Study On The Mechanism Of MCA Combined With G-CSF On Cardiac Function In Rats With Diastolic Heart Failure

Objective:Through the observation of effect of meglumine cyclic adenosine monophosphate(MCA)combined with granulocyte colony stimulating factor(G-CSF)on cardiac function in diastolic heart failure(DHF)rats,and the influence on cAMP/PKA signal pathway and cardiomyocyte-specific genes expression in bone marrow mesenchymal stem cells(BMSCs),and the influence on serum markers of BNP,CA125,hs-CRP and ANG-2,to explore the effects of MCA combined with G-CSF on heart function of DHF rats and its possible mechanism.Methods:60 SD male rats modeled were divided into 4 groups,control group(Control),diastolic heart failure model group(Model),MCA treatment group(MCA)and MCA combined with G-CSF treatment group(MCA+G-CSF),each group of which was given different treatment for 15 days.After the intervention,cardiac function was measured by BL-420F physiological recorder and echocardiography,myocardial changes of rats were detected by immunohistochemical detection.RT-PCR and Western blot were used to identify the expression of cardiac specific genes GATA-4,Cx43,cTNI and c-kit in BMSCs,ELISA was used to determine the level of cAMP and BNP,CA125,hs-CRP,ANG-2 in cells,flow cytometry was used to detect the differentiation of BMSCs cells.Results:First,the hemodynamic analysis of modeled rats showed that MCA and MCA+G-CSF treatment significantly improved the cardiac contractile function,at the same time improved their diastolic function.We found that MCA and G-CSF mainly decreased the LVDd and LVM index to restore the cardiac function of rats through echocardiography checking.The histological findings of myocardial tissue of rats with diastolic heart failure showed fibrosis and necrosis,edema,muscle fiber fracture,arrangement of chaos,while MCA can reduce myocardial cell edema,and G-CSF promote myocardial cell regeneration,which worked together to recover the cardiac function.Secondly,by detecting the changes of cardiomyocyte-specific genes and their proteins,we found that the expression of GATA-4 and Cx43 gene in DHF model rats was significantly higher than that in Control group(P<0.05),and they were significantly higher in MCA group and MC A+G-CSF group than that in Model group(P<0.05).The trend of cTNI and c-kit protein of stem cells was consistent with GATA-4 and Cx43,which indicated that MCA and G-CSF could promote the expression of cardiomyocyte-specific genes and proteins,and promote the development of cardiomyocytes,thus restore the heart function of DHF rats.MCA and G-CSF had a significant effect on increasing intracellular cAMP levels,which could improve the viability ofcardiomyocytes.Thirdly,the anti-apoptotic gene BcL-2 and its protein expression decreased,while the expression of apoptosis gene Bax was significantly increased,indicating that the change of the expression explained on the emergence of myocardial cell apoptosis,necrosis,edema,muscle fibers,fibrous rupture,arrhythmia and myocardial fibrosis.MCA could promote the expression of BcL-2 at mRNA level and protein level,inhibit the expression of Bax,the combination of MCA and G-CSF played a more important role in regulating the expression of the two genes,which could reduce the apoptosis of cardiomyocytes and improve the cardiac function of heart failure rats.Finally,the levels of serum BNP,CA125,hs-CRP and ANG-2 were the highest in the heart failure rats of Model group.When using MCA alone and MCA combined with G-CSF,the levels of the serum factors declined significantly,which was consistent with their functions.MCA combined with G-CSF decreased the most significantly.It showed that the degree of differentiation of BMSCs was the lowest in the Control group,and the degree of Model group,MCA group and MCA+G-CSF group increased in turn,which was consistent with the expression of the cardiomyocyte-specific genes.Conclusion:G-CSF can regulate cell apoptosis,MCA combined with G-CSF can significantly improve the cardiac function of rats with diastolic heart failure,and the effect of the combination treatment is obviously superior to MCA alone.MCA and G-CSF can regulate the differentiation of BMSCs through cAMP/PKA signaling pathway,promoting the expression of GATA-4 and Cx43 in the early stage of cardiomyocyte development and the differentiation of BMSCs into cardiomyocytes.MCA combined with G-CSF can regulate the expression of apoptosis gene,delaying myocardial cell apoptosis,besides,they can regulate the expression of serum related factors.The efficacy of MCA combined with G-CSF in the treatment of DHF rats was significantly better than that of MCA alone.