Protective Effect Of Sesamin On Myocardial Remodeling In SHRs And The Potential Mechanisms Implicated

Aim: To investigate the effect of sesamin on myocardial remodeling in spontaneously hypertensive rats(SHRs) and the possible mechanisms involved.Methods: 1. Animal experiment: Twenty-eight male SHRs were randomly allocated to SHR group, Ses160 group(sesamin 160mg/kg), Ses80 group(sesamin 80mg/kg) and Cap30 group(captopril 30 mg/kg). Seven male WKY rats were used as control. After 1 week adaptive feeding, rats in treatment group were given sesamin and captopril intragastrically respectively for 12 weeks, with the rest given the same volume of 0.5% sodium carboxymethyl cellulose. Blood pressure was measured using noninvasive tail arterial blood pressure analysis system. Left ventricular mass index was calculated as the ratio of left ventricular weight to body weight(mg/g). Histopathological changes and collagen deposition of cardiac tissue were observed by HE or Masson staining. Protein expression of transforming growth factor-β1(TGF-β1) in cardiac tissue was detected by immunohistochemical staining. The content of angiotensinⅡ(AngⅡ) in serum as well as contents of AngⅡ, TGF-β1, typeⅠand type Ⅲ collagen in cardiac tissue was detected by ELISA; The serum lactate dehydrogenase(LDH) content as well as total antioxidant capacity(T-AOC) and malondialdehyde(MDA) level in cardiac tissue was measured by colorimetric assay. Cardiomyocyte apoptosis was evaluated by TUNEL assay. The protein expression of Bax, Bcl-2, p47 phox, superoxide dismutase(SOD), matrix metalloproteinase-9(MMP-9), tissue inhibitor of matrix metalloproteinase(TIMP-1), Smad7 and phosphorylation level of Smad3 in cardiac tissue were determined by western blot. 2. Cardiac fibroblasts experiment: Sesamin containing serum was prepared using serum pharmacological method. Neonatal rat cardiac fibroblasts(CFs) were separated and purified by compound enzyme digestion and differential adhesion. Cells were treated with AngⅡ(10-7mol/l) for 24 h after preincubated with sesamin-containing serum(10%, 20%) or blank serum(20%) for 12 h. CFs proliferation was determined by MTT assay. The secretion of TGF-β1, typeⅠand type Ⅲ collagen were determined by ELISA. T-AOC and MDA content were determined by Colorimetric assay. Phosphorylation level of Smad3 were determined by western Blot method. 3. H9C2 cardiomyocytes experiment: H9c2 rat cardiomyocytes were preincubated with sesamin(10%, 20%) containing serum or blank serum(20%) for 12 h, followed by incubation with AngⅡ for 24 h. Cell viability was assessed by MTT assay and cell apoptosis was evaluated by flow cytometric analysis. Protein expression of Bax, Bcl-2, Caspase-3, p22 phox and SOD was determined by western blot analysis. Levels of intracellular reactive oxygen species(ROS), T-AOC and MDA were measured colorimetrically.Results: 1. Animal experiment:(1) Captopril significantly reduced systolic blood pressure and AngⅡ levels in SHRs(P <0.05 or P <0.01), accompanied by a marked attenuation of left ventricular hypertrophy and collagen deposition(P <0.05 or P <0.01). Though sesamin had no significant influence on AngⅡ levels, and the hypotensive effect was also significantly inferior to that of captopril(P <0.05 or P <0.01), however, the improvement of LVH and collagen deposition was similar to that in captopril group.(2) Sesamin and captopril markedly reduced TGF-β1 content in cardiac tissues, with Smad3 phosphorylation decreased and Smad7 protein expression increased notably(P <0.05 or P <0.01). Protein expression of TIMP-1, typeⅠcollagen and type Ⅲ collagen, target genes of Smad3, was down-regulated markedly by sesamin and captopril(P <0.05 or P <0.01).(3) Supplementation with sesamin and captopril markedly down-regulated protein expression of MMP-9 in SHRs(P<0.05 or P<0.01).(4) Supplementation with sesamin and captopril significantly reduced cardiomyocyte apoptosis in SHRs, with protein expression of Bax down-regulated and Bcl-2 up-regulated markedly(P<0.05 or P<0.01).(5) In addition, Sesamin effectively enhanced T-AOC in cardiac tissue, with MDA content reduced notably(P<0.05 or P<0.01). 2. Cardiac fibroblasts experiment:(1) Preincubation with sesamin containing serum significantly supressed AngⅡinduced cardiac fibroblasts proliferation and collagen secretion(P <0.05 or P <0.01);(2) Preincubation with sesamin containing serum significantly supressed TGF-β1 protein expression and Smad3 phosphorylation(P <0.05 or P <0.01).(3) T-AOC was effectively increased in sesamin groups, while MDA contents were decreased evidently(P <0.05 or P <0.01). Blank serum has no influence on the above mentioned measurements. 3. H9C2 cardiomyocytes experiment:(1)Preincubation with sesamin containing serum significantly improved cell viability and suppressed cell apoptosis in H9C2 rat cardiomyocytes exposed to AngⅡ(P <0.05 or P <0.01), with the expression of Bax and Caspase-3 protein down-regulated and Bcl-2 protein up-regulated markedly(P <0.05 or P <0.01).(2) Levels of SOD protein and T-AOC were effectively increased in sesamin containing groups, while the levels of p47 phox protein, intracellular ROS and MDA contents were decreased evidently(P <0.05 or P <0.01). Blank serum has no influence on the above mentioned measurements.Conclusion: Sesamin effectively ameliorates myocardial remodeling in spontaneously hypertensive rats, which might be attributed to suppression of cardiac myocyte apoptosis and myocardial fibrosis. The beneficial effect of sesamin on myocardial remodeling might be derived from its antioxidant capacity.