| ObjectiveCardiovascular disease(CVD)is a major menace to human health,with cardiac hypertrophy(CH)emerging as an independent pathogenic factor.Although CH can optimally maintain the heart’s physiological functions,persistent pathological CH will results in abnormal cardiac structure and function,remodeling of the cardiac tissue,and eventually Heart failure(HF).Therefore,ameliorating CH is of prime importance for the treatment of HF.Traditional Chinese medicine exhibits an unparalleled edge in HF and CH.Previous investigations demonstrated that Huoxin pill(HXP)treated chronic heart failure in rats.HXP having the capacity of supplementing Qi and activating blood,as well as warming and unblocking meridians.Nevertheless,fewer studies have reported on the application of HXP in treating CH.On this basis,the present study further investigated the inhibitory mechanism of HXP on CH.Methods1.A Pharmacodynamic study was performed to investigate the effects of HXP treatment on abdominal aortic constriction(AAC)and intraperitoneal injection of isoproterenol(ISO)-induced CH in rats and in vitro myocardial hypertrophy cell model.In vivo,two CH models were established with AAC,and with ISO injection.In the rat model of CH induced by AAC,it was divided into 6 groups: Sham operation group(Sham),model control group(MC),captopril control group(CAP,10 mg/kg),low dose group of HXP(HXPL,12 mg/kg),medium dose group of HXP(HXPM,24 mg/kg)and high dose group of HXP(HXPH,48 mg/kg).After successful model establishment,CAP and each dose of HXP were continuously given via gavage for 8 weeks.In the ISO-induced CH model,rats were divided into five groups: normal group(NC),model group(ISO),low dose group(HXPL),medium dose group(HXPM),and high dose group(HXPH).CH was established by intraperitoneal injection of 15 mg/kg ISO for 7 consecutive days.After the CH model established,HXP was given by gavage for 14 days.The protective effect of HXP on CH in rats were evaluated by detecting various indexes.In the vitro ISO-induced H9c2 cell hypertrophy model,the cytotoxicity of HXP on H9c2 cell was investigated by CCK8.Then,the appropriate dose concentration was selected,and according to the level of ANP,BNP andβ-MHC m RNA expression and myocardial cell surface area,the anti-CH effect of HXP were evaluated.2.Metabonomic study of HXP on treating CH induced by ISO in rats.The serum samples of NC group,ISO group and HXPH group in the ISO-induced CH model in rats were selected for extensive targeted metabonomics analysis.Subsequently,the metabolites were obtained by LC-MS database contrast scanning.Finally,The anti-CH effect of HXP was evaluated by multiple biological analysis of metabonomics.3.Mechanism investigation of network pharmacologyFor further explored the anti-CH mechanism of HXP,a network pharmacology approach was utilized to carry out biological analysis on the active components of HXP and the commonly targeted molecules of CH.Active ingredients and action targets of HXP were queried via TCMSP.BATMAN-TCM and other databases,and the official name were connected with Uniprot.Genecards and OMIM database were selected to identify the target of CH,while Venn was applied to screen the common target of HXP-CH.The protein interaction network(PPI)and signaling pathway and function analysis of the common target of HXP-CH were also established.4.The mechanism of HXP in treating CH in rats.In CH model in vivo: in the aspect of autophagy detection,immunohistochemical staining of LC3 protein was performed on paraffin tissue sections,and Western blot was conducted for autophagy-related proteins(LC3,Beclin-1,p62).For apoptosis detection,TUNEL staining was performed on paraffin sections,and Western blotting was implemented to detect apoptosis-related proteins(Bax,Bcl-2,cleaned-capase3).Furthermore,Western blot was used to evaluated the PI3K/Akt/m TOR pathway-related proteins(p-PI3 K,PI3K,p-Akt,Akt,p-m TOR,m TOR)were detected by.In the ISO-induced H9c2 cardiomyocyte hypertrophy model,autophagy detection was conducted via Adm Cherry-GFP-LC3 B transfection,LC3 immunofluorescence staining and Western blot for autophagy-associated proteins.Apoptosis was surveyed through mitochondrial membrane potential assays,TUNEL detection,flow cytometry and Western blot for apoptosis-related proteins.Subsequently,rapamycin(RAPA)was utilized to induce excessive autophagy of cardiac myocytes and these techniques were utilized for analysis.The expression of p-PI3 K,PI3K,p-Akt,Akt,p-m TOR,m TOR proteins related to the PI3K/Akt/m TOR pathway were detected by western blotting.The PI3 K inhibitor LY294002 was used to inhibit the phosphorylation of PI3 K.Western blot was then employed to investigate the regulatory effect of HXP on PI3K/Akt/m TOR pathway related proteins after PI3 K inhibition.Results1.HXP had cardioprotective effect in rat model of CH.In the models of CH caused by ISO and AAC,the findings indicated that HXP augments echocardiographic data and symptoms of CH,mitigates the severity of fibrosis,and reverses serum markers of CH(ANP,BNP,NT-pro BNP).Moreover,in the myocardial cell hypertrophy induced by ISO,HXP significantly decreases indexes of CH(ANP,BNP,NTpro BNP m RNA)and myocardial cell surface area.2.HXP improved CH by regulating metabolomics.HXP modulates the production of detrimental cardiac harmful DIHOME by regulating the levels of 9,10-Ep OME and 12,13-Ep OME metabolites,thereby performing a protective role in the heart.Additionally,it alleviates the manifestations of CH by regulating bile secretion,primary bile acid biosynthesis pathway,related receptor expression and PI3K/Akt signaling pathway affected by multiple pathways.3.HXP explored the mechanism of treating CH through network pharmacology.HXP is composed of quercetin,ursodeoxycholic acid,luteolin,kaempferol and chenodeoxycholic acid,which have demonstrated anti-CH activity.HXP exerts its beneficial effects by acting on multiple biological processes,such as cardiac process and cardiac contraction.KEGG analysis shown that HXP alleviated the symptoms of CH by regulating multiple signaling pathways such as MAPK and PI3K/Akt signaling pathways.4.HXP affected autophagy and apoptosis to improve CH in rats through PI3K/Akt/m TOR signaling pathway.In two types of CH models in vivo,the results demonstrated that HXP decreased the expression of LC3 in the heart and reduced cardiomyocyte apoptosis.Western Blot experiment also showed that HXP could inhibit the expression of autophagy-related proteins and apoptosis-related proteins.HXP affected the expression of p-PI3 K,PI3K,p-Akt,Akt,pm TOR and m TOR,indicating that HXP serves to inhibit autophagy through regulating the PI3K/Akt/m TOR pathway.In order to further verify the ability of HXP to modulate autophagy and exert a protective role against CH,this study utilized RAPA to induce excessive autophagy in cardiac myocytes.The results of autophagy flux and Western Blottiong showed that HXP could mitigate the excessive autophagy induced by RAPA and ISO.In addition,the results of immunofluorescence test also indicated that HXP could attenuate CH by decreasing the expression of LC3.The results of mitochondrial membrane potential,TUNEL,flow cytometry and Western Blotting showed that HXP could reduce the apoptosis of myocardial cells induced by RAPA and ISO.In order to further verify that HXP affected autophagy and played an anti-CH role through PI3K/Akt/m TOR signaling pathway,this study used PI3 K inhibitor LY294002 to inhibit signaling pathway transduction.Western Blot experiment showed that HXP improved the expression of p-PI3 K,PI3K,p-Akt,Akt,p-m TOR and m TOR inhibited by LY294002,thereby implying that regulation of HXP on autophagy is mediated by the PI3K/Akt/m TOR pathway.ConclusionTaken together,HXP was found that having pharmacodynamic effect against CH in this study.Through the analysis of metabolomics and network pharmacology,it was further verified that HXP mitigated CH by regulating PI3K/Akt/m TOR pathway to inhibit autophagy and apoptosis.The results of the present study provide experimental evidence for the application of HXP in the treatment of HF and CH. |
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