Study Of The Role And Mechanisms Of Silibinin Mediates Cardiac Hypertrophy

Background:Cardiac hypertrophy is an adaptive process in response to increased hemodynamic overload, characterized by an increase in the size of individual cardiac myocytes and whole-organ enlargement. Although it may be compensatory initially, sustained pathologic hypertrophy is deleterious and frequently decompensate into congestive heart failure. It is well known that the effects of hypertrophic stimuli such as mechanical stretch and angiotensin II (Ang II) on cardiac intracellular signaling cascades are crucial to elucidate the molecular mechanism underlying cardiac hypertroph. These signaling pathways include the epidermal growth factor receptor (EGFR) pathway, the mitogen-activated protein kinases (MAPK) pathway, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, the NF-κB pathway, TGF-β1/Smad pathway, and Insulin/IGF-1 signaling pathway. Therefore, pharmacological interventions of these signaling pathways may provide promising approaches in treating cardiac hypertrophy and heart failure.Silibinin is a natural polyphenolic flavanoid extracted from fruits and seeds of milk thistle (Silybum marianum), which has been extensively used clinically as an anti-hepatotoxic agent for treatment of liver diseases. In addition, anti-tumor efficacy of silibinin is shown in prostate, skin, renal, colon, and bladder cancer models. It is also reported that silibinin possesses anti-oxidant, anti-apoptotic, anti-inflammatory and anti-fibrotic properties. However, the effect of silibinin on cardiac hypertrophy and the related signaling mechanisms still remain unclear. Although silibinin has been shown to inhibit EGFR activation, very little is known about whether this inhibitory effect is related to the protective role in cardiac hypertrophy. Therefore, we aimed to determine whether silibinin attenuates cardiac hypertrophy in vitro and in vivo by interfering EGFR-dependent pathways. Methods:Part one:Cardiomyocytes were treated with different dose of silibinin for 60 minutes and then incubated with 1μ,M Ang II for 48 hours. Ang II-induced. cardiac myocytes were incubated with 1μM Ang II or pretreated with 20μM silibinin for 60 minutes and then incubated with 1μM Ang II for indicated time. To evaluate the hypertrophy of cardiomyocyte, we measured [3H]-leucine incorporation and quantificated cell cross-sectional area by measuring 50 random cells. Moreover, we observed ANP, BNP, andβ-MHC protein expression levels by Western blot.Part two:Adult C57BL/6 male mice (8-10 weeks old) were randomly assigned into four groups, sham and AB group, sham and AB group treated with silibinin. Aortic Banding (AB) was performed as animal model. Treatment with 50 mg/kg/d of silibinin or vehicle for 8 weeks after AB surgery or sham operation allowed for critical evaluation. HE staining were used to detect the myocyte area. Doppler and hemodynamics was performed to analysis the cardiac function. Real-Time PCR and Western Blot were used to analysis the expression of transcripts of hypertrophic, fibrosis, inflammation markers in vivo and in vitro.Part three:First, effects of silibinin on EGFR transactivation in vitro and in vivo.Western Blot was used to analysis the protein expression levels of transactivation of total EGFR and phosphorylation of Tyr1068 and Tyr1173. Moreover,Western Blot were used to analysis the expression of transcripts of major molecular of MAPKs, PI3K/Akt/GSK3β,TGF-β1/Smad, and NF-κB signaling in vivo and in vivo.At last, cardiomyocytes were treated with SU1428 or vehicle, [3H]-leucine and [3H]-proline incorporation in response to Ang II-induced protein and collagen synthesis were measured. Western blot analysis of the effect of SU 1428 on AngⅡ-induced activation of ERK1/2, AKT, P85, IκBα, IKKβ,Smad2, Smad3 and Smad4.Results:Silibinin was non-cytotoxic for cardiomyocytes in all tested concentration and no significant differences in cell viability were found between normal cardiomyocytes and cardiomyocytes treated with 20μM silibinin for 48 hours. Silibinin reduced the increase of [3H]-leucine incorporation and the size of cardiomyocyte induced by AngⅡin a dose-dependent manner. Additionaly, silibinin inhibited the activities of atrial natriuretic peptide (ANP) promoter and Western blotting showed the increasedβ-MHC, ANP and BNP protein expression were blocked by silibinin treatmentTreatment with silibinin prevented ventricular dysfunction, as evidenced by improvements in LVESD, IVSD, LVPWD, LVEDD, and FS. Furthermore, silibinin pretreatment significantly decreased heart HW/BW ratio, LW/BW ratio, and cardiomyocyte cross-sectional area in AB mice. No significant changes were observed in the sham-operated mice treated with silibinin or vehicle. Silibinin attenuated the observed increase in hypertrophic, fibrotic and inflammation marker expression caused by AB.Silibinin almost inhibited EGFR transactivation and phosphorylation of Tyr1068 and Tyr1173 both induced by AngⅡ-treated cardiomyocytes and in AB mice (P＜0.01).Western blotting showed the increased ERK1/2、GSK3β、Akt、p85、IκBα、IKKβ、Smad2、Smad3、Smad4 protein expression were blocked by silibinin treatment in vivo and in vivo (P＜0.01).SU1428 almost completely abrogated both [3H]-leucine and [3H]-proline incorporation in response to AngⅡ. SU1428 remarkably attenuated AngⅡ-induced activation of ERK1/2, AKT, P85, IκBα, IKKβ, Smad2, Smad3, and Smad4 by Western Blot analysis (P＜0.01)Conclusion:Our results demonstrate for the first time that silibinin inhibits cardiac hypertrophy in vitro and in vivo by blocking EGFR-dependent hypertrophy, inflammation and fibrosis. We further confirm that EGFR-dependent ERK1/2, PI3K/Akt, NF-κB and Smad signaling pathways are targets of silibinin’s inhibitory actions.