The Effects And Mechanisms Of Berberin On Cardiac Hypertrophy And Fibrosis

Background:Cardiac hypertrophy is an adaptive process in response to increased hemodynam icoverload, characterized by an increase in the size of individual cardiac myocytes and wholeorgan enlargement. It is a common complication and pathological basis of a variety of cardiovascular diseases, and eventually lead to serious arrhythmias, heart failure. prevention and reversal of this change is an important goal of treatment. AMPK(Adenosine monophosphate activated protein kinase), is an protein kinases performancing regulation effect of energy metabolism in cell. Present study of AMPK focused on the energy metabolism. Furthermore, AMPK plays a crucial role on metabolic diseases of human, such as diabetes and obesity. Recent studies have demonstrated that AMPK gene mutation can lead to cardiac hypertrophy and arrhythmia, as well as activation of AMPK can negatively regulate mTOR / p70 S6K signaling pathway.Therefore, AMPK is expected to become a new therapeutic target to therapy cardiac hypertrophy and fibrosis.Berberine, a natural isoquinoline alkaloids, is an important component of Chinese medicine,such as Coptis chiensis.As a detoxification and antibacterial drugs , it was used in clinical medicine for long time, Past decade , western countries have demonstrated that berberine has a wide range of pharmacological effects on diabetes, metabolic diseases and cardiovascular diseases.It possessed anti-atherosclerotic, anti-Ⅱdiabetes, anti-obesity, anti-arrhythmia properties, anti-myocardial ischemia, and has a positive inotropic effect on the heart, and its mechanisms is that berberine directly activate AMPK.. In view of AMPK in cardiac hypertrophy and fibrosis, we hypothesized that activation of AMPK by berberine may inhibit the cardiac hypertrophy and fibrosis development.AMPKα2 is the main catalytic subunit in the heart, where it is predominantly expressed in cardiac myocytes. Therefore, in this study, we examined the effect of berberine on cardiac hypertrophy and fibrosis using neonatal cardiac myocytes and fibroblasts induced by PE and animal model by aortic banding (AB) AMPKα2 Ko mice., and clarify the molecular mechanism of the effect of berberine on cardiac hypertrophy and fibrosis. It is expected to provide sufficient scientific evidence for berberine as a new durg to therapy cardiac hypertrophy and fibrosis , and provide a new therapeutic target to therapy cardiac hypertrophy and fibrosis.Objective:To obtain the reliable evidence of the inhibitional effects of berberine on cardiac hypertrophy and fibrosis, and clarify its mechanism; To provide sufficient scientific evidence for AMPK as a new therapeutic target to therapy cardiac hypertrophy and fibrosis, and adequate theory for berberine as a new durg to therapy cardiac hypertrophy and fibrosis.Methods:1 The effect of Berberine on Cardic hypertrophy induced by PECardiomyocyttes were treated with different dose (0, 5, 10, 20, 30μM) of berberine for 60 minutes and then incubated with 100μM PE for 0, 6, 12, 24,48 h.To evaluate the hypertrophy of Cardiomyocyttes,we measured [3H] - leucine incorporation, quantificated cell cross-sectional area by measuring 50 random cells and quantificated the ANP, BNP,expression of transcripte of hypertrophic by real-time PCR2 The effect of Berberine on Cardic hypertrophy and fibrosis induced by aortic banding? ?C57BL / 6 male wild-type mic (8 weeks old, 24-26g weight) were randomly assigned into eight groups , sham and AB group, sham and AB group treated with different dose (40, 80, 100 mg/kg) of berberine. Aortic Banding(AB)was performed as animal model. One week before and 8 weeks after AB surgery , all animal were treated with different dose (40, 80, 100 mg/kg) of berberine or vehicle.Doppler and hemodynamics was performed to analysis the cardiac function. HE ,WGAand PSR staining were used to detect the myocyte area and collagen. Real-time PCR were used to analysis the expression of transcripts of hypertrophic markers, ANP,BNP,α-MHC,β-MHC,and fibrosis markers, CTGF,CollagenI,CollagenIII, in vivo.3 To explore the molecular mechanisms of the inhibition effect of Berberine oncardiac hypertrophy and fibrosis Cell experiment: Cardiomyocyttes were treated with 30μm of berberine for 60 minutes and then incubated with 100μmPE for,48 h. Animal experiment: C57BL / 6 male wild-type mic (8 weeks old, 24-26g weight) were randomly assigned into four groups , sham and AB group, sham and AB group treated with 100 mg/kg of berberine. Aortic Banding(AB)was performed as animal model. Western Blot was used to analysis the expression levels of transzctivation of total and phosphorylation of the protein of AMPK signal pathways.4 The effect of Berberine on Cardic hypertrophy and fibrosis induced by animal model by aortic banding AMPKα2 Ko miceAMPKα2 Ko male mic (8 weeks old, 24-26g weight) were randomly assigned into four groups , sham and AB group, sham and AB group treated with 100 mg/kg of berberine. Aortic Banding(AB)was performed as animal model. One week before and 4 weeks after AB surgery , all animal were treated with 100mg/kgof berberine or vehicle.Doppler and hemodynamics was performed to analysis the cardiac function. HE ,WGAand PSR staining were used to detect the myocyte area and collagen. Real-time PCR were used to analysis the expression of transcripts of hypertrophic markers(ANP,BNP,α-MHC,β-MHC,Acat1,AT1,GATA4,Mef2c,MCIP1.4,SERCA2a,ACE),and fibrosis markers(TGFβ1,TGFβ2,Collagen I,CollagenⅢ, CTGF, Fibronection, MMP-2,MMP-9,OPN), in vivo. Results:1 The effect of Berberine on Cardic hypertrophy induced by PEData revealed that berberine reduced the increase of [3H]-leucine incorporation, augmentation of the size of Cardiomyocytte and up-regulation of the expression of transcripts of hypertrophic markers, ANP and BNP, induced by PE in adose-dependent manner., 30μM of berbernie reches the best inhibitor effects.2 The effect of Berberine on Cardic hypertrophy and fibrosis induced by aortic bandingTreatment with berberine prevented cardical dysfunction , as evidenced by improvements in LVEDD ,LVESD,LVPWd, and FS. Furthermore, berberine pretreatment significantly decreased Cardiomyocyttes cross-sectional area and collagen volume in AB mice. Additionaly, berberine attenuated the increase in hppertrophic and fibrotic marker expression caused by AB. No significant changes were observed in the sham-operated mice treated with berberine or vehicle.3 To explore the molecular mechanisms of the inhibition effect of Berberine on cardiac hypertrophy and fibrosisWestern blot showed the increased p-AMPK,p-ACC ser79,p-mTOR,p-70S6K thr389 , decreased p-eEF2 thr56 and no significant changes of totalAMPK,ACC,mTOR,p70S6K eEF2 proten expression induced by PE-treated Cardiomyocytes and in AB mice. Berberine augmented the increase of p-AMPK,p-ACC ser79, attenuated the increase of p-mTOR,p-70S6K thr389, and increased the decrease of p-eEF2 thr56 proten expression induced by PE-treated Cardiomyocytes and in AB mice. No significant changes of total AMPK,ACC,mTOR,p70S6K eEF2 proten expression were observed in the AB-operated mice treated with berberine or vehicle.4 The effect of Berberine on Cardic hypertrophy and fibrosis induced by animal model by aortic banding AMPKα2 Ko miceNo significant changes were observed in the AB-operated AMPKα2 Ko mice treated with berberine or vehicle, as evidenced by the cardiac function performed by Doppler and hemodynamics, the myocyte area and collagen evalued by HE ,WGAand PSR staining , and the expression of transcripts of hypertrophic markers and fibrosis markers measured by Real-time PCR in vivo.Conclusions:Our results demonstrate that berberine inhibits cardicac hypertrophy in vitro and in vivo by directly activating AMPKα2 and subsequently negative regulating mTOR/ p70 S6K signaling pathways.