The Regulatory Mechanisms Of Yulangsan MHBFC Reversing Ventricular Remodeling Rats Based On ENOS-NO Signaling Pathway

Objective: To investigate the regulatory mechanisms of 17-methoxyl-7-hydroxy-benzene-furanchalcone(MHBFC)reversing ventricular remodeling rats by e NOS-NO signaling pathway,and elucidate the molecular mechanisms of MHBFC reversing ventricular remodeling.Methods: The abdominal aorta of the male SD rats was narrowed to the outer diameter of 0.7mm to induce cardiac hypertrophy and ventricular remodeling.The rats in the sham-operated group were only free of abdominal aorta,but they were not narrowed.Three days after the operation,the living rats were randomly divided into 6 groups,10 in each group as follows: sham-operated group,model group,MHBFC 6 mg﹒kg-1 group,MHBFC 12 mg﹒kg-1 group,MHBFC 12 mg﹒kg-1 + Nω-Nitro-L-arginine methyl ester hydrochloride(L-NAME)50 mg﹒kg-1 group and L-NAME 50 mg﹒kg-1 group.After grouping,the corresponding drugs were given to the stomach,1 times a day for 6 weeks.(1)Hematoxylin-eosin stain(HE)and Masson?s stain were used to observe the pathological changes of myocardium.The nitric acid reduction method was used to detect the content of serum nitric oxide(NO).Serum endothelial nitric oxide synthase(e NOS)level was detected by ELISA method.Hemorheological analyzer was used to test hemorheologic changes.The expression of phosphorylated-endothelialnitric oxidesynthase(p-e NOS),phosphorylated-phosphoinositide 3-kinase(p-PI3K)and phosphorylated-serine/threonine protein kinase(p-Akt)proteins in myocardial tissue were detected by immunohistochemical method.Real-time fluorescence quantitative polymerase chain reaction(RT-PCR)method was used to detect the gene expression levels of e NOS,PI3 K,and Akt in myocardial tissue.Western blotting(WB)was used to detect the expression of e NOS,p-e NOS,PI3 K,p-PI3 K,Akt,and p-Akt proteins in myocardial tissue.(2)Transmission electron microscope was used to observe morphological changes of myocardial microvascular endothelial cells(MMVEC).The terminal deoxynucleotidyl transferase d UTP nick end labeling(TUNEL)method was used to detect the apoptosis rate of MMVEC.(3)The chemical digestion method was used to isolate the left ventricular tissue to obtain MMVEC,and then the Dil-acetylated low-density lipoprotein(Dil-Ac-LDL)phagocytosis test was used to identify MMVEC.RT-PCR was used to detect the gene expression levels of MMVEC e NOS,PI3 K and Akt.WB was used to detect the expression of MMVEC e NOS,p-e NOS,PI3 K,p-PI3 K,Akt,and p-Akt proteins.Results: 1.MHBFC preconditioning reversed ventricular remodeling in rat by activating e NOS-NO signaling pathway.Compared with the model group,the MHBFC preconditioning group could significantly inhibit the increase of cross sectional area of cardiac myocytes,inhibit the increase of myocardial fibrosis and inhibit the increase of cardiac collagen volume fraction(P<0.05).In addition,Serum NO and e NOS levels were significantly increased and hemorheology was improved after MHBFC administration(P<0.05).The expression of p-e NOS(Ser1177),p-PI3 K p85(Tyr607)and p-Akt(Ser473)proteins and the expression of e NOS,PI3 K and Akt genes were obviously increased after MHBFC administration(P<0.05).Compared with the model group,the cross sectional area of cardiac myocytes,myocardial fibrosis and cardiac collagen volume fraction in the L-NAME group were increased(P>0.05)and the level of serum NO and e NOS decreased significantly(P<0.05),indicating that it induced more serious ventricular remodeling than the model group.In addition,blood viscosity,plasma viscosity and hematocrit were significantly increased,and the phosphorylation of e NOS protein and the expression of e NOS gene were obviously downregulated in L-NAME group(P<0.05).Compared with the L-NAME group,the downregulation of the above index was obviously reversed after the combination of MHBFC(P<0.05).2.MHBFC preconditioning inhibited MMVEC apoptosis in rats with ventricular remodeling.Compared with the model group,the electron microscopic results showed that the endothelial cells were closely connected,the vacuolization of the cytoplasm was improved,and the structure of mitochondria was intact and TUNEL results showed that the MMVEC apoptosis rate was significantly reduced in the MHBFC pretreatment group(P<0.05).In addition,the organelle was dissolved and the apoptotic body was appeared in L-NAME group.The apoptosis rate of MMVEC in L-NAME group was significantly higher than that in model group(P<0.05),indicating that L-NAME induced more serious myocardial injury than model group.Compared with the L-NAME group,the MMVEC apoptosis was obviously antagonized after the combination of MHBFC(P<0.05).3.The regulatory mechanisms of MHBFC preconditioning on MMVEC in rats with ventricular remodeling by e NOS-NO signaling pathway.The results of the Dil-Ac-LDL phagocytosis test showed that the extracted cells were MMVEC.Compared with the model group,the expression of MMVEC p-e NOS(Ser1177),p-PI3Kp85(Tyr607)and p-Akt(Ser473)and the expression of MMVEC e NOS,PI3 K and Akt m RNA were significantly increased in the MHBFC pretreatment group(P<0.05).In addition,the phosphorylation of MMVEC e NOS protein and the expression of e NOS gene were obviously downregulated in L-NAME group(P<0.05).Compared with the L-NAME group,the downregulation of the above index was obviously reversed after the combination of MHBFC(P<0.05).Conclusion: This study indicated that MHBFC preconditioning increased e NOS protein phosphorylation and e NOS gene expression by increasing PI3 K and Akt protein phosphorylation and PI3 K and Akt gene expression,and activated the e NOS-NO signaling pathway,increased e NOS enzyme activity,catalyzed the generation of protective NO,improved hemorheology,and counteracted MMVEC apoptosis induced by ventricular remodeling,thereby protecting against myocardial damage and reversing ventricular remodeling.