| Objective: Doxorubicin(DOX) is an effective superior andbroad-spectrum anthracycline antibiotic, widely used in the treatment ofnumerous malignancies. It is one of the most effective chemotherapeutic drugs.However, dose-dependent cardiotoxicity caused by DOX is a major limitationto the application. The mechanisms underlying DOX-induced cardiotoxicity iscomplex and mainly involves increased oxidative/nitrosative stress. Reductionof DOX in cardiomyocytes may generate semiquinone DOX, the latterreduces molecular oxygen(O2) to superoxide anion(·O-2) and H2O2。Theincreased free radical levels in cardiomyocytes may lead to lipid peroxidation,DNA and protein damage. Inflammation and apoptosis may happen in thecardiaomyocytes, which limit the heart function. Recent year, more and moresyudies suggested that the main toxic mechanism of DOX is oxidatice stressand inflammation, and both NO produced by iNOS and·O-2are the keycellular composition, however, the molecular mechanisms are still unknown.Since the inflammation process of tissue injure induced by oxidativestress is mediated by kallikrein–kinin system (KKS). Bradykinin(BK), theeffective end production of KKS system, is an important pro-inflammataryreaction pepetides，mediated the vascular and inflammatory reaction in tissueinjury. Bradykinin acts as agonist of two subtypes of GPCR, B1and B2receptor. The B2receptor is constitutively expressed in cardiac tissue，whichhas high affinity with BK, while B1receptor is almost no expression at basialcondition in cardiac tissue，and has low affinity with BK,buty is increasedafter inflammation and tissue injury，so it belongs to inducible receptor. Andmultiple factor for example pro-inflammatroy cytokines（IL-1）, growth factor（EGF、TNF-α and LPS）and agonists up-regulate B1receptor expression.Both our and other people’s studies have shown that the cardiac tissue in DOX-induced cardiacinjury mainly expressed as hyperaemia andinflammation. The cardiac injury induced by DOX in B1receptor genedeletion(B1-/-) mice is attenuated compared to control. Since there is still existB2receptor in B1-/-mice，it is difficult to judge the whole effects of BK system,and we are still unkown the exact mechanism mediated by BK receptors inDOX induced acute cardiac injury. Therefore, we investaged the effects of BKreceptor double knockout（B1B2-/-）on the devepment of DOX induced acutecardiac injury and subsequent effects on the expression of FABP4, iNOSandeNOS.Methods:1B1B2-/-mice validationB1B2-/-mice were a gift from Professor Hailin Zhang. Two kinds ofprimers were used in examing the genotype through PCR method. mice DNAswere extracted from tail tissues seperately, then PCR were performed usingthe B2Receptor primers provided by Professor Wu Yi(SuZhou University).Since the gene may be lost during the breeding periods, PCR also performedwith B1R and B2R primers at the same time.2DOX-induce acute myocardial injury in mice15healthy female wild-type C57/BL6mice and10B1B2-/-female micewere used. Wild-type mice were divided randomly into three groups:wild-type blank control(Con-A) group, the wild-type DOX10mg/kg(DOX-B)group, wild-type DOX20mg/kg(DOX-C) group, each group has5animals;B1B2-/-mice were divided randomly into2groups: black control(Con-D)group, DOX20mg/kg(DOX-E) group, each group has5animals. DOX(10mg/kg or20mg/kg) was given seperately in DOX group by i.p. for onlyonce, and saline was given to the control group at the same volme of0.2mL/10g.3cardiac enzymes detection and cardiac tissue histopathologyThe mice were weighed then anesthesiaed After5days given DOX,serums were collected to detect the myocardial enzymes(CK, CK-MB, AST,ALT and LDH levels) activity. the heart was weighed and cardiac tissues were fixed for histopathological examination.4The detection of BK receptor expression, FABP4, eNOS and iNOSexpressionQuantitative analysis of B1and B2receptor gene obtained from tail tisuewere performed by PT-PCR method. eNOS, iNOS and FABP4expression incardiac tissue were detected by immunohistochemical methods and westernblot assay.Results:1B1B2-/-mice validationThere is B2R expression in all the wide type animals while there is no B2receptor gene but NEOKi gene expression in the gene knockout mice,Results showed that the knockout out mice lack the gene of B2receptor.Because of the close chromosomal position of both kinin receptor genes, it isthought that B2receptor detection itself is enough to ensure the genetype ofthe mice. To prevent mutations or deletions of the genes during the breedingprocess, both B1R and B2R gene were detected with PCR again, and there arethe mRNA of both B1and B2receptors in wild type and the bands for B1andB2gene were missed in gene knockout mice, The results indicated that thegene deletion mice we used are B1B2-/-mice2The general conditions of miceThere are no significant differences in food intake, body weight and thegeneral activities between Con-A and Con-D groups. There are no significantdecrement or worse for body weight and the general conditions in DOX-B andDOX-E group compared to Con group except that in DOX-C group. NOsignificant differences were observed among the groups of heart organ index（p>0.05respectively）.3Mice cardiac enzyme activity detectionThe serum AST, CK and LDH levels were elevated in Con-B groupCompared with those in Con-A group（p <0.05respectively）, and ALT andCK-MB levels have no significant change compared with those of Con-Agroup（p>0.05respectively）. The serum AST, ALT, CK, LDH and CK-MB levels were higher obviously than those of Con-A group（p <0.05or0.01）.There were no obvious difference in cardiac enzymes levels in Con-D groupcompared to those of Con-A except to the lower levels of CK-MB（p <0.05）.The AST, ALT, CK, LDH and CK-MB levels were all elevated（p <0.05or0.01）in DOX-E group compared with those on Con-D group.. The elevationratios in DOX-E group were lower those in DOX-C group.4Cardiac tissue histopathological examinationThe myocardial cells arranged in neat rows, there were no capillarycongestion, edema and inflammatory cell infiltration observed in Con-A andCon-D group. There are increasing degrees of inflammatory cell infiltration,capillary congestion and vacuolar degeneration of cardiomyocytes observed inDOX-B and DOX-C group especislly in DOX-C group. Inflammation cell andcapillary congestion could found occationlly in DOX-E compared with Con-Dgroup but is much better than those in DOX-B and DOX-C groups.5The gene expression of BK receptorResults from RT-PCR showed th gene expression of B1and B2receptorsin DOX-B and DOX-C group increased significantly compared with Con-Agroup（p<0.01respectively）, while no gene expression of B1or B2receptorwas observed in Con-D and DOX-E group.6The expression of FABP4in cardiac tissueThere is almost no expression for FABP4in the microvascular endothelialcells and cardiamyocytes in Con-A and Con-D groups，While there were alarge amount of expression observed in endothelial cells in DOX-B, DOX-Cand DOX-E group. Western blots of cardiac tissue showed that FABP4levelsincreased dose-dependently in DOX-B and DOX-C compared with Con-A（p<0.01respectively）. FABP4level also increased in DOX-E comparedwith Con-D（p <0.01）。7The expression of eNOS and iNOS in cardiac tissueeNOS was expressed mainly in the cardiomycyte in Con-A and Con-D.The eNOS expression was decreased dose-dependently in DOX-B andDOX-C. There was almost no expression in DOX-C. There were some expression of eNOS in DOX-E group. The Western blot results showed thatthe eNOS expression in DOX-B and DOX-C group were decreseddose-dependently than those in Con-A（p <0.01）. And the expression inDOX-E group also decreased compared with that in DOX-D group（p <0.01）。iNOS mainly expressed in vascular endothelial cells lacated in theinterstinum of cardiomycytes. There were almost no expression in Con-A andCon-D group, while iNOS expression increased dose-denpendently in DOX-Band DOX--C group（p<0.01respectively）. iNOS expression also increased inDOX-E group compared with that in DOX-D group（p<0.01）.Conclusions:1. Our PCR results showed that the gene knockout mice we used wereB1B2-/-mice;2. Compared with wild type mice, there were no obvious changes in thegeneral condition, serum cardiac enzyme levels(except for CK-MBdecrement) and cardiac tissue hispathology in Con-D group comparedwith those in Con-A group.3. DOX caused dose-dependent cardiac injury in mice，up-regulated both thegene expression of B1and B2receptor and the protein expression ofiNOS and FABP4，meanwhile down-regulated the protein expressionof eNOS in cardiac tissue；4. The activation of both B1and B2receptorinvolved in DOX-induced acutecardiac injury，this maybe related with the NO levels mediated by theB1/iNOS and B2/eNOS signal transduction pathway. It seems to noobvious effect on FABP4signal by BKreceptor activation... |
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