Study On The Effects Of Sodium-hydrogen Exchanger Inhibitor (HOE642) In Heart Transplantation Of Dead Rat Caused By Warm Ischemia

Objectives:Setting up a heterotopic heart transplantation model of heart transplantation of warm ischemia death rat,infuse HOE642 (concentration 20μmol/ L)into the rat hearts at different stage after the rat death.Detecting the myocardial cell ultrastructural organization of transplant donator:the change of myocardial cell ATP,SOD,MDA;the expression of Bcl-2,Bax genes through RT-PCR;Bcl-2,Bax,Caspase-3 proteins through immunohistochemistry as well,so as to study the possible mechanism of HOE642 on protecting dead rat myocardium suffered from warm ischemia.Methods:The experiment include two parts.One part is to establish a model of a heterotopic heart transplantation of warm ischemia death rat:25 pairs of rats were studied and treated heterotopic heart transplantation with the method of neck Cuff.Another part is to study the influence of sodium-hydrogen exchanger inhibitor(HOE642)to heart transplantation of dead rats because of warm ischemia:112 clearing SD masculinity rats were divided into 7 groups at random,each group contains 8 rats.The groups are:C,the control group(normal hearts),S₁₀ (the group of transplanted hearts after 10 min of asystole),SH₁₀(group of transplanted hearts after 10 min of asystole and infused with HOE642), S₃₀(group of transplanted hearts after 30 min of asystole),SH₃₀(group oftransplanted hearts after 30 min of asystole and infused with HOE642), S₄₅(the hearts after 45 min of asystole),SH₄₅(the hearts after 45 min of asystole and infused with HOE642).The rats divided in to experimental groups were killed totally by warm ischemia,then,the donators of the groups(S₁₀,S₃₀,S₄₅)were infused with STH-1 for 30 min,and the dead rats in group SH₁₀,SH₃₀and SH₄ were infused with STH-1and HOE642 (20μmol/L)for 30 min,then,heterotopic heart transplantation were processed by the method of neck Cuff.The heart samples of groups (S10,SH10,S30,SH30)were taken after 48 hours of transplantation,and the heart samples of groups(S45,SH45)were taken just after transplantation.Then the samples were detected as follows:observing ultrastructural organization of myocardium through transmission electron microscope;measuring the change of myocardial cell ATP,SOD,MDA through high efficiency liquid chromato-graphy,the expression of Bcl-2,Bax genes through RT-PCR,the expression of Bcl-2,Bax,Caspase-3 proteins through immunohistochemistry and the apoptosis of myocardial cell through TUNEL.Data were expressed by(?)±s.To calculate of means and SD with the software of SPSS13.0,Comparison of groups were performed with student t test,difference between multiple groups were studied with analysis of variance;Treatment of statistic results:P＜0.05 was considered as significant difference statistically,p＜0.01 was considered as extreme difference statistically,P＞0.05 was considered as meaningless statistically.Result:1.Observing the death time of shock caused by blood loss:the rats were discerned death when cardiac electric wave vanished after 9～11 minutes of bloodletting by transsection of abdominal aorta.All the operations were cooperated by 2 staffs,cumulative time of transplantation was 54～75(54±3)min;time of preparing donated hearts was about 4～8(6±2)min,time of transplanting donated hearts is about 3～10(4±5)min, time of receptor preparation was about 12～25(17±7)min.2.The recipient rats in experimental groups of S₁₀,SH₁₀,S₃₀and SH₃₀ all survived the operation of heart transplantation through cervical part over 48 hours;the hearts trans-planted in experimental groups of S₄₅and SH₄₅could not beat again after operation by same ways mentioned above;3.electron microscope test:collagen fibers among the muscle fiber hyperplasy,chondriosome edema as well as myofilament structure of muscle fiber getting vague in the experimental group of S₁₀;most of chondriosome in the group of SH₁₀were normal,only small part of chondriosome getting edema slightly,muscle fiber arrangement was in regular pattern,small part of muscle fiber structure getting vague, sarcoplasmic reticulum getting enlarged,compared with group S₁₀,the changes of group SH₁₀were slighter.In the experimental group of S₃₀. inflammatory cell infiltrate muscle interstitium,karyopycnosis, ambonuclear edema,tunica muscularis chondriososme getting vacuolus, myofilament structure getting vague,chondriosome arrangement disorder, sarcoplasmic reticulum was enlarged obviously,muscle interstitial edema, muscle fibrous structure getting vague,by comparison,the change in SH₃₀group was a little bit slitht.4.Compared with S₁₀group,the contents of ATP in group SH₁₀was higher,the discrepancy was significant(P＜0.05),the contents of ATP in group SH₃₀was higher then S₃₀one,the discrepancy was significant as well,however,there was no significant difference between group S₄₅and SH₄₅(P＞0.05).5.The contents of SOD in the experimental group of S₁₀,SH₁₀, S₃₀,SH₃₀,S₄₅and SH₄₅were lower then the control group,however,the contents of MDA were higher then the latter,among the total,the SOD in group S₁₀was lower then which in group SH₃₀,then,the MDA was higher,and the difference was significant(P＜0.05).Comparing group S₃₀ and SH₃₀,there was extreme difference(P＜0.01).6.The apoptosis of cardiac muscle increased obviously with the warm ischemia time by the means of TUNEL test.Based on the RT-PCR test;the BcL-2 genes detected by electrophoretogram in control group expressed strongly,and ROD value was maximum,meanwhile,the electrophoretogram of Bax genes displayed most faint in the control group,and ROD value was minimum,otherwise,compared with the control group based on immunohistochemistry detection,Caspase-3 protein expression in the experimental groups was on the rise,the level of BcL-2 protein expression decreased.Bax protein expression increased and there was direct correlation with warm ischemia time.7.By the means of TUNEL test,cardiac muscle cells with TUNEL positive in group S₁₀were more then which in group SH₁₀,the difference was significant(P＜0.05),the same,cardiac muscle cells with TUNEL positive in group S₃₀were more then which in group SH₃₀,the difference was also significant(P＜0.01).Based on the RT-PCR test,the ROD value of BcL-2 genes in group S₁₀was less then group SH₁₀,meanwhile,which in group S₃₀was more then in group SH₃₀.however,the ROD value was just opposite,the difference mentioned above was all significant.8.compared with the group S₁₀based on immunohistochemistry detection,the level of BcL-2 protein expression was less then group SH₁₀, and the group S₃₀was more then SH₃₀on the aspect of BcL-2 protein expression.However,the level of Bax,Caspase-3 protein expression was just opposite,the difference was totally significant statistically,there was no statistical difference between group of S₄₅and SH₄₅.Conclusion:1.The model of a heterotopic heart transplantation on the cervical part is convenient,easy,stable and reliable.It's a kind of ideal animal model for base researches,such as ischemia/reperfusion injury,immune tolerance of transplantation,cardiac muscle protection and so on.2.HOE642 can increase the composition of ATP in hearts of dead rats(within 30 min)caused by warm ischemia,improve energy metabolism of hearts.3.HOE642 can increase the contents of SOD in hearts of dead rats (within 30 min)caused by warm ischemia,raise the capability of clearing oxygen free radicals,and decrease MDA so as to relieve lipid peroxidation injury.4.HOE642 can suppress rat cardiac muscle cells apoptosis(within 30 min)after death caused by warm ischemia.5.Its almost useless for rat hearts to use HOE642 after the death out of 45 min. new ideas:1.It`s initiated to carry out heart heterotopic graft of death rats caused by warm ischemia domestically2.The researches on HOE642 protecting transplanted hearts of death rats were never reported both home and abroad.Shortage:because of restriction of funds and equipment,detected data may not be overall and observing time may be short...