The Effect And Mechanism Of Metabolic Regulation In Atherosclerosis Related Diseases

BackgroundCardiac rehabilitation based on exercise training improves the prognosis of patients with coronary heart disease(CHD).Exercise training has been confirmed to enhance exercise tolerance,reduce cardiovascular mortality and hospitalization rates and significantly improve the quality of life.CHD patients should perform appropriate exercise training after controlling angina pectoris,and the standard and effective drugs should be prescribed.Statin-based lipid-lowering therapy is the cornerstone of primary and secondary prevention of CHD.Whereas,statins offset the positive effect of exercise training on exercise tolerance.The decreased exercise tolerance resulted from statin-induced adverse effects on muscles.Statin-associated muscle symptoms manifested as aches fatigue and spasms,and 38%of patients with statin-related myalgia avoided moderate physical exertion.Epidemiological study showed that 7%to 29%of patients complained of statin-associated muscle symptoms.Statin-associated muscle symptoms could lead to decreased treatment adherence.Discontinuation from statin therapy had a marked impact on cardiovascular benefit.Thus it is needed to explore the mechanism of statin-induced exercise intolerance and search for solutions.Mitochondrial dysfunction is the major mechanism of statin-related muscle injury.In vitro study found that statins binded to the Q0 site of mitochondrial complex III and blocked the oxidation-phosphorylation system,which in turn reduced ATP production and aggravated oxidative stress.Insufficient energy supply and oxidative stress eventually led to muscle fibers atrophy and impaired skeletal muscle function.Therefore,it is essential to protect on mitochondria for reducing the statin-related skeletal muscle injury.Trimetazidine regulated mitochondrial function and enhanced skeletal muscle performance.Studies shown that trimetazidine could protect mitochondria from palmitic acid induced division and improve mitochondrial function.Trimetazidine protected cardiomyocytes by enhancing mitochondrial complex activity and inhibiting mitochondrial electron leakage in heart failure model.Trimetazidine also protected skeletal muscles.Studies found that trimetazidine protected muscle cells from starvation or inflammation factors-induced atrophy.In addition,clinical studies showed that early combination of trimetazidine in traditional pharmacotherapy of stable CHD patients improved the exercise tolerance and reduced angina frequency.Exercise training combined with trimetazidine significantly increased exercise tolerance in patients with ischemic heart disease.However,no research reported whether trimetazidine reversed the statin-related exercise intolerance.We established the statin-induced muscular damage mode with high fat-fed ApoE-/-mice and elucidated the role of trimetazidine in energy modulation,which could improve statin related exercise intolerance.Objectives1.To investigate whether trimetazidine can reduce statin-induced skeletal muscle injury and increase exercise capacity during exercise training.2.To determine whether trimetazidine reduces skeletal muscle damage by improving mitochondrial function and mediating energy metabolism.Methods1.AnimalsFour-week old male ApoE-/-were fed a high-fat diet for 4 weeks and weighed.They were randomized into five groups:sedentary group,exercise group,exercise+simvastatin(SMV)group,exercise+trimetazidine(TMZ)group and exercise+SMV+TMZ.Mice in the exercise group,exercise+SMV group,exercise+TMZ group and exercise+SMV+TMZ group were subjected to 8 weeks of running training and 5 days per week.In the exercise+SMV group,20mg/kg/d simvastatin was intragastrically administered.Mice in exercise+TMZ group were given gavage of 30mg/kg/d trimetazidine orally.Mice in exercise+SMV+TMZ group received gavage of 20mg/kg/d simvastatin and 30mg/kg/d trimetazidine.The mice received gavage for 8 weeks.2.Exercise capacity assessmentAt the end of the experiment,each group of mice was tested for exercise capacity.Horizontal bar experiment:The barbed wire(45cm x 45cm,2mm wire thickness and 18mm grid)was suspended 50cm above the cushion.The mice were placed in the center of the barbed wire,and the barbed wire was flipped over with the head declining first.Record the time of drop.Data of all three tests were averaged,and each test interval was more than 20 minutes.Forelimb grip strength test:The two upper limbs of the trained mice strained the dynamometer.The mouse tail was gently pulled and the mice were emptied until the upper limbs left the dynamometer.Record the dynamometer display value.Measured three times to calculate the average.Running endurance test:Running treadmill were used for endurance testing.The initial movement speed was 13m/min,and the speed increased 2m/min every 3 minutes.The initial runway slope was 0° and the slope increases by 2° every 3 minutes up to 14°.The exercise time and distance were recorded when the mice were exhausted.3.Plasma detectionApproximately 1 mL of blood was drawn at the apex of the mouse and added into heparinized tubes.Centrifuge for 15 min at 2 000 rpm 4? and separate the plasma.The Bayer 1650 Blood Biochemistry Automatic Detector was used to measure the glucose,total cholesterol,low-density lipoprotein cholesterol,high-density lipoprotein cholesterol,triglyceride and free fatty acid levels.Kits were used to measure plasma CK and lactate levels.4.Pathology analysesHE staining was performed to observe morphology of gastrocnemii.Central nuclei fibers were detected by immunofluorescence staining with laminin.Fiber type was detected by immunofluorescence staining with slow or fast MyHC.PAS staining was performed to detect glycogen content of skeletal muscle.Modified Gomori staining was performed to detect ragged red fibers.5.Skeletal muscle mitochondrial assayMitochondria of gastrocnemius were isolated with the Mitochondria Isolation Kit for Tissue,Complex III activity was determined by measuring the reduction of cytochrome c.Citrate synthase(CS)activity was determined by measuring coenzyme A formation.Mitochondrial membrane potential was determined by investigating fluorescence of JC-1.6.Oxidative stress of skeletal muscleMitochondrial hydrogen peroxide level was measured using Amplex red.Frozen muscle samples were homogenized and total SOD activity was detected by xanthine oxidase method with SOD Assay Kit.Glutathione redox state was measured by total and oxidized glutathione(GSH and GSSG)respectively using a commercially available kit.Results1.Simvastatin lowered plasma lipid and elevated CK level.Plasma lipid levels were significantly reduced in ApoE-/-mice in exercise+SMV group.Compared with sedentary,the plasma CK level in exercise-trained mice was not significantly changed.Compared with exercise group,the plasma CK level was increased in simvastatin-treated mice.It was shown that simvastatin could reduce blood lipid levels in exercise-trained ApoE-/-mice and increase plasma CK level.2.Simvastatin disturbed exercise capacity of training ApoE-/-mice.Exercise induced an increase of exercise capacity.Compared with exercise group,exercise capacity was decreased in exercise plus simvastatin group.The above results indicated that simvastatin counteracted the enhancement of exercise performance in ApoE-/-mice.3.Simvastatin induced muscle damage in training ApoE-/-mice.Compared with the sedentary group,the cross-sectional area of the skeletal muscle fibers of the exercise group increased in the ApoE-/-mice,and the muscle fiber cross-sectional area of the ApoE-/-mice in the exercise+SMV group was decreased compared with the exercise group.Central nuclear fibers were the manifestation of repair after injury of skeletal muscle fibers.Compared with the sedentary group and the exercise group,the skeletal muscle core fibers in the ApoE-/-mice of exercise+SMV group increased.The above results showed that simvastatin can cause skeletal muscle fiber atrophy and injury.4.Simvastatin counteracted the change in MyHC stimulated by exercise in ApoE-/-mice.Exercise had upregulated slow-twitch fibers.Compared with exercise group,slow-twitch fibers significantly decreased in simvastatin group.In addition,compared with the exercise group,the cross-sectional area of the slow-fiber and fast-twitch fibers in ApoE-/-mice of exercise+SMV group was significantly reduced.It was shown that exercise training increased the content of slow muscles,and simvastatin reduced the content of slow muscle fibers in exercise-trained ApoE-/-mice and resulted in atrophy of fast and slow muscle fibers.5.Simvastatin induced aerobic metabolism dysfunction of skeletal muscle.Compared with the sedentary group,the skeletal muscle glycogen content was decreased in the exercise group ApoE-/-mice.Compared with the exercise group,the skeletal muscle glycogen content was increased in the exercise+SMV group ApoE-/-mice.In addition,the increase of plasma lactic acid content in ApoE-/-mice in exercise+SMV group indicated that simvastatin inhibited the aerobic metabolism of skeletal muscle in exercise-trained ApoE-/-mice.Compared with the control and exercise groups,the RRF of skeletal muscle in ApoE-/-mice was significantly increased in exercise+SMV group,suggesting that simvastatin caused mitochondrial dysfunction.6.Trimetazidine treatment restored the plasma CK elevation induced by simvastatin without effecting plasma lipid.Compared with the exercise group,there was no significant change in plasma glucose and lipid levels in the exercise+TMZ group ApoE-/-mice.Compared with exercise+TMZ group,ApoE-/-mice in exercise+SMV+TMZ group had significantly lower blood lipid levels.Simvastatin resulted in elevated plasma CK levels during exercise training.Compared with exercise+SMV group,CK levels ApoE-/-mice in exercise+SMV+TMZ significantly reduced.The above results suggested that trimetazidine neither affected plasma lipid levels nor impacted the lipid-lowering effect of simvastatin and trimetazidine can significantly reduce plasma CK levels.7.Trimetazidine counteracted simvastatin-induced exercise intolerance.Compared with the exercise group,the exercise ability of ApoE-/-mice was significantly reduced in the exercise+SMV group.Compared with the exercise+SMV group,the exercise ability of ApoE-/-mice was increased in the exercise+SMV+TMZ group.Trimetazidine protected exercise ApoE-/-mice from the decline of exercise capacity induced by simvastatin.8.Trimetazidine improved muscle damage.Compared with the exercise group,the cross-sectional area of the skeletal muscle fibers of the ApoE-/-mice In the exercise plus simvastatin group was reduced.Compared with the exercise+SMV group,the cross-sectional area of skeletal muscle fibers was significantly increased in the exercise+SMV+TMZ group.This indicated that trimetazidine improves the skeletal muscle fiber atrophy in ApoE-/-mice induced by simvastatin.Compared with exercise group,the percentage of central nuclear fibers in ApoE-/-mice of exercise+SMV group increased.Compared with exercise+SMV group,the percentage of skeletal central nuclear fibers in the exercise+SMV+TMZ group mice was significantly reduced.These showed that trimetazidine reduced skeletal muscle fiber damage in ApoE-/-mice.9.Trimetazidine terminated inhibitory effect of simvastatin on fiber type shift in ApoE-/-mice.Compared with simvastatin treatment group,simvastatin plus trimetazidine treatment induced an increase of slow-twitch muscle fibers.Trimetazidine restored the responsiveness of slow and fast fibers to the growth promoting effect of exercise.10.Trimetazidine improved simvastatin-induced skeletal muscle aerobic metabolism dysfunction.Muscle glycogen content was decreased upon trimetazidine treatment in exercise ApoE-/-mice with simvastatin gavage.Compared to simvastatin treatment alone,simvastatin and trimetazidine treatment reduced the plasma lactic acid level of exercise ApoE-/-mice.Trimetazidine treatment decreased simvastatin-induced RRF in exercise ApoE-/-mice.These results demonstrated that the aerobic metabolism of skeletal muscle in ApoE-/-mice was restored after treated with trimetazidine.11.Trimetazidine reversed the inhibitory effects of simvastatin on mitochondrial function.Compared with sedentary group,complex ? activity was increased in exercise group.Complex ? activity of simvastatin-treated mice decreased significantly compared to mice in exercise group.Trimetazidine restored the decrease of complex? activity induced by simvastatin.Mitochondrial complex activity is an important factor in maintaining mitochondrial membrane potential.Compared to mice in exercise group,the membrane potential decreased after simvastatin treatment and trimetazidine improved mitochondrial membrane potential.We measured the biochemical activity of citrate synthase to obtain an index of mitochondrial mass.Exercise induced an increase of citrate synthase activity.Citrate synthase activity of simvastatin-treated mice decreased compared to mice in exercise group.And trimetazidine ameliorated simvastatin-induced decrease of citrate synthase activity.The above results suggested that trimetazidine counteracted simvastatin-induced mitochondrial dysfunction.12.Trimetazidine inhibited oxidative stress induced by simvastatin.Mitochondrial hydrogen peroxide was lower in simvastatin plus trimetazidine treated mice when compared to mice treated with simvastatin alone.SOD activity and GSH/GSSG in skeletal muscle of simvastatin-treated mice were significantly decreased compared to the trained mice.Compared with exercise+SMV group,the activity of SOD and GSH/GSSG were increased in exercise+SMV+TMZ.These results indicated that simvastatin could induce oxidative stress in skeletal muscles of exercise-trained ApoE-/-mice,and that oxidative stress was reduced in simvastatin plus trimetazidine treated mice.Conclusions1.Trimetazidine could mitigate simvastatin-induced skeletal muscle injury in exercise training ApoE-/-mice and increase exercise capacity;2.Trimetazidine reduced skeletal muscle damage by protecting mitochondrial function.BackgroundCoronary heart disease(CHD)accounts for increasing death in China.Diabetes is one of the major risk factors of CHD.Diabetes is associated with younger prevalence and incident CHD which is the composite of heart failure,cardiac death.It was reported that 76.9%of CHD patients suffered from glycometabolic disorders.Therefore,it is needed to explore the mechanism of atherosclerosis aggravation in diabetic patients.Chronic inflammation is the common mechanism of atherosclerosis and diabetes.Diabetes-related systemic chronic inflammation was the main cause of atherosclerotic lesions.Systemic chronic inflammation originates from adipose tissue.Adipose tissue contains almost all kinds of immune cells.Adipose tissue not only stores lipids,but also regulates the inflammatory response.The inflammatory response in adipose tissue of lean people tends to be inhibited.However,when energy is overloaded,the immune system in adipose tissue was activated.Insulin resistant adipocytes and activated immune cells secreted adipokines and inflammatory cytokines which mediated glycometabolic disorders and systemic chronic inflammation by endocrine.Adipose tissue macrophages are regulatory and effector cells in chronic inflammation.The number of macrophages was more than other immune cells in adipose tissue.In insulin resistant adipose tissue,macrophages could make up about 50%of total cell population.Under conditions of chronic inflammation,the adipose tissue macrophages not only increased but also changed in phenotype.Macrophages could be divided into M1 type(classically activated)or M2 type(alternatively activated)macrophages.Macrophages in insulin resistant adipose tissue polarized to the M1 type.The M1 macrophages promoted Th1 type immune response.Ml macrophages were the major source of chemokines and proinflammatory cytokines which promoted immune response and blocked insulin signaling.Thus,the underlying modulator of macrophage M1 polarization might be explored.Adipocyte insulin resistance launched the macrophage polarization.Exosomes from adipocyte mediated activation of macrophages.Exosomes are extracellular vesicles released from cells when multivesicular bodies fuse with the plasma membrane.Carrying varieties of proteins and RNA,exosomes activated signaling pathways in target cells with ligand-receptor binding or membrane fuse,which resulted in the change of biological function.Exosomes are the semantide of communication between adipocytes and macrophages.Adipocyte exosomes(ADEs)could recruit macrophages,and exosomes from diabetic mice adipocyte tissue promoted inflammatory activity of macrophages.Systemic chronic inflammation can be reduced by regulating the ADEs carrying signal molecules or components of signaling pathways.Whereas the mechanism of ADEs-mediated macrophage activation remains to be investigated.Shh is a secreted morphogen carried by exosomes.The Shh protein was post-translationally modified by cholesterol at the C-terminus and palmitate at the N-terminus which acted as membrane anchors.Exosomes mediate the long-lived effects of Shh signals.Exosome-associated Shh was able to activate signalling pathways and target genes.Shh regulated proliferation and differentiation of types of immune cells in adults.Components of the Shh signaling pathway are expressed in macrophages.Recombinant preparation of Shh upregulated cytokine expression in macrophages,suggesting that Shh was involved in macrophage activation.The expression of Shh was increased under high glucose conditions.Whereas,ADEs from insulin resistant adipocytes carrying Shh may play a role in adipose tissue Ml macrophage polarization and induce insulin resistance.Our study will establish a model of adipocyte insulin resistance and investigate the effect of Shh carried by ADEs on the phenotypic transformation of macrophages and the role of macrophage polarization in insulin resistance of adipocytes.Objectives1.To construct insulin resistance model with 3T3-L1 adipocyte and explore the effects of ADEs on macrophages M1 polarization.2.To explore the effects of ADEs carrying Shh and Ptch/PI3K signaling pathway on macrophage polarization.3.To explore the effects of exosome in macrophage induced adipocyte dysfunction.Methods1.Construction of insulin resistant adipocyte modelAdipocytes were divided into control and insulin resistance groups.Adipocytes in insulin resistance group were stimulated by high glucose and high insulin for 24 hours.Western Blot was used to detect p/t-IRS1,p/t-Akt and cells membrane and cytoplasm levels of Glut4.2NBDG was used to detect glucose uptake.Oil red O staining was used to detect lipid content.The insulin resistance was measured by the above method.Shh was up-regulated in adipocytes transfected with Shh cDNA adenovirus in the control and insulin resistance groups.Insulin resistance group adipocytes were transfected with Shh ShRNA adenovirus to downregulate Shh expression.The transfection efficiency was observed under an inverted fluorescence microscope.Western blot and RT-PCR were used to detect the expression of Shh.2.Isolation of ADEsADEs were isolated from conditioned media of differentiated 3T3-L1 adipocytes.The media was ultra-centrifuged and the pelleted vesicles were resuspended in PBS.The ultrastructure of ADEs was observed with electron microscope.The expression of Tsg101,CD63,CD81,Calnexin and Grp94 was determined by Western Blot.The size homogeneity of ADEs was checked using a Zetasizer Nano ZS90.ADEs carrying Shh was quantified by Western blot and flow-cytometry.3.Macrophages were stimulated with ADEsBMDM or RAW 264.7 macrophages were starved in FBS-free DMEM for 24 hours and incubated with 50 ?g/mL ADEs for 24 hours.To determine whether Ptch and PI3K participated in ADEs-mediated macrophage phenotype transformation,20?M Ptch inhibitor cyclopamine was added 2 hours before ADEs stimulation or 10?M PI3K inhibitor LY294002 was added 1 hour before ADEs stimulation.4.Macrophages polarization analysisThe M1 macrophage(CD11c positive)and M2 macrophage(CD206 positive)was quantified by Flow-cytometry.And the adherence of macrophages was detected.5.Pathway analysisWestern blot was used to detect the expression of Ptch?Gli and p/t-PI3K.EMSA was used to detect Gli binding activity.6.Coculture experiment3T3-L1 adipocytes were cultured in the upper compartment of transwell(0.4?m,Millipore,USA),and ADEs treated RAW 264.7 macrophages were cultured in the lower compartments of the same wells.The adipocytes and macrophages were cultured for 24 h prior to the insulin signaling study.7.Adipocytes were stimulated with exosome from macrophages3T3-L1 adipocytes were differentiated in 24-wells plates and then treated with exosomes isolated from macrophage-conditioned media for 24 h before the insulin signaling study.8.Insulin resistance of adipocytesThe expressions of IRS-1 and HSL were determined via Western blot.9.Serum exosome examinationThe serum exosomes were isolated by ultracentrifugation.Then the Shh-positive exosomes were analyzed by flow-cytometry.Results1.Construction of insulin resistance adipocyte modelAfter adipocytes incubated with high glucose and insulin for 24 hours,serine phosphorylation of IRS-1 was dramatically enhanced,and the expression of IRS-1 was decreased after 6 to 24-hour exposure.Akt phosphorylation decreased in a time-dependent manner.Subsequently,insulin-triggered Glut4 translocation was decreased.These resulted in a decrease in insulin-simulated 2NBDG uptake.And adipocytes lipid accumulation increased.The above results showed that we constructed adipocyte insulin resistance model.2.ADEs derived from insulin-resistant adipocyte carried increased Shh(1)Characterization of ADEsADEs were visualized by TEM.The images revealed membrane-enclosed vehicles.The average size of exosomes was between 50-100 nm.Exosome-associated proteins like CD63,CD81 and Tsg101 could be detected in exosomes,but not endoplasmic reticulum-residing proteins including Calnexin and Grp94.(2)Detection of ADEs carrying ShhShh was stably expressed during the differentiation of preadipocytes into mature adipocytes.The Shh content in ADEs released from adipocytes was significantly higher than that in ultracentrifuge supernatants,indicating that adipocytes secreted Shh mainly in the form of exosomes.Compared with exosomes secreted by control adipocytes(CtrlADEs),exosomes secreted by insulin-resistant adipocytes(IRADEs)carried increased Shh.3.ADEs carrying Shh induced macrophage M1 polarizationCompared with CtrlADEs,IRADEs promoted M1 differentiation of BMDM and RAW 264.7 macrophages and inhibited M2 differentiation.After the neutralizing antibody blocked Shh carried by IRADEs,macrophage M1 polarization was significantly reduced and M2 polarization was increased compared with IRADE-stimulated macrophage.Downregulation of IRADE-carrying Shh by adenovirus transfection reduced IRADE-mediated macrophage M1 polarization.CtrlADEs overexpressing Shh(AdV-Shh CtrlADEs)promoted macrophage M1-type polarization compared to CtrlADEs.Compared to IRADEs,IRADEs overexpressing Shh(AdV-Shh IRADEs)further promote macrophage Ml-type polarization.The above results indicated that Shh carried by ADEs promoted M1 type differentiation of macrophages and inhibited M2 type differentiation.4.ADEs carrying Shh mediated macrophage M1 polarization through Ptch/PI3K/Gli pathway(1)ADEs carrying Shh activate Ptch/Gli pathwayIRADEs increased Ptch and Gli protein levels in RAW 264.7 macrophages and Gli binding activity was increased.Ptch signaling was inhibited by neutralizing antibodies or ShRNA of Shh and promoted by adenovirus-induced Shh overexpression.(2)Cyclopamine,a specific Ptch inhibitor,blocked the ADE-mediated M1 macrophage polarizationCyclopamine abolished the IRADEs AdV-Shh CtrlADEs or AdV-Shh IRADE-induced M1 type polarization and macrophage adherence.(3)ADEs carrying Shh activate PI3KIRADEs increased PI3K phosphorylation in macrophages.PI3K phosphorylation was inhibited by neutralizing antibodies or ShRNA of Shh and promoted by adenovirus-induced Shh overexpression.(4)LY294002 blocked the ADE-mediated M1 macrophage polarizationLY294002 decreased the IRADEs AdV-Shh CtrlADEs or AdV-Shh IRADE-induced Mlpolarization and adherence of macrophages.(5)PI3K mediate Ptch/Gli pathwayIn the IRADEs AdV-Shh CtrlADEs or AdV-Shh IRADE-treated macrophages,cyclopamine leaded to decreased PI3K activation and LY294002 decreased Gli expression.These data indicated the involvement of the PI3K signaling pathway in ADE-carried Shh-induced Ptch pathway activation.5.Macrophage M1 Polarization Mediates Adipocyte Insulin Resistance.(1)Co-culture of adipocytes and ADEs-treated macrophagesAdipocytes which were cultured with ADE-treated macrophages showed markedly reduced IRS-1 and HSL expression.(2)Exosomes from macrophages mediate adipocyte dysfunction:The expression of IRS-1 and HSL were lower in the adipocytes cultured with the exosomes released from macrophages treated with ADEs.6.Shh positive exosome increased in diabetes patientsCompared to healthy volunteers,the Shh-positive exosomes in the diabetes serum were increased significantly.Conclusions1.Insulin-resistant adipocytes derived exosomes carrying increased Shh and could promote macrophage M1 polarization.2.Adipocytes derived exosomes carrying Shh mediated macrophage M1 polarization through the Ptch/PI3K/Gli signaling pathway.3.Adipocytes derived exosomes carrying Shh mediated macrophage M1 polarization,and subsequently M1 macrophages promoted adipocytes insulin resistance.