Function Of Receptor Operated Calcium Chnnel In Daunorubicin Myocardial Injury

To investigate the role of receptor-operated channels in daunorubicin-induced myocardial injury and its pathophysiological significance in animals and cell models.Method:?Animal Model?Sprague Dawley rats(110-130 g,male)were randomly divided into two groups(n=10 each)as follows: normal control group(Control),model group(DNR).The DNR group was intraperitoneally injected with daunorubicin once a week for 4 weeks at 2.5 mg/kg.In the normal group,normal saline was intraperitoneally injected once a week for 4 weeks,and every 5 ml/kg was administered;One week after the last administration,rats in each group were intubated into the left ventricle.The histological structure of the myocardium was observed by HE staining.The expression of TRPCs in the myocardium was detected by Real-time PCR and Western Blot.?Cell Model? Cardiomyocytes from neonate(1-3d)were isolated and cultured in vitro,20 males and females,experimental group: normal control group,daunorubicin group(1?M DNR treatment for 24h),cell culture supernatant was measured.For the purpose of analysis in LDH and CK,3 segments were detected as follows:immunofluorescence,Real-time PCR detection and Western Blot.Furthermore,detect cell proliferation and TRPCs expression were extracted in each group.They were further grouped into DNR +OAG group(100?M OAG,ROCC receptor agonist pretreatment 2 h,1?M DNR treatment for 24 h),DNR + CPA group(10?M CPA,SOCC receptor agonist pretreatment 2 h,1?M DNR treatment 24 h),In the DNR+SKF group(5?M SKF96365,ROCC receptor blocker was pretreated for 2 h,followed by 1?M DNR for 24 h),THE cell proliferation was assessed by MTT.RESULTS:?Animal Model?Rats with intraperitoneal injection of daunorubicin for 4 weeks exhibited myocardial injury,manifested marked decreased activity,lethargy,poor mental status,slow weight gain,diarrhea,and death.Myocardial damage enzymes increased,left ventricular systolic and diastolic function decreased,myocardial parenchymal cells decreased,and structural damage changed.Compared with the normal group,the levels of LDH and CK in the daunorubicin group increased(p<0.05).In comparison with the normal group,the rat left ventricle was cannulated,the m LVDP in the daunorubicin group decreased(P<0.05),the heart rate slowed(P<0.05),the LVP-dp/dtmax decreased(P<0.05),and the LVP+dp The /dtmax decreased(P<0.05).There was no significant difference between m LVSP and m LVP(P>0.05)HE staining of rat myocardial tissue showed that the myocardial fibers of the normal rats were regular and well-distributed,with no inflammatory cell infiltration;Myocardial cells in the mycin group had large nuclei,malformations,deep staining,and disordered myocardium fibers.The cytoplasm was dissolved and the number of inflammatory cells was increased.In the daunorubicin group,the Real-time PCR showed that the expression of TRPC3 and TRPC6 was significantly higher than that in control group(P<0.05),and there was no significant difference in the m RNA expression of TRPC 7(P>0.05).The results of Western Blot in daunorubicin group indicated that,compared with the normal group,the expression of TRPC6 protein in daunorubicin group was up-regulated(P<0.05),and there was no significant difference of expression between TRPC3 and TRPC 7 protein(P>0.05).?Cell Model?Microscopic observation:Primary cultured neonatal rat cardiomyocytes,myocardial cells can be intertwined into a network,showing that myocardial cells with a unified rhythm into pieces pulsed,good growth state can be used to prepare models.Compared with the normal group,the24-hour survival rate of daunorubicin 1?M was 75.08±1.26%(P<0.05),so the concentration of daunorubicin in the injury group was set at 1?M,and a cell injury model was established.At the same time,it was observed that the morphological changes found that after treatment with 1?M DNR for 24 h,the cells lost their normal spindle or star shape,their nucleus was pyknosis,and the cytoplasm vacuolated.Compared with the normal group,the levels of CK and LDH in the cardiomyocyte culture fluid of the daunorubicin group increased(P<0.01).The results of real-time PCR showed that compared with the normal group,the expression of TRPC3,TRPC6 and TRPC 7 m RNA in the cardiomyocytes of the daunorubicin group showed increased(P<0.05).The results of Western Blot in cardiomyocytes of neonatal rats in the DNR group showed that the expression of TRPC6 protein was up-regulated in the DNR group compared with the control group(P<0.05),and there was no significant difference in the expression of TRPC3 and TRPC7 proteins(P>0.05).Compared with the normal group,OAG,CPA and SKF96365 alone had no significant effect on the survival rate of cardiomyocytes in normal neonatal mice(P>0.05).Compared with the normal group,the survival rate of suckling mice in the DNR group was decreased(P<0.05).Compared with the DNR group,the cell survival rate was increased in the DNR+SKF group(P<0.05),and the cell survival rate was decreased in the DNR+OAG group(P<0.05).Compared with DNR group,the cell survival rate of DNR+CPA group had no significant difference(P>0.05).Observation by immunofluorescence showed that the TRPC6 polyclonal antibody was green fluorescence,while the DAPI-stained nuclei showed blue fluorescence.Compared with the control group,TRPC6 immunofluorescence in the neonatal rat cardiomyocytes in the DNR group was positive in the injured cardiomyocytes,and the immunoglobulin in the control group was weakly positive in the normal cells.