| Atherosclerosis（AS）is a chronic inflammatory disease of arteries caused by dyslipidemia,characterized by endothelial dysfunction,inflammatory infiltration of vascular wall,lipid accumulation and plaque formation.At present,atherosclerotic cardiovascular disease（ASCVD）is still one of the major diseases with high imorbidity and mortality worldwide,which has brought huge economic burden to families and society.Therefore,it is necessary to continuously optimize existing strategies and explore new strategies for preventing and treating ASCVD.Non-alcoholic fatty liver disease（NAFLD）is a clinical syndrome characterized by hepatocyte steatosis and fat accumulation,ranging from simple steatosis to Non-alcoholic steatohepatitis,cirrhosis,and hepatocellular carcinoma.In clinical cases,NAFLD often occurs simultaneously with insulin resistance（IR）,metabolic syndrome,and AS,constituting an early risk factor for AS.AS lesions are widely affected by NAFLD-induced dyslipidemia,oxidative stress,inflammatory reactions,and liver factor secretion.These findings suggest that the prevention and treatment of NAFLD may delay the progression of AS.Sodium glucose cotransporter-2（SGLT-2）inhibitors are novel hypoglycemic drugs that selectively inhibit SGLT-2 to reduce renal tubular glucose reabsorption and increase urinary glucose excretion.In addition to its hypoglycemic effect,more and more studies have confirmed that SGLT-2inhibitors have protective effects on cardiovascular system.Canagliflozin,a SGLT-2 inhibitor,has been found to delay the onset of NAFLD,effectively improve its symptoms,and even improve blood lipids and regulate cholesterol metabolism.Other studies have shown that Canagliflozin also has protective and therapeutic effects on AS.In order to further explore possible mechanisms of SGLT-2 inhibitors in preventing and treating AS,we constructed a model of AS and liver steatosis with ApoE-/-mice fed on Western diets,and integrated liver transcriptomics and targeted metabolomics to clarify effects of Canagliflozin on metabolic disorders in mice,and to further explore the possible mechanism of Canagliflozin in improving AS and liver steatosis.In addition,in clinical research,we collected patients with stable coronary artery disease（SCAD）,a common ASCVD,as a case group,and used Plin5,a differentially expressed molecule discovered in animal experiments,as a detection point.That is,by detecting the level of Plin5 in patients’serum,we further explored its correlation with cardiac function,providing a theoretical basis for the prevention and treatment of SCAD with SGLT-2 inhibitors.Part One Effects of Canagliflozin on atherosclerosis and hepatic steatosis in ApoE-/-miceObjective:To establish the model of atherosclerosis and hepatic steatosis in ApoE-/-mice fed with western diet,and to give Canagliflozin early,in order to explore the effects of Canagliflozin on atherosclerosis and hepatic steatosis in ApoE-/-mice.Methods:1.Western diet-fed ApoE-/-mice were randomly divided into two groups:model group(ApoE-/-,n=10):given 0.5%sodium carboxymethyl cellulose solution by gavage,and Canagliflozin group(ApoE-/-+Cana,n=10):given 20 mg/Kg·d of Canagliflozin by gavage;C57BL/6J mice were fed a normal diet as a control group（Control,n=10）.2.Amount of food eaten,mental state,and physical appearance of the mice were recorded daily.Their body weights were recorded weekly.The intervention period was 14 weeks.3.At the 14th week of the experiment,5 mice in each group were randomly selected for intraperitoneal glucose tolerance test（IPGTT）.The remaining mice were tested for metabolic indicators including 24-hour food intake,water intake,activity distance,and urine volume through a metabolic cage.4.At the end of the experiment,after overnight fasting,fasting blood glucose in the tail vein of mice was measured using a rapid blood glucose meter,and serum was collected from blood samples retrieved from posterior orbital vein.Automatic biochemical analyzer was used to detect triglyceride（TG）,total cholesterol（TC）,low density lipoprotein cholesterol（LDL-C）,high density lipoprotein cholesterol（HDL-C）,superoxide dismutase（SOD）,glutathione peroxidase（GSH-PX）,alanine aminotransferase（ALT）,and aspartate transferase（AST）levels.ELISA was employed to measure fasting insulin（FINS）,adiponectin（ADP）,interleukin-6（IL-6）,interleukin-1β（IL-1β）,,tumor necrosis factorα（tumor necrosis factorα,TNFα）,and monocyte chemokine protein-1（MCP-1）levels in mice serum.5.Heart,aorta,and liver tissues were preserved.The liver tissues were weighed and the liver indices calculated.HE staining,oil red O staining,and immunohistochemical staining were performed for pathological examination.6.Supernatant of Liver tissue homogenate was collected,and tissue TC,TG,free fatty acid（FFA）,malondialdehyde（MDA）were measured by colorimetry.7.Real-time fluorescence quantitative PCR was used to detect the m RNA expression of MCP-1 and MMP-9 in aortic tissue.Results:1.Compared with the ApoE-/-group,the ApoE-/-+Cana group showed lower body weight,higher water consumption and urine production,though no significant differences in food consumption and activity level were observed between the two groups.2.IPGTT results showed that the peak blood glucose in the ApoE-/-group mice was delayed,and its AUC was significantly greater than that in the Control group.The AUC decreased after the intervention of Canagliflozin;Similar differences and changes were also observed in HOMA-IR,and the differences were all statistically significant.3.Serum TC,TG,LDL-C,IL-6,IL-1β,TNFα,MCP-1 and ADP were significantly higher in the ApoE-/-group than in the Control group;Intervention with Canagliflozin could significantly reduce the levels of the above serum indicators4.In ApoE-/-group,plaque was observed in the aorta and aortic root,with increased infiltration of F4/80 positive cells in the aortic root,and increased m RNA expression of MCP-1 and MMP-9 in the aorta;Intervention with Canagliflozin can reduce the area of plaque in ApoE-/-mice,and significantly improve other indicators mentioned above.5.In ApoE-/-group,the hepatocytes showed ballooning degeneration,lipid deposition increased significantly,and the contents of TC,TG,FFA,and MDA in liver tissue increased significantly;Intervention with Canagliflozin can reduce liver cell lipid deposition,and liver tissue homogenate indicators also have varying degrees of improvement,with statistical differences.Conclusions:Western diet-fed ApoE-/-mice for 14 weeks can be used to establish AS and NAFLD models.Early intervention with Canagliflozin attenuated the progress of AS and NAFLD significantly in ApoE-/-mice after14 weeks of intervention,and had the effects of reducing body weight,reducing fat,improving IR,and inhibiting inflammatory reactions.Part two Effects of Canagliflozin on liver transcriptomics in ApoE-/-miceObjective:To analyze the differentially expressed genes（DEGs）and their functional associations in the liver of three groups of mice using liver tissue transcriptomics,and to explore possible mechanisms of Canagliflozin in its prevention and treatment of AS and liver steatosis.Methods:1.Extracted total RNA from mouse liver tissue.2.Selection of total RNA with a total amount≥1μg for transcriptomic analysis.3.Validation of DEGs using qRT-PCR.4.Detection of amino acids contents in the liver by LC-MS.Results:1.RNA-Seq analysis showed that 43 down-regulated genes in the ApoE-/-group were up-regulated in the ApoE-/-+Cana group,whereas 44 genes that were upregulated in the ApoE-/-group were down-regulated in ApoE-/-+Cana group.These 87 DEGs may be related to the improvement of liver steatosis and AS by Canagliflozin.2.GO enrichment analysis and KEGG pathway enrichment analysis of DEGs showed that Canagliflozin had a significant effect on liver amino acid metabolism in mice.3.qRT-PCR assay identified 11 DEGs（CPS1,ASS1,ASL,ARG1,MATLA,GLS2,GOT1,SREBP1,Plin5,Retreg1,and C/EBPβ）whose expressions were consistent with their results from the RNA-Seq analysis.4.Compared with the Control group,mice in the ApoE-/-group exhibited significantly higher levels of glutamine,L-serine,L-citrulline（Cit）,L-aspartic acid（Asp）,L-tyrosine,and L-hydroxyproline in the liver,while compared with the ApoE-/-group,mice in the ApoE-/-+Cana group showed statistically lower levels of amino acids above except for glutamine.Conclusions:In the study of liver transcriptomics,it was found that Canagliflozin can regulate amino acid metabolism in ApoE-/-mice,possibly promoting the ornithine cycle.Part three Canagliflozin improves atherosclerosis and hepatic steatosis in ApoE-/-mice through ASS1/AMPK pathwayObjective:Based on the transcriptomic analysis results of mouse liver,it is hypothesized and verified that Canagliflozin may regulate amino acid metabolism by enhancing ASS1 activity,and then activate AMPK,improve lipid metabolism and promote hepatocyte autophagy.Methods:1.Ultrastructure of liver tissue was observed under electron microscope.2.ELISA method was used to detect the serum Cit level.3.The protein expression levels of ASS1,AMPK,p-AMPK,SREBP1,FAS,ACC1,CPT1,HMGCR,Plin5,Retreg1,C/EBPβ,p-m TOR,m TOR,p-ULK1,ULK1,Beclin1,LC3 II/LC3 I,and p62 in liver tissue were detected using Western Blot.4.The gene expression levels of ASS1,ASL,and e NOS in aorta were detected using q RT-PCR.Results:1.Electron microscopy showed that compared to the Control group,the ApoE-/-group exhibited in its liver tissues nuclear heteromorphism of hepatocytes,swollen mitochondria,dissolved mitochondrial cristae,increased lipid droplets,and visible autophagic bodies.After treatment with Canagliflozin,heterochromatin decreased,mitochondrial swelling and cristae lysis were improved,lipid droplets decreased,and autophagic bodies significantly increased in number.2.Serum Cit was significantly increased,the protein expression levels of ASS1 and p-AMPK in liver tissue decreased,and the ratio of p-AMPK to AMPK decreased in ApoE-/-group;intervention with Canagliflozin can reduce the level of serum Cit and increase the expression of above proteins.3.The protein expression levels of SREBP1,FAS,ACC1,and HMGCR increased significantly in liver tissue of ApoE-/-group,while the protein expression levels of CPT1 decreased;Canagliflozin partially revised changes in the indicators above with statistical differences.4.The expression of Plin5 and Retreg1 in liver tissue of ApoE-/-group increased,while C/EBPβdecreased.Canagliflozin could improve the above indicators.Moreover,Canagliflozin significantly downregulated the levels of p-m TOR/m TOR,p-ULK1/ULK1,and p62 in the liver tissue of ApoE-/-mice,and upregulated the levels of Beclin1 and LC3Ⅱ/LC3Ⅰ（P<0.01）.5.Canagliflozin elevated the aortic endothelial function-associated markers including ASS1,ASL and e NOS,which were all downregulated in the aortic tissues in mice of the ApoE-/-group.Conclusions:1.Canagliflozin may attenuate hepatic steatosis by improving lipid metabolism,enhancing autophagy,and reducing inflammatory response through ASS1/AMPK pathway.2.Canagliflozin may effectively improve aortic endothelial function by increasing the m RNA expression of ASS1,ASL,and e NOS in the aorta of ApoE-/-mice.Part four The expression levels of serum Perilipin5 in patients with stable coronary heart disease and their correlation with cardiac functionObjective:The objective of the study was to analyze the expression levels of serum Perilipin5（Plin5）in patients with stable coronary artery disease（SCAD）,and to explore their correlation with cardiac function.Methods:1.Patients were selected from those hospitalized in the Department of Cardiology,Hebei General Hospital from March 2021 to June 2021,including45 cases in the control group and 86 cases in SCAD group.Then,according to the LVEF value,SCAD patients were divided into two groups,namely,LVEF≥50%group（n=49）and LVEF<50%group（n=37）.2.Clinical data of all patients were collected,including age,gender,height,weight,blood pressure,heart rate,smoking history,chronic disease history,clinical medication history,relevant serum indicators（of liver and kidney function,blood lipids,myocardial enzymes,NT-pro BNP and Plin5）,and cardiac ultrasound results.3.The serum level of Plin5 was detected using ELISA.4.Statistical analysis of data was conducted using software SPSS 23.0.Results:1.There were no statistical differences in gender,BMI,systolic blood pressure,diastolic blood pressure,heart rate,smoking history,history of hypertension and hyperlipidemia,and the medication history ofβreceptor blockers,ACEI/ARB and statins between the three groups;but differences in the age,history of diabetes and medication history of anti-platelet drugs were significant.2.There were no significant differences in serum ALT,AST,creatine kinase-MB（CK-MB）,myoglobin,blood glucose,HDL-C,and LDL-C levels among the three groups;However,there were significant differences in the levels of serum albumin,lactate dehydrogenase,creatinine,TC,TG,and NT-pro BNP among the three groups of patients.3.The serum Plin5 level was the highest in the control group,followed by the group with LVEF≥50%in SCAD patients,and the group with LVEF<50%had the lowest level,with a statistically significant difference.4.Correlation analysis showed that serum Plin5 level was positively correlated with LVEF in SCAD patients.Multivariate Logistic regression analysis showed that Plin5 is a negative factor in affecting the occurrence of reduced left ventricular systolic function after accounting for confounding factors including age,history of diabetes,anti-platelet drug,serum albumin,lactic dehydrogenase,creatinine,total cholesterol and NT-pro BNP.Conclusions:The serum level of Plin5 is significantly reduced in SCAD patients and is positively correlated with LVEF,which may be was a negative influencing factor in the occurrence of reduced left ventricular systolic function. |
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