| Objective Deficiency of both qi and Yin is an important syndrome type of chronic heart failure(CHF).Shengmai Yin(SMY)is a classic prescription for Qi Yin deficiency.Previous research results showed that SMY can significantly improve the cardiac function of CHF model rats,but its mechanism is not clear.Therefore,the purpose of this study is to replicate the rat model of CHF,on the basis of clarifying the treatment of CHF and inhibiting myocardial fibrosis by SMY,and explore the mechanism of inhibiting myocardial fibrosis and then treating CHF with metabonomics technology,so as to provide experimental basis for the basic research and clinical treatment of CHF,and enrich the scientific connotation of Shengmai Yin.Method1.The CHF model was established by ligation of left anterior descending coronary artery(LAD)combined with exhaustive swimming.SMY was used as the research drug,and Sacubitril valsartan sodium tablets(S)was used as the positive control drug.The CHF model was identified and preliminary evaluation of drug efficacy by the combination of cardiac function,serum NT-pro BNP level,HE staining and Masson staining;The m RNA and protein expression levels of collagen I,MMP-2/3/9 and TIMP-2,which are key players in cardiac fibrosis,were combined with serum galectin-3(Gal-3)levels to clarify the efficacy of SMY treatment on CHF and inhibit cardiac fibrosis.2.Serum metabolic data were collected by UPLC-Q/TOF-MS technique,principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA)were performed by multivariate statistical method,the resulting variable weights(VIP)were subjected to a t test,and substanceswith VIP>1 and P<0.05 were screened as metabolic markers of disease in CHF model rats.Comparative analysis of addback markers in CHF model rats after SMY intervention as metabolic markers for treatment of CHF by SMY,a drug target for inhibition of myocardial fibrosis,combined with metaboanalyst 5.0website mapping into sets and mapping metabolic pathways.Meanwhile,the target metabolic markers of SMY were separately subjected to Pearson correlation analysis with NT-pro BNP and Gal-3 to finally obtain the potential targets of SMY treatment in CHF to inhibit myocardial fibrosis.ELISA,RT-q PCR and Western blotting were used to validate the predicted targets and elucidate the mechanism by which treatment with SMY inhibited cardiac fibrosis in CHF.Result1.“Left anterior descending coronary artery ligation” combined with “exhaustive swimming”can reproduce a stable and reliable CHF modelUsing a combination of cardiac function,serum NT-pro BNP levels,HE staining and Masson staining to identify CHF models,we found that the left ventricular ejection fraction(LVEF)of rats in the model group was significantly decreased(P<0.01)and LVEF≤45% compared with the sham group,and the serum NT-pro BNP levels of rats in the model group were significantly increased(P<0.01)compared with those in the sham group.Morphological observation after HE staining revealed that the morphological structure of rat myocardium in sham group was almost normal,and the myofibers were arranged orderly and evenly colored,without inflammatory cell infiltration.In the model group,there were obvious old myocardial infarction foci in the anterior wall of the left ventricle(under low magnification),and the arrangement of residual muscle fibers was disordered,with a large amount of connective tissue distributed among them.(high magnification)myocytes are markedly reduced and fibrocytes are markedly increased,with inflammatory cell infiltration seen between fibrocytes.Masson staining and morphological observation found that only a few collagen fibers were found in the gap between cardiomyocytes of rats in sham group;Morphological structure of myocardium was basically normal.In the model group,large sheets of myocardial collagen fibrils surrounded by aniline blue and blue were found in the anterior wall of the left ventricle,which showed a cord like distribution and inclusion of a few irregularly arranged and preserved cardiomyocytes.2.Evaluation of the efficacy of Shengmaiyin treatment in the inhibition of cardiac fibrosis in CHF2.1 Shengmaiyin improves mental state and symptoms of CHF ratsCompared with the sham group,the mental state of rats in the model group was poor,the respiratory rate was rapid,different degrees of edema occurred in the limbs,and the consciousness of defiance was strong when grasping,but the strength of defiance was weak;Compared with the model group,the above symptoms of rats in the SMY group and the S group were all alleviated to different degrees,and the mental state and symptoms of rats in the SMY group were comparable with those in the S group.2.2 Shengmaiyin has the effect of improving cardiac function in CHF rats(1)Compared with sham group left ventricular anterior walldiastolic(LVAWd),left ventricular anterior wall systolic(LVAWs),left ventricular posterior wall diastolic(LVPWd),LVEF and left ventricular fractional shortening(LVFS)in model group were significantly decreased(P<0.01),while Left ventricular end diastolic diameter(LVEDd)and Left ventricular end systolic diameter(LVESd)were significantly increased(P<0.01);compared with model group,LVESd in SMY group and S group decreased significantly(P<0.05),LVEF and LVFS increased significantly(P<0.05).(2)Compared with sham group,the serum NT-pro BNP level of model group was significantly higher(P<0.01);compared with model group,the serum NT-pro BNP levels in SMY group and S group were significantly increased(P<0.05);the level of NT-pro BNP in S group was significantly lower than that in SMY group(P<0.05).2.3 Shengmaiyin improves myocardial structural changes in CHF rats(1)HE staining:In sham group,the morphology and structure of myocardium were normal,the muscle fibers were arranged orderly and stained evenly,and no inflammatory cell infiltration was found.Old infarct lesions of myocardium in the anterior left ventricular wall of rats in the model group were obvious,and the arrangement of myofibers was disordered and the gap was widened;Myocytes were markedly reduced and fibrocytes were markedly increased,and few inflammatory cells were seen.Compared with the model group,old infarct foci of myocardium were observed in the anterior wall of the left ventricle in rats of the SMY group and the S group,and the myofibers were arranged in a relatively orderly and uniform manner,with more cardiomyocytes and a few inflammatory cells visible.(2)Masson staining:a small number of collagen fibers were seen in the myocardium of rats in the sham group,and the morphological structure of the myocardium was normal;In the model group,large areas of myocardial collagen fibrils surrounded by aniline blue and visible in the anterior left ventricle wall myocardial tissue were significantly increased in CVF(P<0.01);Compared with the model group,the myocardial tissue in the anterior left ventricle wall of rats in the SMY and s groups was narrowed by myocardial collagen fiber areas stained with aniline blue,and CVF was significantly downregulated(P<0.05);There was no difference in CVF levels between the SMY group and the S group.(3)Compared with sham group,Gal-3 level in serum of model group increased significantly(P<0.01);Compared with the model group,Gal-3 levels in the SMY and S groups were significantly decreased(P<0.05).(4)Compared with sham group,collagen Ⅰ protein level in myocardial tissue of model group increased significantly(P<0.01);Compared with the model group,the levels of collagen I protein expression in the SMY and S groups were significantly decreased(P<0.05).(5)Compared with the sham group,the m RNA and protein levels of MMP-2/9,and TIMP-2 in the myocardial tissue of the model group were significantly increased(P<0.05),and the m RNA level of MMP-3 was significantly increased(P<0.05);Compared with the model group,the m RNA and protein levels of MMP-2 and MMP-9 in the SMY and S groups were significantly downregulated(P<0.05),and the m RNA level of MMP-3 was significantly downregulated(P<0.05).3.Predicting and validating targets for Shengmaiyin treatment of CHF to inhibit myocardial fibrosis3.1 using metabolomics technology to predict the therapeutic targets of pulse drinking in CHF to inhibit myocardial fibrosisUPLC-Q/TOF-MS technique combined with multivariate statistical analysis method identified 19 metabolic markers in serum of CHF rats,among which 12 metabolites were the target metabolic markers for myocardial fibrosis inhibition by treatment of CHF with pinprick drinking.After Met PA metabolic pathway combined with Pearson correlation analysis,we found that the target of pinogenic diet treatment in CHF to inhibit myocardial fibrosis was tryptophan metabolic pathway.Therefore,starting with the tryptophan metabolic pathway,follow-up studies were performed to further validate the metabolomics results.3.2 Validation of metabonomics results in vivo(1)The Kyn/Trp ratio in serum increased significantly in the model group compared with the sham group(P<0.05).(2)Compared with the sham group,the m RNA and protein levels of IDO in myocardial tissue in the model group were significantly increased((P<0.05);Compared with the model group,the m RNA level and protein level of IDO in the SMY group were significantly downregulated(P<0.05).(3)Compared with sham group,the serum levels of IL-1β,IFN-γ,IL-18,TNF-α,IL-4 and IL-10 in model group were significantly increased(P<0.05),and the m RNA expression levels of IL-1β,IFN-γ,COX-2,TNF-α,IL-18,IL-6 and IL-10 in model group were significantly increased(P<0.05);compared with model group,the expression levels of IL-1β,IFN-γ,IL-18 and TNF-α in serum of SMY group were significantly down regulated(P<0.05),the expression levels of IL-10 were significantly up-regulated(P<0.05),the m RNA expression levels of IL-1β,IFN-γ,COX-2 and TNF-α in myocardial tissue of SMY group were significantly down regulated(P<0.05),and the m RNA expression levels of IL-10 were significantly up-regulated(P<0.05).Conclusion1.“left anterior descending coronary artery ligation ” combined with “exhaustive swimming”replicates a stable and reliable CHF model;Shengmaiyin improved cardiac function and thus treated CHF in CHF rats by inhibiting myocardial fibrosis;2.The mechanism of action of Shengmaiyin in the treatment of CHF to inhibit myocardial fibrosis may be associated with regulating the tryptophan metabolic pathway and alleviating the inflammatory response. |
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