| Objective:1.Investigating the effects of ?3-adrenoceptors(?3-AR)on myocardium structure of rat hearts using coronary artery ligation.2.Exploring the effect of SR 59230 A on MicroRNAs expression in rat myocardium and observing the related mechanisms of ?3-AR on MicroRNAs expression in rat hearts.Methods:1.Establishment of Chronic Hearts Failure: 100 Male Sprague-Dawley(SD)Rats were randomly divided into Sham operated group(40)and Chronic Heart Failure group(60).Coronary artery ligation was used to induce Chronic Heart Failure(CHF).The left anterior descending coronary artery was occluded with a black braided silk suture of ~2mm below its origin in CHF group.The ligature was placed but not tied in the sham-operated animals.Normal Electrocardiogram(ECG)was recorded before operation.During operation,we have monitored the ECG changes in real-time.Eight weeks after operation,the experimental rats were performed in Echocardiography,Left ventricular Internal Dimension Systole(LVIDS)and Left ventricular Internal Diastolic Diameter(LVIDD)were measured.The ejection fraction(EF)and fractional shortening(%FS)were calculated.Rats with ejection fraction(EF)values in echocardiography ranging from 40-65%(heart failure)were selected to be the CHF group rats for further studies.2.Animal Grouping: the rats in Sham group were divided into Sham control group(Sham)and Sham+SR group(Sham+SR).Rats of the CHF group were also randomly divided into CHF control group(CHF)and CHF+SR59230A group(CHF+SR).3.The effect of ?3-AR inhibition on MicroRNAs expression: the Sham+SR group and CHF+SR group received intraperitoneal injection of 1 ml SR59230A(85 mmol/l in0.9% saline)twice a day for seven weeks,while the control groups were injected with same amount of saline for seven weeks separately.After killing of the rats,extraction of rat myocardial RNA was extracted,using NanoDrop2000 C to detect the concentration and the quality of RNA,miScript miRNA PCR Arrays were used to measure the expression profile of MicroRNAs.4.The changes of heart structure and related signal transduction pathways: the heart tissue specimen of left ventricle was soaked in 10% formalin,then dehydrated and embedded in paraffin,5?m-thick slices were obtained from the section and stained with hematoxylin-eosin(H&E).The morphological and structural changes of the rat hearts were obtained from microscopic observation.Immunohistochemistry was carried out to evaluate the distribution of the related proteins ?3-AR,NF-?B and p53 in the heart tissues.Weston Blot was used to evaluate the proteins expression of the offline ligation part of rats myocardial tissue,quantitative analysis of ?3-AR,NF-?B,p53 and p53-Phospho-Serine 15 proteins were carried out to define the possible signaling pathway of the structural and functional changes by ?3-AR.Results:1.Results of Echocardiography: Echocardiography was used to measuring LVIDD,LVIDS and EF,FS values were also calculated.The results showed that the heart function of CHF rats injured obviously compared with the Sham group(P<0.05,P<0.05),whereas EF and FS were significantly decreased(P<0.01,P<0.01),which proved the CHF model was succesful.2.SR 59230 A on the expression of MicroRNAs in rat hearts: MicroRNA microarray analysis showed that,after in vivo blocking of ?3-AR by SR 59230 A,there were 18 MicroRNAs collectively down-regulated in Sham control group and CHF control group.Within them,6 MicroRNAs were related to NF-?B signaling pathway,they were mi R-125b-5p,mi R-143-3p,miR-145-5p,miR-26a-5p,miR-30a-5p and miR-320-5p.3.H&E staining of cardiac structure and histopathological changes: In sham control group,the rats myocardial tissue was intact,no damage was seen and the and myocardial cells rowed neatly,the nuclear staining was clear.However,in CHF control group,myocardial tissue was shrinkaged in large extent,myocardial fibers swelled,fractured,interstitial edema was also observed.In addition,disordered arrangement of myocardial cells were observed and cells arranged loosely.In vivo SR 59230 A application,the myocardial tissue of disordered arrangement cells in CHF group were alleviated,myocardial fibers fracture and inflammatory cells were decreased,there were less inflammation cells and inflammation was alleviated.4.Immunohistochemical results: The result showed in CHF control group,the?3-AR expression was significantly increased(P<0.01)and NF-?B p65 was expressed almost equally in the nucleus and cytoplasm compared with the sham control group;p53 was expressed mainly in the cytoblast in CHF control group.The immunoreactive score(IRS)result showed an increased expression of NF-?B p65 and p53 in cardiac tissue of CHF control group compared with sham control group(P<0.01,P<0.01).The expression of NF-?B p65 and p53 proteins in CHF+SR group decreased both in nucleus and cytoplasm compared with the CHF control group(P<0.05,P<0.05).The NF-?B p65 and p53 proteins expression increased in Sham+SR group compared with the sham control group.(P<0.01,P<0.01).5.Western Blot results: The expression of ?3-AR,NF-?B p65 and p53-Phospho-Serine 15 proteins were significantly increased in CHF control group compared with the Sham control group(P<0.05,P<0.05,P<0.05).p53 protein expression in CHF control group were also higher than the sham control group,but there was no significant difference.After in vivo SR 59230 A application,expression of NF-?B p65 and p53-Phospho-Serine 15 protein were significantly decreased in CHF+SR group compared with the CHF control group(P<0.05,P<0.01),but they were still higher than those in the Sham control group(P<0.05,P<0.05).There was nosignificant difference on p53 protein expression in CHF+SR 59230 A group compared with the CHF control group.However,the expression of NF-?B p65,p53 and p53-Phospho-Serine 15 protein increased significantly in the Sham+SR group compared with the Sham control group(P<0.05,P<0.05,P<0.05).Conclusions:1.After in vivo blocking the ?3-AR,the abnormal morphological structure and the tendency of inflammation on Chronic Heart Failure rats were alleviated,indicating higher ?3-AR expression is related to the damage of cardiac structure.2.In vivo SR 59230 A application caused the change of MicroRNAs expression,in which miR-125b-5p,mi R-143-3p,miR-145a-5p,miR-26a-5p,miR-30a-5p and miR-320-5p,they were related to NF-?B signaling pathway.3.The expression of ?3-AR,NF-?B p65 and p53-Phospho-Serine 15 proteins increased in Chronic Heart Failure rats.Aftre blockade of ?3-AR with SR 59230 A,the NF-?B p65 and p53-Phospho-Serine 15 proteins expression in CHF group were decreased,showing that the effect of ?3-AR in heart is related to NF-?B expression and p53 phosphorylation. |
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