Study On The Protective Effect Of Liraglutide On Myocardial Cell Injury Induced By High Sugar And High Fat And The Prediction Of Cardiovascular Events In Patients With Diabetes And Coronary Heart Disease By High-density Lipoprotein Particle

Part 1 Protective mechanism of liraglutide on cardiomyocytes injury induced by high glucose and high fatBackground and objective:Diabetes mellitus(DM)is a complex metabolic disorder syndrome.Insulin resistance(IR)and insufficiency of insulin secretion can cause abnormal glucose and lipid metabolism,and secondary damage to a variety of macrovessels and microvessels.Previous studies have shown that the cardiovascular protective effect of the novel hypoglycemic drug liraglutide may be related to anti-inflammatory,anti-fibrotic,and improved lipid metabolism.Our previous studies have found that liraglutide could significantly regulate the lipid profile and improve cardiac function in DCM rats,but the specific mechanism by which liraglutide ameliorates cardiomyocyte injury has not been fully clarified.This study was aimed to find the underlying molecular protective mechanism of liraglutide in cardiomyocytes injury induced by high glucose and high fat and provide some experimental evidence for the cardiovascular benefit effect of liraglutide in clinical treatment.Methods:Firstly,different concentrations of glucose(5.5,10,25,30,50 mM)were used to stimulate neonatal rat cardiomyocytes for 24h,CCK8 was used to detect cell viability and the optical high glucose concentration was determined.And then,treatment cardiomyocytes with the optical high glucose concentration and different concentrations of palmitic acid(PA)(100,250,500,1000 μM)for 24h.CCK8 was used to detect cell viability and the optical high fat concentration was determined.Finally,different concentrations of liraglutide(50,100,200,300,400,500 nM)were used to treat cardiomyocytes combined with high glucose and high fat for 24h,CCK8 was used to detect cell viability and select the appropriate treatment concentration of liraglutide.The subsequent experiments were divided into four groups:control group(Con),high glucose and high fat group(HG+PA),liraglutide group(Lira),AMPK inhibitor group(Compound C).The effect of liraglutide on mitochondrial dysfunction in injured cells was assessed by four indicators:cellular ROS content was detected by cellular ROS staining,mitochondrial membrane potential changes were detected by JC-1 staining,respiratory chain related proteins(NDUFB8,UQCR2,SDHB,MTCO2,ATP5A1)expression were detected by Western blot and ATP content was detected by chemiluminescence.Then,the alteration of mitochondrial morphology in injured cells by liraglutide was observed by Mito-tracker red staining.The effect of liraglutide on content of cardiolipin(CL)in injured cells was assessed by fluorometric detection.The effects of liraglutide on the expression of proteins involved in mitochondrial dynamics(MFN1,MFN2,OPA1,DRP1,FIS1)and cell apoptosis(cleaved-caspase 3,and cytochrome C)in injured cells was assessed by Western blot analysis.The effect of liraglutide on mitophagy and signaling pathway proteins in injured cells were detected by Western blot(PINK1,Parkin,LC3II/LC3I,AMPK,p-AMPKα)and Mito-tracker red stained with Lyso-tracker green.Subsequently,the effects of AMPK inhibitor Compound C on the expression of cell apoptosis and related proteins(cleaved-caspase3,cytochrome c)were examined by TUNEL staining as well as Western blot analysis.The effect of Compound C on mitophagy were determined by Western blot analysis(Parkin,LC3II/LC3I,and FIS1)and Mito-tracker red and Lysotracker green staining.Besides,the effect of Compound C on FIS1 expression was also examined by immunofluorescence.Results:We use 25 mM high glucose+250 μM palmitic acid as the experimental treatment concentration for high glucose and high fat injury model of cardiomyocytes,and 200 nM as the experimental treatment concentration of liraglutide.Compared with Con group,HG+PA group showed that significantly increased cell ROS content,significantly decreased mitochondrial membrane potential,respiratory chain related proteins(NDUFB8,UQCR2,SDHB,MTCO2,ATP5A1)expression and ATP content,significantly increased mitochondrial fragmentation,significantly decreased mitochondrial dynamics related protein FIS1 expression and CL content;besides,cell apoptosis related proteins cleaved-caspase 3,cytoplasmic cytochrome C expression significantly increased while mitochondrial cytochrome C significantly decreased,mitophagy related proteins Parkin,LC3II/LC3I expression and co-location of mitochondrial with lysosome significantly decreased,cell signaling protein p-AMPKa expression also significantly decreased.When compared with HG+PA group,Lira group showed significantly decreased cell ROS content,significantly increased mitochondrial membrane potential,respiratory chain related proteins(NDUFB8,UQCR2,SDHB,MTCO2,ATP5A1)expression and ATP content,significantly improved mitochondrial fragmentation,significantly increased mitochondrial dynamics related protein FIS1 expression and CL content;besides,cell apoptosis related proteins cleaved-caspase 3,cytoplasmic cytochrome C expression significantly decreased while mitochondrial cytochrome C significantly increased,mitophagy related proteins Parkin,LC3Ⅱ/LC3Ⅰexpression and co-location of mitochondrial with lysosome significantly increased,cell signaling protein p-AMPKa expression also significantly increased.While compared with Lira group,Compound C group showed that significantly decreased mitochondrial dynamics related protein FIS1 expression and immunofluorescence intensity,significantly decreased mitophagy related proteins Parkin,LC3II/LC3 expression and co-location of mitochondrial with lysosome,the expression of cleaved-caspase 3,cytoplasmic cytochrome C significantly increased while mitochondrial cytochrome C significantly decreased.Conclusions:The protective effects of liraglutide on improving cardiomyocyte injury and inhibiting cell apoptosis may be related to promoting mitophagy mediated by AMPKParkin pathway.The present study identified the molecular mechanism by which liraglutide ameliorated cardiomyocyte injury via mitophagy,and provided strong evidence and theoretical basis for elucidating the mechanism of the cardioprotective effects of liraglutide.Part 2 High density lipoprotein particles and cardiovascular outcome in diabetic patients with coronary artery diseaseBackground and objective:Atherosclerotic cardiovascular disease(ASCVD)is one of the major causes of cardiovascular death worldwide.Type 2 diabetes mellitus(T2DM)is an independent risk factor for ASCVD such as coronary artery disease(CAD).Dyslipidemia is the most significant metabolic feature of T2DM and usually present with elevation of fasting and postprandial triglyceride(TG),reduction of high-density lipoprotein(HDL)and high-density lipoprotein cholesterol(HDL-C),whereas elevation of low-density lipoprotein(LDL)and low-density lipoprotein cholesterol(LDL-C)and small dense low-density lipoprotein cholesterol(sdLDL-C).Although epidemiological studies revealed that plasma HDL-C concentrations are inversely associated with CAD,multiple drug clinical experiments have shown that raising HDL-C concentrations alone does not reduce cardiovascular risks,for reason that may be related to the high heterogeneity of HDL particles.Recent studies have found that the size of HDL particle is closely related to the pathogenesis of CAD and T2DM.However,the distribution characteristics of different size HDL particles in T2DM patients with CAD,and their association with cardiovascular events(CVEs)have not been fully elucidated.Given this background,we amid to investigate the distribution of HDL particles,explore its correlation and predicting value of clinical outcomes in T2DM combined with stable coronary artery disease(SCAD)patients.Methods:From October 2012 to April 2018,a total of 185 SCAD and 185 T2DM with SCAD patients were enrolled and followed up for a median of 37.7 months.The baseline characters and laboratory data were collected from each patient and the incidence of cardiovascular events(CVEs)during follow up was recorded.The CVEs defined with hospitalized unstable angina;non-fatal myocardial infarction(MI);stroke;unplanned percutaneous coronary intervention or coronary artery bypass grafting;and cardiovascular death.The Lipoprint Lipoprotein Classifier classifies HDL particles into three categories,large HDL particles,medium HDL particles and small HDL particles.The results were showed as the concentration of different HDL-C particles,and the percentage of different HDL-C particles concentration in total HDL-C concentration,respectively.Logistic regression model was used to evaluate the relationship between HDL particles and the disease status of T2DM combined with SCAD.Cox regression analyses were used to evaluate the associations of HDL particles and CVEs in this population.The event-free survival rate was estimated by and compared with log-rank test in Kaplan-Meier curves.Results:Compared to SCAD patients,T2DM with SCAD patients had increased concentration and percentage of small HDL-C particles(P=0.002,P<0.001,respectively),while the concentration and percentage of large HDL-C particles decreased(P=0.001,P<0.001,respectively).During a median 37.7-month follow-up,T2DM with SCAD patients had a higher incidence of CVEs compared to SCAD patients(P=0.039).Then divided T2DM combined SCAD patients into two groups based on with or without CVEs.Compared with without CVEs group,patients of CVEs group had increased concentration of medium HDL-C particles,small HDL-C particles and the sum of medium and small HDL-C particles(P<0.001,P=0.028,P<0.001,respectively).Moreover,the concentration of medium HDL-C particles and the sum of medium and small HDL-C particles was significantly related to the CVEs incidence in T2DM with SCAD patients[odds ratio(OR)95%confidence interval(CI):1.154(1.056-1.262),P=0.002;OR(95%CI):1.095(1.029-1.166),P=0.004,respectively],and they were still independently associated with increased CVEs risks even after adjusting for the variables such as age,gender,hypertension,family history of CAD,current smoking,body mass index and alcohol consumption[adjusted hazard ratio(HR)(95%CI):1.097(1.042-1.155),P<0.001;HR(95%CI):1.061(1.024-1.100),P<0.001,respectively].Finally,we divided the sum of medium and small HDL-C particles concentration into two groups according to its median level:the subgroup of lower concentration(≤28 mg/dL)and higher concentration(>28 mg/dL).Kaplan-Meier curve analysis indicated that T2DM with SCAD patients in the subgroup of higher concentration(>28 mg/dL)had lower event-free survival rates compared to ones in the lower subgroup(≤28 mg/dL)(P=0.008).Conclusions:The present study revealed that the correlation between the sum of medium and small HDL-C particles concentration and incidence of CVEs in T2DM combined SCAD patients,furthermore,elevated concentration of the sum of medium and small HDL-C particles was independently associated with worse outcomes among these patients.