The Role Of(Pro)renin Receptor On Myocardial Injury In Diabetic Cardiomyopathy Rats

Objective1.To observe the expression of(Pro)renin receptor(PRR),a novel component of the renin-angiotensin system(RAS),in myocardium in diabetic cardiomyopathy rats.Meanwhile the pathological changes in myocardium are also detected.2-To detect the effect of PRR RNA interference silencing on myocardial injury in diabetic cardiomyopathy rats,especially in cardiac function,myocardial fibrosis and so on.The underlying mechanism is also explored.Method1.PRR shRNA construction:Three adenovirus carried PRR shRNA(Ad-PRR-shRNA),including Ad-PRR-shRNA-547,Ad-PRR-shRNA-691 and Ad-PRR-shRNA-274,and adenovirus-scrambled-shRNA(Ad-SC-shRNA)were successfully constructed by GenePharma Company(Shanghai,China).H9C2 cell linewas used to choose the most efficient PRR-shRNA fragment,the Ad-PRR-shRNA-547 was selected for further experiments and its sequence of PRR is GCT CCG TAA TCG CCT GTT TCA.2.Animal model establishment:Sixty male Wistar rats weighing 200 g to 250 g were randomly divided into normal control group(n = 15)and model group(n = 45).The rats in the treatment group were used to construct a DCM model by a single intraperitoneal injection of streptozotocin(STZ)(65 mg/kg).The normal control group was injected with the same dose of citrate buffer.One week after STZ injection,rats with blood glucose levels>11.1 mmol/L together with classic diabetic symptoms were regarded as successful diabetes model.Then,12 weeks after the STZ administration,the diabetic rats were further divided into three sub-groups,the DCM group,the Ad-SC-shRNA group and the Ad-PRR-shRNA group(n=15 per group).The rats in the Ad-SC-shRNA group,Ad-PRR-shRNA group and DCM group received intravenous injection of recombinant adenovirus containing Scramble-shRNA(1×109pfu),adenovirus containing PRR-shRNA(1×109pfu)or PBS respectively via the tail vein.2 weeks after adenovirus injection,5 rats in each group were randomly chosen for detecting the efficiency and efficacy of virus transfection.At 4 weeks after the adenovirus injection,all rest rats were anaesthetized with an intraperitoneal injection of 10%chloral hydrate(300 mg/kg)for cardiac function tests and the myocardium of the left ventricle was collected for pathological and biochemical analyses.3.Blood glucose and weight measurement in rats:Body weight and blood glucose in 4 groups rats were measured before and after modeling and at the end of expriments respectively.4.Cardiac function test:At the end of experiments,the heart function of each group was detected by echocardiography under anesthesia condition by intraperitoneal inj ection of 10%chloral hydrate 3ml/kg.5.Masson staining observed myocardial fibrosis6.Immunohistochemical staining:The 4.5 ?m-thick sections obtained from paraffin-embedded myocardial tissues from the 4 groups were used to detect the expressions of collagen ?,collage ?,transforming growth factor(TGF-?)in myocardium.7.Real-time PCR:PRR gene fluorescence quantitative detection.8.Western blotting(Western blot):The total protein was extracted from the myocardium of rats or cardiac fibroblasts and the expression of PRR proteinand ERK1/2 pathway proteins were detected by electrophoresis.9.Cell culture and treatments:Cardiac fibroblasts were extracted from the myocardium of 1-to 3-day-old neonatal rats.After fully removed the myocardial matrix with collagenase and based on the difference in growth rates,cardiac fibroblasts were distinguished from myocardial cells.The cardiac fibroblasts were identified by cardiac fibroblast vimentin expression detection,and second-to third-generation cardiac fibroblasts were transplanted into six-well cell plates for further experiments.To determine the influence of high glucose on PRR expression in cardiac fibroblasts,the cardiac fibroblasts were divided into 3 groups,including the normal glucose group(5.5mM glucose),high glucose group(25mM glucose)and mannitol high permeability control group(19.5mM mannitol+5.5mM glucose),and PRR expression levels were detected by immunofluorescence staining.10.Ad-shRNA transfection and treatments in vitro:The cardiac fibroblasts from the high glucose group were divided into 4 groups,including the high glucose group,Ad-SC-shRNA group,Ad-PRR-shRNA group and losartan group.The cardiac fibroblast(1×108)was transplanted into six-well plates.The cells were cultivated in culture medium without serum for more than 12 hours first,and then Ad-PRR-shRNA and Ad-scrambled-shRNA were transfected into the Ad-PRR-shRNA group and Ad-SC-shRNA group,respectively with MOI value up to 150:1,and in the losartan group,the cardiac fibroblasts were first pretreated with losartan(20 ?M)in normal glucose culture medium for 1 h and then treated with 25mM glucose and drugs for another 12 h.11.Immunofluorescence staining:The cardiac fibroblasts were seeded in 24-well chamber slides and treated with normal glucose medium,mannitol high permeability medium and high glucose medium for 24 h.PRR protein were detected by immunofluorescence staining.12.Enzyme linked immunosorbent assay(ELISA):Cardiac fibroblasts in 4 groups,including the high glucose group,Ad-SC-shRNA group,Ad-PRR-shRNA group and losartan group.The cell supernatant from each group was collected for ELISA to detect the expression of collagen ?,collagen ? and transforming growth factor-?(TGF-?)in the different groups.All measurements were performed according to the instructions from the manufacturers of the ELISA Kit(Abcam,Cambridge,MA).13.Statistical analysis:Statistical analyses were performed using SPSS 19.0 software.All data are expressed as the mean ± standard deviation of at least three representative independent experiments.The differences were analysed by one-way ANOVA.A P value<0.05 was considered significant.ResultsThe results showed that firstly,RRR protein expression was significantly increased in DCM rats.PRR RNAi silencing alleviated myocardial fibrosis and also improved cardiac function in DCM.We also observed the effects of PRR RNAi silencing on high glucose stimulated cardiac fibroblasts,and the results showed that PRR RNAi silencing down-regulated the expressions of collagen ?,collagen ? and TGF-?.ConclusionWe conclude that high glucose condition could up-regulate PRR expression in myocardium and PRR plays a key role in the pathological progression of DCM and that inhibition of PRR expression achieved by specific PRR RNAi silencing offers a new therapeutic approach for DCM.The underlying mechanisms of these effects may be associated with the ERK signalling pathway.