| Objective:1. To observe the expressions of myocardial fibrosis indexs, such as hydroxyproline(HYP), transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA) in myocardial tissue with pressure overload rats and to explore the effect of TanshinoneⅡA on ventricular remodeling.2. To investigate the effect of Tanshinone IIA on HO-1 protein and HO-1 mRNA expression, as well as products of oxidative stress, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA) content changes, evaluate the the relationship between oxidative stress and ventricular remodeling and explore the mechanisms of Tanshinone ⅡA antioxidant.Methods:1. Forty male 6-week-old male SD rats, randomly selected 8 as sham-operated (Sham group), and the remaining 32 rats were preparaed with abdominal aortic banding by left ventricular remodeling model.4 weeks after operation, there was no dead in Sham group(n=8), the 22 survival model rats were randomly divided into three groups, namely Model group (n=7), Tanshinone ⅡA group (TanⅡA group,n=7) and Tanshinone ⅡA+Zinc protoporphyrin group (TanⅡA+ZnPP group, n=8). Sham and Model groups were given normal saline(4ml/kg·d, i.p.); TanⅡA group were given Sodium Tanshinone IIA Sulfonate (STS)(20mg/kg· d, i.p,); TanⅡA+ HO-1 inhibitor(ZnPP) group(STS 20mg/Kg·d+ZnPP injection lmg/Kg·d,i.p.). Every group were given continuously for 4 weeks.2.8 weeks later, all rats were killed and accurately weigh body weight (BW), heart weight (HW), left ventricular weight (LVW).Calculate theHWI= HW/BW, left LVWI= LVW/BW. HWI and LVWI are the cardiac hypertrophy index; HE and Masson staining to observe the myocardial morphological changes; the contents of hydroxyproline(HYP) in myocardial were detected using alkaline hydrolysis; the expression of a-SMA was analysized by immunohistochemistry; Western blotting was used to measure myocardial HO-1 and TGF-β1 protein 1; Real-time quantitative-PCR method to detect the expression of HO-1mRNA; Determination of plasma SOD, GSH-Px, MDA.Results:1. Comparison of HWI and LVWI in each groupThe HWI and LVWI in Model group were (3.80±0.18)mg/Kg and (3.40±0.16)mg/Kg, compared with Sham group[HWI(2.98±0.15)mg/Kg、LVWI(2.56±0.12)mg/Kg] were significantly increased (P<0.01). The HWI and LVWI in TanⅡA group were (3.27±0.18)mg/Kg and (2.91±0.21)mg/Kg, in TanllA+ZnPP group were (3.49±0.16)mg/Kg and (3.12±0.20)mg/Kg. compared with Model group, TanⅡA and TanⅡA+ZnPP group both decreased (P<0.01 or P<0.05), while TanⅡA group was decreased more significantly.2.Comparison of myocardial tissue HE and Masson staining in each groupThe HE staining showed that in Sham group cardiomyocytes arranged in neat rows, dyeing uniformity, no focal necrosis, myocardial interstitial and microvascular normal.Compared with the Sham group, Model group myocardial fibers significantly enlarged, arranged in disorder, myocardial nuclear pyknosis and deep staining, more collagen fibers encircled, interstitial inflammatory cells infiltration. TanⅡA group and TanllA+ZnPP groups had different degrees of improvement. The Masson staining showed that myocardial cells were red and collagen fibers were blue. In Model group, collagen deposition was increased significantly compared with Sham, but TanⅡA group and TanⅡA+ZnPP group alleviated in different degree.3.Comparison of cardiac HYP of rats in each groupCompared with the Sham group, Model group was significantly increased myocardial HYP (P<0.01); Compared with Model group, TanⅡA group and TanⅡA+ZnPP group HYP decreased (P<0.01 or P<0.05), while TanⅡA group decreased more significantly.4. Comparison of the expression of a-SMA in ventricular myocardium of rats in each groupCompared with the Sham group, the positive expression of ventricular myocardial a-SMA immunohistochemistry staining were increased markedly in model group (P<0.01); Compared with the Model group, the positive expression of TanⅡA group and TanⅡA+ZnPP group a-SMA decreased(P<0.01 or P<0.05); TanlⅡ group decreased more obviously.5. Comparison of TGF-β1 protein expression in myocardium of rats in each groupCompared with the Sham group, myocardial TGF-β1 protein expression increased significantly in Model group(P<0.01), the expression ofTanⅡA group and TanⅡA+ZnPP group TGF-β1 protein level decreased, compared with the Model group (P<0.01 or P<0.05); TanⅡA group decreased more obviously.6. Comparison of HO-1 protein expression in myocardium of rats in each groupCompared with the Sham group, myocardial HO-1 protein expression increased in Model group (P<0.01); TanllA group protein expression was the highest (compared with Model group P<0.05), when used the HO-1 inhibitor ZnPP, HO-1 protein expression decreased significantly compared with TanⅡA group (P<0.01).7. Comparison of HO-1mRNA expression in myocardium of rats in each groupCompared with the Sham group, the expression level of HO-1mRAN in Model group was increased (P<0.01), while the highest expression level of HO-1mRNA group was TanⅡA (compared with Model group P<0.05), When the HO-1 inhibitor ZnPP was used, the expression of HO-1mRNA was significantly lower than that of the TanⅡA group (P<0.01).8. Comparison of SOD, GSH-Px and MDA in plasma of rats in each groupCompared with the Sham group, Model rats Plasma SOD and GSH-Px content decreased, while MDA content increased (P<0.01); compared with the Model group,TanⅡA and TanⅡA+ZnPP group, SOD and GSH-Px increased, MDA content was decreased (P<0.01 or P<0.05); while TanⅡA group decreased more significantly.Conclusion:1. TanⅡA can reduce the level of myocardial fibrosis and delay the process of ventricular remodeling, it may concern with down regulating the expression of TGF-β1 and α-SMA in rats with pressure overload.2. TanⅡA play an important role in cell protection, and it is helpful to promote the recovery of cardiac function, it may relate to the expression of oxidative stress sensitive factor HO-1. |
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