathological basis for many other complications.Blood vessels are an important part of many organs of the human body.Vascular aging is an important pathophysiological basis for the aging of multiple organ systems in the human body,and a high risk factor for the occurrence of vascular related diseases.Therefore,it is imperative to study diabetic vascular aging and its complications to delay diabetic vascular diseases and improve the health of the elderly.Diabetic vascular aging is divided into diabetic macrovascular aging and diabetic microvascular aging,in which the thoracic aorta and common carotid arteries are the main damage sites of vascular aging,and the thoracic aorta and common carotid arteries are the main blood supply sites of the heart and brain.Two vascular aging can exacerbate myocardial fibrosis,brain cognitive dysfunction such as injury of heart head blood-vessel,preserving leads to aging Vascular Smooth Muscle Cell(VSMC)is the main component of the vascular wall membrane.Its abnormal proliferation,migration,cell cycle change,and phenotypic transformation are important pathological factors that lead to the aging of VSMC,accelerate Vascular aging,and ultimately induce diabetic Vascular aging.The AMP-activated protein kinase(AMPK)/Mammalian target of rapamycin(mTOR)pathway is the main signaling pathway affecting aging.A large number of studies have found that AMPK/mTOR pathway in diabetic vascular injury,diabetes heart disease,diabetes encephalopathy,diabetes kidney disease and diabetes vascular lesions of diabetic retinal damage etc.,it has important prevention and protection.Therefore,it is of great significance to explore the action mechanism of Yiqihuoxue decoction on delaying vascular aging and its complications in diabetic patients based on AMPK/mTOR pathway.Extracts from Ginseng Radix et Rhizoma,Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma(GSC)is an alcoholic extract of Ginseng,Panax Notoginseng,and Chuanxiong.The previous pharmacological studies have shown that GSC has good anti-aging effect,to a certain extent,can be as a whole,vascular tissue,cell and molecular physiology delaying the physiology of rats vascular aging and replicating aging of vascular endothelial cells and VSMCs.However,the research on the mechanism of pathological vascular aging and its complications caused by diseases and the intervention of GSC on this type of vascular aging are still in the preliminary stage.Therefore,in this study,the in vivo diabetic vascular aging and its complications model and the high glucose-induced human aortic smooth muscle cell(HASMC)aging model in vitro were used to observe the protective effects of GSC on common carotid artery and thoracic aorta vascular aging,cardio-cerebral aging and high glucose-induced HASMC aging.By using AMPK inhibitors and gene transfection technology to inhibit AMPK expression in HASMC,the role and mechanism of GSC based on AMPK/mTOR pathway on diabetic vascular aging were exploredPart I In vivo experimental study of Yiqihuoxue decoction on Delaying Vascular Aging and its complications in DiabetesExperiment 1:Mechanism of Yiqihuoxue decoction on delaying vascular aging of common carotid artery and thoracic aorta in diabetic miceObjective:To investigate the mechanism of GSC on the aging of common carotid artery and thoracic aorta in diabetic mice.Methods:Intraperitoneal injection of Streptozotocin(STZ)combined with high-fat diet for long-term feeding induced diabetes in mouse models.After 7 months of modeling,they were randomly divided into a control group(control),a diabetes group(HG),GSC-L group,GSC-H group and metformin group(Met),continuous administration for 9 weeks.A new batch of mice was purchased and fed normally for 7 days before sampling as the young group.Observe the general state of the mice,detect the random blood glucose of the mice.Hematoxylin-eosin(HE)staining,collagen fiber(Masson)staining,and transmission electron microscopy(TEM)were used to detect the pathological and morphological changes of the common carotid artery of the mice.Staining(Von Kossa)was used to detect calcium deposition in the common carotid artery and thoracic aorta of mice.Immunohistochemistry(IHC-P)and Western blot(WB)were used to detect matrix metal Matrix metalloproteinases 2(MMP-2),cyclin-dependent kinase inhibitor 2A(p16),cycle-dependent protein kinase inhibitor 1A(p21),runt-related transcription factor-2(Runt-related transcription factor-2,Runx2),Osteopontin(OPN),α-smooth muscle actin(α-smooth muscle actin,α-SMA),and smooth muscle 22α(SM22α)protein expression.Results:Young and Control mice were moderate in size,sensitive in response,active and active,black and shiny hair,low blood sugar,mild changes in pathological morphology of common carotid artery and thoracic aorta,and normal expression of aging proteins;Compared with the Control group,the mice in the HG group were thin,sluggish,dorsal arched,debilitated,and hair was matted with yellowish white hair,and the blood sugar increased significantly(P<0.01).Common carotid artery fibrosis,vascular wall endothelial cells and VSMC damage was serious,cytoplasmic lysis,protrusion,intracellular vacuoles and lysosomes increased significantly,and vascular pathomorphological changes such as the thickness of the sino-foreign membrane,the degree of calcium salt deposition in the common carotid artery and thoracic aorta were aggravated(P<0.01).The expression of OPN protein was increased in the common carotid artery and thoracic aorta,and the expression of SM22α protein was decreased(P<0.01).Runx2,p16,p21,and MMP2 proteins were highly expressed in the common carotid artery,and α-SMA was poorly expressed(P<0.01).Compared with HG goup,GSC-L,GSC-H,and Met groups had significantly improved aging status,significantly decreased blood glucose(P<0.05).The common carotid artery vascular fibrosis,ultrastructure of the intima layer of the vascular wall,thickness of the medial and adventitia,and the degree of calcium salt deposition in the common carotid and thoracic aorta were significantly reduced(P<0.01).The expression of OPN protein in the common carotid artery and thoracic aorta was decreased,and the expression of SM22α protein was enhanced(P<0.05).The expression of Runx2,p16,p21 and MMP2 proteins in the common carotid artery was inhibited,and the expression of α-SMA was enhanced(P<0.05).Conclusion:High glucose can induce aging of the common carotid and thoracic aorta in mice by stimulating general carotid artery vascular fibrosis,ultrastructure of the intima layer of the vascular wall,thickness of the medial and adventitia,and the extent of calcium salt deposition in the common carotid and thoracic aorta,changing the general state of mice,and adjusting MMP2,p16,p21,Runx2,OPN,α-SMA,and SM22α protein expression.GSC can reduce the blood glucose level of diabetic mice,improve the pathological changes of the external aging state and vascular aging in vivo,and delay the vascular aging of the common carotid artery and thoracic aorta of diabetic mice.The mechanism of action may be through regulating the expression of MMP2,p16,p21,Runx2,OPN,α-SMA and SM22α protein.Experiment 2:Based on AMPK/mTOR pathway to explore the effect of Yiqihuoxue decoction on heart aging in diabetic miceObjective:To observe the effect of GSC on AMPK/mTOR pathway in the heart aging of diabetic miceMethods:Because in vivo experiment 1 showed that there was no significant pathological changes of aging in the blood vessels between the young group and the control group,diabetic myocardial fibrosis was only divided into the control group,HG group,GSC-L group,GSC-H group and Met group.The remaining groups are the same as in vivo experiment 1.HE,Masson,Von Kossa combined with TEM were used to detect the pathological morphological changes of mouse heart tissue.IHC-P and WB were used to detect collagen types Ⅰ(Collagen types Ⅰ,Collagen Ⅰ),collagen types Ⅲ(Collagen types Ⅲ,Collagen Ⅲ),transforming growth factor-β1(TGF-β1)protein expression.The expressions of MMP-2,tumor suppressor p53(p53),phospho-suppressor p53(p-p53)and AMPK/mTOR pathways were detected by WB.Results:The myocardial fibers and cardiomyocytes of Young and Control mice were arranged uniformly,densely,regularly and with clear and complete structure.Cardiac microvessel staining was uniform,and no significant calcium deposition was observed.The expressions of MMP2,p53,p-p53 and AMPK/mTOR pathways were not significant;Compared with the control group,the cardiomyocytes in the HG group increased in size,nuclear shrinkage,mitochondrial swelling,degeneration,mitochondrial vesicle vacuolation,and even breakage.The intercellular space is widened and disorganized,with inflammatory cell infiltration and focal necrosis.The normal structure of the cardiac microvessels was destroyed,a large amount of brown calcium salt particles were deposited between the vascular elastic fibers,and a large amount of blue-dyed collagen fibers were deposited in the myocardial interstitium,especially around the microvessels(P<0.01).Collagen Ⅰ,Collagen Ⅲ,TGF-β1,MMP2,p53,and p-p53 proteins were highly expressed in the myocardium,and the protein ratios of p-LKB1/LKB1,p-AMPK/AMPK were significantly reduced,and the protein ratios of p-mTOR/mTOR,p-p70S6K/p70S6K increased significantly(P<0.05).Compared with HG group,the pathological morphology of heart tissue of mice in GSC-L,GSC-H and Met group was significantly improved(P<0.05),and Collagen I,Collagen Ⅲ TGF-β1,MMP2,p53,p-p53 protein expression are decreased.levels can effectively increase The protein ratios of p-LKB1/LKB1 and p-AMPK/AMPK are increased,and the protein ratios of p-mTOR/mTOR,p-p70S6K/p70S6K are down-regulated(P<0.05)Conclusion:High sugar induced heart aging in mice by stimulating myocardial fibrosis,cardiac ultrastructural changes,cardiac microvascular fibrosis and calcification in mouse heart,increasing the expression of myocardial fibrosis markers Collagen Ⅰ,Collagen Ⅲ,TGF-β1 and aging protein,activating the expression of MMP2,p53,p-p53,and promoting the abnormal expression of various proteins in AMPK/mTOR pathway.GSC can improve the pathological and morphological changes of the heart of diabetic mice and delay the aging of the heart.The mechanism may be through regulating the expression AMPK/mTOR pathway and inhibiting the expression of Collagen Ⅰ,Collagen Ⅲ,TGF-β1,MMP2,p53 and p-p53Experiment 3:Based on AMPK-mTOR pathway to explore the effect of Yiqihuoxue decoction on brain aging in diabetic miceObjective:To observe the effect of GSC on AMPK/mTOR pathway in brain aging of diabetic mice.Methods:Because in vivo experiments 1 and 2 showed that there was no significant pathological changes of aging in the diabetic mice blood vessels of the young group and the control group,the pathological changes of the young group were not detected when detecting the number of Nissl bodies and neuronal damage in the hippocampus of diabetic mice.The remaining groups are the same as in vivo experiment one.Open field experiments were used to detect behavioral changes in mice.HE,Nissl,Von Kossa and TEM were used to detect the histopathological changes in the hippocampus of mouse brain tissue.WB and IHC-P was used to detect advanced glycation end products(AGE)and β-amyloid(Aβ)protein in the hippocampus.WB detected the expression of AGE,Receptor of advanced glycation end-products(RAGE),Aβ,MMP2,p53,p-p53 and AMPK/mTOR pathways in hippocampal tissue.Results:There were no significant changes in behavior,brain micro vascular pathology,and ultrastructure in the Young and Control groups,and there were no significant differences in the expression of aging protein and AMPK/mTOR pathway.Compared with the Control group,the total time of central activity and total travel distance of mice in the HG group were significantly decreased(P<0.01),and the number of cells in the brain micro vessels was reduced and the arrangement was disordered,indicating that the vascular lumen was narrow and there was brown particle deposition on the vascular wall.The nuclear membrane of neurons was wrinkled,the chromatin distribution was uneven,the electron density was low,the intracytoplasmic organelles partially dissolved,and the structure of brain microvascular wall was not clear.The number of Nissl bodies,neurons and pyramidal cells decreased(P<0.01)The expression levels of AGE,RAGE,Aβ,MMP2,p53,and p-p53 in the hippocampus increased significantly,and AMPK/mTOR pathway expression was significantly different(P<0.01).Compared with the HG group,the behavioral,the pathological morphology of cerebral microvessels were significantly improved,neuron degeneration in hippocampal CA1,CA3 and DG areas was reduced.The number of Nissl bodies,neurons and pyramidal cells increased(P<0.01),and AGE,RAGE,Aβ,MMP2,p53 and p-p53 protein activity decreased significantly,and the AMPK/mTOR pathway expression was improved in hippocampal area(P<0.05).Conclusion:High glucose can induce the reduction of Nissl bodies in the hippocampus of mice,lead to neuronal damage and ultrastructural changes,accelerate pathological changes such as cerebral microvascular calcification,and increase the expression of AGE,RAGE,Aβ,MMP2,p53 and p-p53 proteins,promote abnormal expression of various proteins in AMPK/mTOR pathway,change the behavioral changes of mice,and cause brain aging.GSC can improve the aging state of diabetic mice from the aspect of behavior,and it can relieve cerebral microvascular calcification and ultrastructural changes in hippocampus by inhibiting the loss of Nissl body and neuron damage in the hippocampus of mice,and give full play to the brain protection.Its protective mechanism may be related to the inhibition of AGE,RAGE,Aβ,MMP2,p53 and p-p53 protein expression and the regulation of AMPK/mTOR pathway.Part Ⅱ Experimental study on Yiqihuoxue decoction delaying high glucose-induced aging of vascular smooth muscle cellsExperiment 1:Establish a high glucose-induced HASMC aging modelObjective:To explore a stable high-glucose-induced pathological HASMC aging model and provide a new cellular experimental model for studying GSC in delaying vascular aging.Methods:After the 6th or 7th generation of HASMC was inoculated and adhered to the wall,serum-free DMEM low-glucose basal medium was used to starve for 24h,and then high-glucose medium with concentrations of 5.5,11,22,33,44,66,88 and 110 mmol/L was selected to intervene for 24h,48h,72h and 96h,respectively.MTT was used to screen the optimal high glucose concentration and intervention time of HASMC aging induced by high glucose.Thenβ-galactosidase(SA-β-gal)staining was used to detect cell aging.Scratch test and Transwell migration test was used to detect cell migration ability.Flow cytometry was used to detect HASMC cell cycle.WB was used to detect aging-related proteins MMP2,p53,p-p53 and phenotypic marker proteins SM22α,α-SMA,Runx2,OPN and BMP-2 expression,further verified the optimal high glucose modeling concentration and intervention time.Results:The MTT results showed that the cell proliferation activity of HG group decreased significantly and the cell proliferation activity of D-M group did not change significantly when 33 mmol/L D-glucose was used to intervene HASMC for 72h.It was established as the optimal high glucose concentration and intervention time for the HASMC aging model;Compared with the Control group,HASMC proliferation activity,migration ability,percentage of S-phase cells and SM22α,α-SMA protein expression levels decreased in the HG group(P<0.05),SA-β-gal blue staining ratio,G0/G1 phase cell percentage and the expression levels of MMP2,p53,p-p53,Runx2,OPN,BMP-2 protein increased(P<0.05)Conclusion:Cell aging is more pronounced when HASMC is treated with 33 mmol/L D-Glucose medium for 72 hours.It is the best high glucose concentration and the best action time for inducing HASMC aging.The aging mechanism of HASMC may be related to altering cell proliferation and migration ability,SA-β-gal expression,cell cycle,phenotypic transformation,and regulating p53,p-53,MMP2,SM22α,α-SMA,Runx2,OPN,BMP-2 protein expression.Experiment 2:Effect of Yiqihuoxue decoction on high glucose-induced HASMC AgingObjective:To observe the effect of GSC on high glucose-induced HASMC agingMethods:Based on a cell model in vivo experiment 1,the optimal intervention concentrations of GSC and Met were selected,and then divided into Control group,HG group,GSC-L group,GSC-M group,GSC-L group and Met group.On the basis of the in vivo experiment 1 detection method,immunofluorescence was used to detect α-SMA and OPN fluorescence expression.Results:Compared with the Control group,HASMC proliferation activity,migration ability,percentage of S-phase cells,α-SMA fluorescence expression and SM22α,α-SMA protein expression levels were decreased in the HG group(P<0.05),and SA-β-gal blue staining Ratio,G0/G1 phase cell percentage,fluorescent OPN expression and MMP2,p53,p-p53,Runx2,OPN,BMP-2 protein expression levels increased(P<0.05);Compared with HG group,GSC and Met intervention group increased cell proliferation activity and migration ability,increased the SA-β-gal blue staining ratio and the percentage of G10/G1 phase cells.Aging protein MMP2,p53,p-p53 and synthetic markers Runx2,OPN,BMP-2 expression were inhibited,contractile markers SM22α and α-SMA protein were increased(P<0.05).Conclusion:GSC can delay high-glucose-induced HASMC aging by improving HASMC proliferation and migration ability,cell cycle,phenotypic transformation and SA-β-gal expression,and its intervention mechanism may be related to reducing MMP2,p53,p-p53,OPN,Runx2,BMP-2 expression levels,and increasing SM22α,α-SMA expression levels.Experiment 3:Based on AMPK/mTOR pathway to explore the effect of Yiqihuoxue decoction on high glucose-induced HASMC AgingObjective:To observe the effect of GSC on AMPK/mTOR signaling pathway in VSMC aging.AMPK inhibitors and gene transfection techniques were used to interfere with AMPK activity and expression,and further explore the mechanism of GSC delaying HASMC agingMethods:Based on a cell model in vivo experiment 1,the gene transfection interference AMPK activity was divided into Control group,HG group,GSC group and Met group,and the rest groups were divided into the same in vivo experiment 2.WB detected AMPK/mTOR pathway activation.Specific indicators were tested using in vivo experiments 1 methods,when AMPK activation was interfered through AMPK inhibitors Compound C and AMPKal lentiviral particlesResults:1)Compared with the Control group,the HASMC p-AMPK/AMPK ratio was down-regulated and the p-mTOR/mTOR ratio was up-regulated(P<0.01).Compared with the HG group,p-AMPK/AMPK and p-mTOR/mTOR ratio tended to be normal(P<0.05).2)After Compound C inhibited AMPK activity,there were no significant differences in proliferation and migration capacity,cell cycle changes,SA-β-gal blue staining ratio,phenotypic transformation,MMP2,p-p53,p53,Runx2,OPN and BMP-2 protein expression and AMPK/mTOR pathway expression,compared with the HG group.3)RNAi technology specifically interfered with AMPKal protein expression.Compared with the SCR shRNA negative control group,the expression levels of AMPK phosphorylation and unphosphorylation were down-regulated in HASMC after transfection of AMPKal shRNA(P<0.01),and mTOR phosphorylation and unphosphorylation expression levels were significantly increased(P<0.01),the proliferation and migration ability of HASMC and the expression of SM22α,α-SMA protein were decreased,and the expression of MMP2,p-p53,p53,Runx2,OPN and BMP-2 protein were up-regulated(P<0.05).Compared with HG group,there was no significant difference in HASMC proliferation migration ability,phenotypic transformation,and expression of aging related proteins and AMPK/mTOR pathway in the drug intervention group.Conclusion:The AMPK/mTOR pathway is involved in the process of high glucose-induced HASMC aging,and GSC can activate AMPK/mTOR pathway and protect HASMC aging.After AMPK expression was interfered with by AMPK inhibitors and RNA interference techniques,the effect of GSC on inhibiting high glucose-induced HASMC aging was significantly reduced.It is suggested that the mechanism of GSC inhibiting high glucose-induced HASMC aging may be partly due to the regulation of AMPK/mTOR pathway.In summary,this study used a vascular aging and its complications model of diabetic mice in vivo and a high-glucose-induced HASMC aging model in vitro to prove that GSC can delay the aging of the common carotid and thoracic aorta in the diabetic mice,cardio-cerebral aging and HASMC aging induced by high glucose.After interfering with HASMC AMPK expression through AMPK inhibitors and RNA interference technology,observe the effect of GSC on HASMC aging,further confirming that the mechanism of GSC delaying vascular aging and its complications in diabetes may play a role by regulating the AMPK mTOR pathway and its upstream and downstream pathways.In the past,the protective effect of Yiqihuoxue decoction on vascular aging and its complications has not been explored from this perspective,which provides a new way for Chinese medicine to prevent and treat diabetic cardiovascular and cerebrovascular diseases. |
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