Effect Of(Pro)renin Receptors On Myocardial Injury In Diabetic Cardiomyopathy By Regulating Pyroptosis-NET Scircuits

BackgroundAs one of the most common cardiovascular complications of diabetes,diabetic cardiomyopathy(DCM)refers to the damage to the structure and function of the heart that is not accompanied by hypertension and coronary artery diseases.The main clinical manifestations are cardiac enlargement,myocardial diastolic and systolic function decline,which is an important cause of heart failure and sudden death in diabetic patients.Studies have shown that(pro)renin receptor(PRR)is a specific receptor for renin.As an important component of RAS system,PRR is a key factor in the onset of diabetes mellitus and its resulting end-organ damage.Pyroptosis,as a novel cell death mode associated with inflammatory cascade reaction,is also involved in the pathological process of DCM.In addition,Netosis,the form of neutrophils that resist external stimuli,is still involved in the pathological process of DCM.However,existing studies on DCM have not yet explored whether PRR is involved in the scorch death of myocardial cells in DCM and whether PRR affects the scorch death of myocardial cells in DCM by influencing the production of Netosis and regulating the scorch death-Netosis loop.Therefore,to explore the effect of PRR on the scorch death of myocardial cells in DCM and its mechanism.It may provide new targets and ideas for the treatment of DCM.Objective1.The expression of PRR,caspase-1,IL-1β and IL-18 in myocardial tissue of DCM was observed by establishing a rat model of diabetic cardiomyopathy.2.Adenovirus vector overexpressing PRR was constructed to observe the effect of PRR overexpression on the expression levels of pyrogenic related molecules such as caspase-1,IL-1β and IL-18 in myocardial tissue of DCM and the cardiac function of DCM.3.Adenovirus vector overexpressing PRR was constructed by extracting primary cardiomyocytes to observe the expression of PRR,caspase-1,IL-1β,IL-18 and other pyrogenic molecules in primary cardiomyocytes,and the cell supernatant was collected to stimulate neutrophils.To observe the effect of PRR overexpression on neutrophil NETs stimulated by high glucose.Then,the pyrogenic pathway was blocked to observe the effect of PRR overexpression on the formation of NETs.4.Neutrophil production of NETs was stimulated by neutrophil strong stimulant PMA,and NETs were extracted and incubated with primary cardiomyocytes to observe the effect of NETs on pyrodeath of cardiomyocytes.Method1.Construction of adenovirus vector overexpressing PRR:query rat PRR gene sequence(GeneID:302526)according to Genbank,entrust Shanghai GenePharma Company to construct adenovirus coated PRR gene overexpression vector,and construct empty viral vector Ad-EGFP as virus control.2.Construction of DCM rat model:60 of 8-week-old wistar rats were randomly divided into 4 groups after adaptive feeding for a week:control group,DCM group,Ad-PRR group and Ad-EGFP group.DCM group,Ad-PRR group and Ad-EGFP group were injected intraperitoneally with high dose of streptozotocin(65mg/kg)12 hours after fasting.One week later,the rats with blood glucose>11.1mmol/L and typical diabetic symptoms such as polydipsia,polyuria and polyphagia were regarded as successful diabetic models.After 12 weeks of feeding,echocardiography was performed to examine the cardiac function of each group.When the cardiac chamber was enlarged and the systolic function was weakened,the DCM model was considered to be successful.Then,DCM group,Ad-PRR group and Ad-EGFP group were injected with phosphate buffer(PBS),Ad-PRR(1×109fu/100 μL,dissolved in PBS)and Ad-EGFP(1×109fu/100 μL,dissolved in PBS)through tail vein,respectively.Two weeks after virus injection,3 rats in each group were randomly selected to detect the efficiency of virus transfection.Four weeks after virus injection,the rats in each group were anesthetized and the cardiac function was detected by ultrasound,and the samples were taken for the follow-up experiments.3.The body weight and blood glucose of rats were measured before modeling,after successful modeling and before animal sampling.4.Cardiac function test:the rats in each group were anesthetized with 3%chloral hydrate(0.3ml/100g),and the hair in front of the chest was shaved and exposed,and the cardiac function was detected by VEVO770 imaging system.5.The expression of PRR and fibrotic proteins such as collagen Ⅰ,Ⅲ and TGF-β in myocardial tissue was observed by immunohistochemical staining.6.The structural changes of myocardial tissue were observed by HE staining.7.The expression levels of caspase-1,IL-1β and IL-18 in myocardial tissue were observed by immunohistochemical staining.8.Extract primary cardiomyocytes:the hearts of 0-3-day-old Wistar rats were washed with precooled PBS,cut into pieces,digested with collagenase,and the supernatant was extracted and centrifuged at 800rpm and 25℃ for 5min.The precipitates containing cardiomyocytes and fibroblasts were taken and suspended in cardiomyocyte culture medium.According to the different attachment speed of cardiomyocytes and fibroblasts,cardiomyocytes and fibroblasts were separated.The cardiomyocyte suspension was inoculated into a six-hole culture plate for further experiments.9.Intervention of primary cardiomyocytes:Firstly,primary cardiomyocytes were divided into normal group,hyperosmotic group and high glucose group to observe the effect of high glucose stimulation on PRR and NLRP3 related proteins.Secondly,primary cardiomyocytes were divided into normal group,high glucose group,AdPRR group and Ad-EGFP group.Among them,Ad-PRR group and Ad-EGFP group were given high glucose stimulation together with high glucose group after transfection of Ad-PRR and Ad-EGFP,respectively,to observe the effect of PRR overexpression on pyroptosis related proteins such as NLRP3,caspase-1,IL-1β,IL-18 and so on.Finally,the primary cardiomyocytes were divided into Ad-PRR group,AdPRR+AMPK agonist(GSK621)group,Ad-EGFP group and Ad-EGFP+AMPK agonist(GSK621)group.After transfection of Ad-PRR and Ad-EGFP into each group,AMPK agonist GSK621 was given to observe whether PRR regulates cardiomyocyte scorch death through AMPK-NLRP3 pathway.10.Western blotting was used to detect the protein expression of PRR,caspase-1,IL1β and IL-18 in myocardial tissue and cardiomyocytes.11.Neutrophilic granulocyte extraction:Fresh whole peripheral anticoagulant blood was extracted from rats,reagents A and B were added,respectively,and centrifuged 700g for 30min.Two layers of circular opalulocyte cells appeared in the centrifuge tube,the upper layer was mononuclear cell layer and the lower layer was neutrophilic granulocyte layer.The neutrophil layer was carefully absorbed into the centrifuge tube with a straw,cleaned and centrifuged for purification.12.NETs activation:PMA was used to stimulate neutrophil-stimulated NETs,and NETs were extracted and purified to stimulate primary cardiomyocytes to detect the expression of pyroxis-related proteins.13.The levels of IL-1β and IL-18 in culture supernatant of primary cardiomyocytes were detected by enzyme linked immunosorbent assay(ELISA)kit.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.The expression level of PRR,caspase-1,IL-1β and IL-18 were increased in the myocardial tissue of DCM rats.2.Overexpression of PRR can significantly increase collagen fiber deposition in myocardial tissue of DCM rats and worsen cardiac function in DCM rats.3.Overexpression of PRR in primary cardiomyocytes promoted the production of neutrophil Nets,which was reduced in cardiomyocytes incubated with pyrodeath closed receptors.4.NETs can increase the expression of pyroxin protein caused by PRR overexpression and aggravate the cardiomyopathy of diabetic cardiomyopathy.ConclusionIn DCM,hyperglycemia stimulated the increase of PRR expression,accompanied by the increase of DCM scorch death and myocardial fibrosis.Overexpression of PRR can significantly increase the level of pyroptosis in myocardial tissue of DCM,and also increase the level of myocardial fibrosis,thus aggravating the cardiac function injury in DCM.Meanwhile,myocardial PRR overexpression can stimulate the production of neutrophil NETs,which in turn aggravate the pyroptosis of cardiomyocytes and block the reduction of neutrophil Nets in the pyroptosis pathway,suggesting that PRR is involved in the myocardial pyroptosis of DCM by regulating the pyroptosis-Nets loop,which aggravates myocardial injury in diabetic cardiomyopathy.This may provide new ideas and targets for the treatment of DCM.