| Objective: To investigate the ameliorated effects and mechanism of Ginkgo Biloba Extract(GBE) on experimental cardiac remodeling of rat in vitro and in vivo. Methods: 1. The cardiac fibroblast proliferation and differentiation were reproduced by culture cardiac fibroblast exposed with TGF- β1. Five groups were randomly allotted into as following: Group A : negative control group, the CFb + drug with the volume of cell culture; Group B: TGF- β1 group, CFb + TGF- beta 1(20 ng/m L); Group C: SB431542(ALK5 inhibitor) group, CFb + SB431542(6 m g/m L) + TGF- beta 1(20 ng/m L); Group D: GBE low-dose group, CFb + GBE(2 m g/ml) + TGF- beta 1(20 ng/ml); Group E: GBE high dose group, CFb + GBE(20 m g/ml) + TGF- beta 1(20 ng/ml). The cardiac fibroblast proliferation and morphology were determined by MTT method and HE staining detection. Immunohistochemical method was used to detect protein expression of TGF- beta 1, collagen type I and III, MMP- 2 and MMP- 9 protein expression, as well as TIMP1 and TIMP2. 2. The injury of rat primary culture cardiac myocytes(RPCCM) was reproduced by Ang II, and then incubated with GBE and ALK5 inhibitor. The experimental groups were randomly divided into 5 groups as following:(1) : group A: negative control group, the RPCCM + equality volume culture medium,(2) group B: Ang II group, RPCCM + Ang II(10-5 mol/L),(3) group C: SB431542 inhibitor action group, RPCCM + SB431542/ALK5 inhibitor(6 mg/m L) + Ang II(10-5mol/L),(4) group D: GBE low-dose group, RPCCM + GBE(2 m g/ml) + Ang II(10-5 mol/L),(5) group E: GBE high dose group, RPCCM + GBE(20 m g/ml) + Ang II(10-5mol/L). The apoptotic morphology was deteremined by HE staining in the cardiac myocytes induced by AngⅡ, the cardiac myocytes apoptosis were detected by flow cytometry and TUNEL staining. The expression of apoptosis related proteins Bcl-2 and Bax in the cardiac myocytes were detected by real-time fluorescence quantitative PCR and laser scanning confocal microscopy(LSCM). 3. The rats were designated into 5 groups(n=20, respectively)by chance after acute cardiac infarction induced by ligated the anterior descending branch of left coronary artery as following: sham operation group(group A), acute myocardial infarction model group(group B), aspirin 10 mg/kg treatment group(group C), captopril 20 mg/kg treatment group(group D) and Ginkgo Biloba extract 100 mg/kg treatment group(group E). The rats were treated with different agents continued 4w and 8w, respectively. The immunohistochemistry methods was used to assay the protein expression of I collagen, MMP-2 and MMP-9, and real time PCR was used to detect the m RNA expression of TGF-β1,MMP-2 and MMP-9. Results:1. Compared with TGF- beta 1 group, high concentration of GBE and inhibiting group I, III collagen protein expression significantly reduced(P < 0.05). Compared with TGF- beta 1 build module, high concentration of GBE group of MMP- 2 and MMP- 9 protein expression was significantly reduced(P < 0.05), Only MMP- 2 protein expression was significantly reduced(P < 0.05) in SB431542. And in low concentration GBE group, MMP 2 and MMP- 9 protein expression had no statistical significance; Compared with TGF- beta 1 build module, TIMP1 and TIMP2 protein expression were decreased in SB431542(P < 0.05). However, only TIMP1 protein expression decreased in high concentrations of GBE group(P < 0.05), and there is not statistical significance in low concentration GBE group. 2. Compared with the negative control group, Ang II made module Bax gene expression is increased, bcl-2 expression decreased, the difference has statistical significance(P < 0.05). B431542 group and high concentration GBE group Bax gene expression decreased(P < 0.05).B431542 group and high concentration GBE group bcl-2 gene expression increased(P < 0.05).Compared with Ang II group, cell apoptosis rate reduced significantly in inhibitors and high concentration of GBE group(P < 0.01), the Bcl-2 protein expression was significantly increased in high concentration of GBE group cells(P < 0.05), Bax protein expression in only significantly reduced high concentration of GBE group cells(P < 0.05), the Bcl- 2 / Bax have significantly higher expression in low concentration group and high concentration of GBE(P < 0.05). 3.Compared with group B, regardless of 4 weeks or 8 weeks, m RNAtranscriptional level of TGF-β1, MMP-2, 9 in group E,C,D and protein expression of I collagen and MMP-2, 9 were significantly lower(P<0.01); the m RNA expression of TGF-β1 in group E,C,D at 8 weeks were significantly less than that at 4 weeks(P<0.01); at 8 weeks, the expression level of I collagen in group E,C,D were lower than that at 4 weeks(P<0.05), and there was no statistical difference in MMP-2, 9 protein expression level between group E and group C. Conclusion: 1. TGF-β1 can obviously promote the process of myocardial fibrosis, GBE could significantly inhibit myocardial fibrosis process induced by TGF- beta 1, resulting a significant reduction in Ⅰ, Ⅲ collagen secretion and deposition, lower levels of MMP- 2 and MMP- 9, and obviously inhibiting TIMP – 1. 2expression.2. Ang II can cause myocardial cell apoptosis, but GBE can significantly inhibit apoptosis caused by Ang II reaction, by raising the Bcl- 2 protein apoptosis gene expression and promoting the expression of apoptosis gene Bax protein. In addition, it can increase the Bcl- 2 / Bax raito. 3. GBE may inhibit rat myocardial remodeling after acute myocardial infarction via reducing the transcription of TGF-β1, MMP-2,9 gene and the expression of I collagen, MMP-2, 9 protein in myocardial cells. GBE can ameliorate the cardiac fibrosis which involved in inhibiting cardiac myocytes apoptosis and ameliorated the proliferation and differentiation of cardiac fibroblast. |
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