(Pro)renin Receptor Regulates Myocardial Pyroptosis In Diabetic Cardiomyopathy Through AMPK-NLRP3 Pathway

BackgroundDiabetic cardiomyopathy(DCM),as one of the most common complications of diabetes,is usually characterized by abnormal myocardial structure and function in diabetic patients without hypertension or coronary heart disease.Due to the complex pathogenesis of DCM and lack of effective treatment,it has been the focus of research in recent years.(Pro)renin receptor(PRR)is a specific receptor of renin.As an important component of renin-angiotensin system(RAS),it has been shown to be a key factor in the pathogenesis of diabetes and terminal organ damage.Pyroptosis,as a new type of cell death accompanied by inflammatory cascade reaction,Studies have shown that nucleotide binding oligomerization domain-like receptor protein 3(NLRP3)mediated pyroptosis is involved in the pathological process of DCM.In addition,activation of AMP-activated protein kinase(AMPK)can inhibit NLRP3-mediated myocardial pyroptosis in DCM.However,it is not clear whether PRR participates in DCM myocardial pyroptosis through AMPK-NLRP3 pathway,so to explore the effect of PRR on DCM cardiomyocyte pyroptosis and its mechanism may provide new targets and ideas for the treatment of DCM.Objective1.To observe the expression of PRR,AMPK,NLRP3 and other pyroptosis related proteins in DCM myocardium by constructing diabetic cardiomyopathy rat model.2.To observe the effects of PRR overexpression on the expression of PRR,AMPK,NLRP3 and other pyroptosis related proteins,the level of fibrosis in DCM myocardium and cardiac function of DCM by constructing adenovirus vector overexpressing PRR(Ad-PRR).3.Cardiomyocytes were stimulated with high glucose to observe the expression of PRR,AMPK,NLRP3 and other pyroptosis related proteins.Ad-PRR and AMPK agonist(GSK621)were used to interfere with cardiomyocytes to observe the effect of PRR overexpression on pyroptosis of cardiomyocytes stimulated by high glucose and its regulatory mechanism.Method1.Construction of PRR overexpression adenovirus:According to the PRR gene sequence of rats(GeneID:302526),entrust Shanghai GenePharma Company to construct adenovirus coated PRR gene overexpression vector(Ad-PRR),and construct empty vector adenovirus(AdEGFP)as virus control.2.Construction of DCM model:After one week of adaptive feeding,a total of 60 8-weekold Wistar rats were randomly divided into 2 groups:control group(n=15)and diabetes group(n=45).And the diabetes group was injected intraperitoneally with streptozotocin(65mg/kg)12 hours after fasting.One week after injection,the rats with 3 random blood glucose levels higher than 11.1mmol/L and symptoms such as polydipsia,polyuria and polyophagia were regarded as successful diabetes modeling.Twelve weeks after the successful modeling of diabetes,echocardiography was performed to examine cardiac function in each group.When the cardiac chamber was enlarged and the systolic function was weakened,the DCM model was successful.Then,DCM rats were randomly divided into 3 groups with 15 rats in each group,including DCM group,DCM+PRR overexpression adenovirus group(Ad-PRR group),DCM+empty vector adenovirus group(Ad-EGFP group),and were injected with 100μL PBS buffer,Ad-PRR(1 × 109PFU,soluble in 100μL PBS buffer),Ad-EGFP(1×109PFU,soluble in 100μL PBS buffer)through tail vein,respectively.Two weeks after adenovirus injection,randomly selected 3 rats from each group to detect the efficiency of adenovirus transfection.Four weeks after adenovirus injection,all the rats were anesthetized and the cardiac function was detected by echocardiography,and the samples were taken for the follow-up experiments.3.Cardiac function test:The rats in each group were anesthetized with intraperitoneal injection of chloral hydrate(0.3mL/100g),then left ventricular ejection fraction(LVEF),left ventricular end systolic diameter(LVESD),left ventricular end diastolic diameter(LVEDD)and E/A ratio were measured by VEVO770 imaging system.4.During the experiment,the body weight,heart weight,blood glucose and blood pressure of the rats were measured regularly.5.Structural changes of myocardial tissue were observed by HE staining.6.Immunohistochemical staining was used to observe the expression of pyroptosis related proteins such as NLRP3,Cysteine aspartate protease 1(Caspase-1),interleukin-1 beta(IL-1β)and interleukin-18(IL-18),as well as Collagen-Ⅰ and Collagen-Ⅲ fibers in myocardial tissue.7.Masson staining kit was used to detect collagen fiber expression in myocardial tissue.8.Extract primary cardiomyocytes:The hearts of 0-3-day-old Wistar rats were washed,cut into pieces,and digested with collagenase Ⅱ.Supernatant was taken and centrifuged,and the precipitation were suspended in cardiomyocyte culture medium.According to the different attachment speed of cardiomyocytes and fibroblasts,cardiomyocytes and fibroblasts were separated.The cardiomyocytes were inoculated into a six well culture plate for further experiments.9.Primary cardiomyocyte intervention:Firstly,the cells were divided into normal group(N group),hyperosmotic group(HP group)and high glucose group(HG group)to observe the effects of high glucose stimulation on PRR,NLRP3,Caspase-1,IL-1β and IL-18.Secondly,the cells were divided into normal group(N group),high glucose group(HG group),high glucose+PRR overexpression adenovirus group(Ad-PRR group)and high glucose+empty vector adenovirus group(Ad-EGFP group).Ad-PRR group and Ad-EGFP group were transfected with Ad-PRR and Ad-EGFP,respectively,and were stimulated with high glucose together with HG group to observe the effect of PRR overexpression on the above proteins.Finally,the cells were divided into high glucose+PRR overexpression adenovirus group(AdPRR group),high glucose+PRR overexpression adenovirus+AMPK agonist GSK621 group(Ad-PRR+GSK621 group),high glucose+empty vector adenovirus group group(Ad-EGFP group)and high glucose+empty vector adenovirus+AMPK agonist GSK621 group(AdEGFP+GSK621 group).After transfection of Ad-PRR and Ad-EGFP respectively,high glucose and GSK621 were given to observe whether PRR regulates cardiomyocyte pyroptosis through AMPK-NLRP3 pathway.10.The mRNA levels of PRR,NLRP3,Caspase-1,IL-1β and IL-18 in myocardial tissue and cardiomyocytes were detected by real-time fluorescence quantitative PCR(RT-PCR).11.Western blot was used to detect the protein expression of PRR,t-AMPK,p-AMPK,NLRP3,Caspase-1,IL-1β and IL-18 in myocardial tissue and cardiomyocytes.12.The contents of IL-1β and IL-18 in rat serum and cardiomyocyte culture medium were detected by enzyme-linked immunosorbent assay(ELISA)kit.13.The levels of reactive oxygen species(ROS)in cardiomyocytes were detected by dihydroethyl chloride(DHE)staining.14.Statistical analysis:The data of each group were processed by GraphPad 8.0 software,the experimental data were expressed by mean±standard deviation,and the data of multiple groups were compared with each other by single factor analysis of variance(ANOVA).The difference was statistically significant when P<0.05.Results1.In DCM myocardium,the expression of PRR increased,the expression of pyroptosis related protein such as PRR,NLRP3,Caspase-1,IL-1β and IL-18 increased,the level of AMPK phosphorylation decreased,and the fibrosis level increased.After overexpression of PRR,the expression of above pyroptosis related protein increased significantly,the level of AMPK phosphorylation decreased significantly,and the fibrosis level was significantly increased,thus aggravating the cardiac function damage of DCM.2.Under the stimulation of high glucose,the expression of PRR in primary cardiomyocytes increased,the phosphorylation level of AMPK decreased and the expression of pyroptosis related protein such as NLRP3,Caspase-1,IL-1β and IL-18 increased.After overexpression of PRR,the level of ROS increased,the level of AMPK phosphorylation decreased significantly,and the expression of above pyroptosis related protein increased significantly,increasing the level of pyroptosis.3.Under high glucose stimulation,after the intervention of AMPK agonist on the basis of PRR overexpression,it was found that the phosphorylation level of AMPK increased,the expression of pyroptosis related protein such as NLRP3,Caspase-1,IL-1β and IL-18 decreased,the level of pyroptosis reduced,reversing the increase of pyroptosis caused by overexpression of PRR.ConclusionIn DCM,hyperglycemia increased the expression of PRR,decreased the phosphorylation of AMPK,and occurred NLRP3 mediated pyroptosis.Overexpression of PRR can significantly reduce the phosphorylation of AMPK and increase the level of myocardial pyroptosis in DCM,Increased levels of fibrosis,thus aggravating the injury of cardiac function in DCM.This suggests that PRR is involved in myocardial pyroptosis in DCM and may be related to the decreased level of AMPK phosphory lation,the increased level of ROS and overactivation of NLRP3 in DCM myocardium.This may provide new ideas and targets for the treatment of DCM.