| IntroductionPost-myocardial infarction(MI)remodeling,including the changes of car-diocytes and extracellular matrix,affects cardiac function and the prognosis for survival.Both cardiac renin-angiotensin-aldosterone system(RAAS)and tumor necrosis factor-alpha(TNF-α)are activated after MI,which might play an important role in the development and progression of ventricular remodeling. Besides,apoptosis of cardiocytes,interstitial fibrosis,genes expression of apop-tosis and matrix metalloproteinases(MMPs)might also play a significant role during the course.Some data demonstrated a significant interaction between the cardiac RAAS and remodeling,therefore,blockading the RAAS with angiotensin converting enzyme inhibitor(ACEI)or angiotensin receptor blocker(ARB) might act as a potential target for the treatment.In the present study,we determined some indices,including cardiac AngⅡ,aldosterone(ALD),Bcl-2,P53,TNF-α,collagen type 1,collagen type 3, Matrix metalloproteinase-9(MMP-9),caspase-3 and collagen volume frac-tion(CVF)in the non-infarct zone in a rat model of MI,and investigated the effects of ramipril,a long acting ACEI,on cardiac interstitial collagen and apop-tosis of cardiocytes.Materials and Methods250 healthy female Wistar rats,weighing 250-300g,were randomly divid- ed into 3 groups.In MI group and drug group,each respectively had 100 rats and the other 50 rats belonged to the sham-operation group.A left thoraeotomy was performed in the fourth intercostal space,the pericardium was opened with a clamp and the heart was exteriorized by applying pressure to the right aspect of the thoracic cage.The left coronary artery was then occluded 2 to 3 mm from its origin with 6-0 silk suture.The chest was closed fleetly and the rat was allowed to recover.All rats of drug group received ramipril solution 1 mg/kg/d in one milliter tap water by intragastric administration.In sham-operation group,50 rats were treated in the same way as previously described except for coronary ligation.The rats surviving 3 hours after operation in the three groups were 53,48 and 48 respectively.The rats with less than 20%of infarction size were exclu ded from analysis.The rats fitting the standard of experiment were allocated to 4 time points.After 24 hours,7 days,14 days and 28 days of operation,all rats were killed.The heart was excised and then the LV including the septum was cut into two parts from apex to base,the apex of heart was immersed into 4% paraformaldehyde in phosphate-puffered saline for more than 24 hours.Subse quently,the heart was embeded in paraffin,transverse serial sections 5μm thick were cut.The changes of left ventricular structure were observed by HE stai ning;Messon staining was used for the determination of collagen volume fraction (CVF);Immunohistochemical staining(SP method)was used for the determi nation of TNF-α,MMP-9 and caspase-3.TUNEL was used for detection of apoptotie cells.The remaining non-infarct myoeardium was cut into two parts, one was immersed into 2.5%Glutaral solution for ultrastructure observation by transmission electron microscope,the other was immediately put in liquid nitro gen and stored at-70℃for cardiac AngⅡand Ald contents by radioimmunoas-say and genes expression of collagen type 1(col-1),collagen type 3(col-3),Bel-2 and p53 by reverse transcriptase-polymerase chain reaction(RT-PCR).The following primer sequences were used;sense 5'-CAGATTGAGA ACATCCGCAGC,antisense TCTAGCTCGAGTCCCCGC-5'(481BP)for col -1;sense 5'-AGGATCTGAGGGCTCGCC,antisense AGTTCTCGCCTCTTAT-GACCC-5'(314bp)for col-3;sense 5'-GACCGTCGGACAGAGGAAGA, antisense GTAGTGGTCGGAGGGGC-5'(379bp)for p53;sense 5'-TGGTG- GACAACATCGCTCTG,antisense CGTTTACCAGTTTAGTCGATAAATG-5' (412bp)for Bcl-2;sense 5'-TGTGCTATGTTGCCCTAGACTTC,antisense TCGTCCTCATGCTACTCAGGC-5'(450bp)forμ-actin.PCR product 8μL underwent electrophoresis on 2%agarose gels,the level of col-1 or col-3 was expressed as the ratio of col-1 mRNA grey level or col-3 mRNA grey level toμ-actin mRNA grey level,the level of Bcl-2 or p53 was expressed as the ra tio of Bcl-2 mRNA grey level or p53 mRNA grey level toβ-actin mRNA grey level by a gels image system(Band Leader Ver 3.0).Statistical analysisAll results were expressed as M±S.SPSS 11.5 software was adopted to ex-amine all values,A value of P＜0.05 is considered statistically significant.Results1.General state of ratsOf the 149 rats surviving 3 hours after operation,22 died during the subse-quent intervention course,the mortality was 14.8%.Within 24 hours after oper-ation,15 rats died,with 10 in MI group,4 in drug group and 1 in sham-opera-tion group;7 days after operation,6 rats died,with 4 in MI group and 2 in drug group;14 days after operation,1 rat died in MI group.Of which,8 rats died from heart failure,the other had no clear reasons.In the end,116 rats were analysed, with 47 in sham-operation,32 in MI group and 37 in drug group.2.Morphometric analysis with HE stainingLeft coronary artery occlusion produced myocardial infarction in 69 rats,the infarction sizes were 23%-35%.The left ventricular absolute and relative weight(LVAW and LVRW),the left ventricular transverse diameter and spheric-ity indices of the three groups were similar(P＞0.05)24 hours after operation; Compared with sham-operation rats,the LVAW,LVRW and left ventricular transverse diameter increased markedly and the left ventricular sphericity indices decreased in MI rats 7 days after operation,especially after 14 days of the opera-tion(P＜0.05～0.001).The above-mentioned indices were improved marked-ly from 14 days to 28 days after treatment with ramipril(P＜0.05). 3.Morphometric analysis with Messon stainingCollagen proliferation was much more obvious in MI group than in sham-operation group 14 days after operation,The CVF increased gradually alonging with the course of infarction(P＜0.05～0.01)and decreased 28 days after treatment with ramipril(P＜0.05).4.cardiocytes ApoptosisThere were no apoptotic cardiocytes in the rats of sham-operation group and in the rats of MI and drug groups 24 hours after surgery(p＞0.05).The yellow-brown-colour apoptotic nuclei were observed and the apoptotic index increased gradually in the rats of MI and drug groups from 1 week to 4 weeks af-ter operation.The apoptotic index decreased markedly in the rats of drug group 4 weeks after treatment with ramipril(P＜0.05).5.Ultrastructure changesThere were changes in different degree in cardiocytes and collagen fibers, some of these changes included nuclei shrinkage,nuclear membrance incisure, mass chromatin and mitochondria swelling in MI and drug groups.The interstitial fibroblasts and collagen fibers increased in MI group and decreased after treat-mere with ramipril.6.Changes of TNF-αThe location of TNF-αwas mainly cardiac interstitium.The tissue from the sham-operation group exhibited weak expression of TNF-α.The expression of TNF-αincreased and grey level was lower in MI group(P＜0.05～0.01);The expression of TNF-αreduced in drug group 14 days after treatment with ramiprilP＜0.05).7.Changes of MMP-9There were not changes of MMP-9 expression between the three groups at each time point(P＞0.05).8.Changes of caspase-3Staining grey levels of positive products of caspase-3 were similar in the three groups from 24 hours to 7 days after operation;Compared with sham-op-eration group,the number of positive cardiocytes of caspase-3 increased in MI and drug groups 14 days after operation(P＜0.05),but the changes of caspase -3 were not obvious after treatment with ramipril(P＞0.05).9.Changes of AngⅡand ALDDuring the duration from 7 days to 28 days,the AngⅡlevel increased grad-ually(P＜0.05～0.001)and decreased only 14 days after treatment with ramipril(P＜0.05).ALD level also increased gradually from 14 days to 28 days (P＜0.05～0.001)and decreased only 28 days after treatment with ramipril(P＜0.05).10.Expression of col-1 and col-3 mRNAThe expression levels of col-1 and col-3 mRNA were similar in the three groups 24 hours after operation.The ratio of col-3 mRNA grey level toβ-ac-tin mRNA grey level was significantly higher from 7 days to 28 days after opera-tion in MI group(P＜0.05);The ratio of col-1 mRNA grey level toβ-actin mRNA grey level was markedly higher from 14 days to 28 days after operation in MI group(P＜0.05～0.01).Ramipril decreased the ratio of col-1 mRNA grey level toβ-actin mRNA grey level 28 days after treatment,but it did not re-duced the ratio of col-3 mRNA grey level toβ-actin mRNA grey level from 7 days to 28 days after treatment(P＞0.05).11.Expression of Bcl-2 and p53 mRNAThe expression levels of Bcl-2 and p53 mRNA were similar in the three groups 24 hours after operation.Compared with sham-operation group,the ratio of p53 mRNA grey level toβ-actin mRNA grey level increased significantly(P＜0.05～0.01)and the ratio of Bcl-2 mRNA grey level toβ-actin mRNA grey level decreased obviously in MI group(P＜0.05～0.01).Ramipril had no effects on expression of p53 mRNA or Bcl-2 mRNA(P＞0.05).ConclusionBased on the results of this study,we conclude;1.Ventricular remodeling happened 7 days after MI in rats.Using ramipril early and persistently had reverse effects on the remodeling.2.There was interstitial fibrosis in non-infarct zone 14 days after MI in rats,which might be associated with augmentation of AngⅡand upregulation of TNF-α3.Apoptosis happened in non-infarct zone 14 days after MI in rats,which might concerned with augmentation of AngⅡand upregulation of TNF-α.In addition,activated caspase-3,upregulation of p53,downregulation of Bcl-2 and excess deposition of interstitial collagen might also play important roles.4.Ramipril might inhibit the interstitial fibrosis and the apoptosis of cardio-cytes by reducing AngⅡlevel and inhibiting expression of TNF-α.Besides, lessening the deposition of interstitial collagen to inhibit the apoptosis of cardio-cytes might also become one of the therapeutic mechanisms of ramipril. |
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