| Objective: To investigate the effects of puerarin on myocardial fibrosis and the expression of connective tissue growth factor (CTGF) in myocardial tissue of the rats induced by isoproterenol (ISO).Methods: In this study, the model of myocardial fibrosis was established by subcutaneous injection (s.c.) with ISO (0.05% ISO, 5 mg/kg per day for ten days) in male Sprague-Dawley rats. Meanwhile, the rats were administrated by intraperitoneal injection (i.p.) with peurarin (100mg/kg per day for 56 or 46 days). The period of the present study of animals was 8 weeks. 48 male Sprague-Dawley rats weighting 150 to 170 g were devided into four groups randomly: model group (n=16, 0.05% ISO 5 mg/kg per day s.c. for ten days, NS 1ml per day i.p. for 56 days), control group ( n=8, NS 10 ml/kg per day s.c. for ten days, NS 1ml per day i.p. for 56 days), early administration with puerarin group (early puerarin-treated group, n=12, 0.05% ISO 5mg/kg per day s.c. for ten days, puerarin 100mg/kg per day i.p. for 56 days) , later administration with puerarin group (later puerarin-treated group, n=12, 0.05% ISO 5mg/kg per day s.c. for ten days, NS 1ml per day i.p. for ten days, puerarin 100mg/kg per day i.p. for 46 days after ten days). At the end of study, left ventricular internal diameter at end-diastole (LVIDd), left ventricular internal diameter at end-systole (LVIDs), left ventricle end-diastole volume (LVEDV), left ventricle end-systole volume (LVESV), left ventricle ejection fraction (LVEF) and fractional shortening (FS) of rats were detected by GE vivid7 diasonography. Then rats were anesthetized with 3% pentobarbital, and left ventricle (LV) weight to body weight ratio was assessed. The cross serial transverse sections of LV of the rats were examined microscopically after Van Gieson (VG) collagen staining, CTGF-antibody immunohistochemstry staining and transforming growth factor beta1 (TGF-β1)-antibody immunohistochemstry staining. The quantitative analyses of collagen volume fraction (CVF) , CTGF protein and TGF-β1 protein were performed using an image analysis computer system. CTGF mRNA level, TGF-β1 mRNA level and ratio of hydroxyproline to the total protein in LV myocardial tissue were measured.Results: There were 32 survival rats at the end of the present experiment.①parameters of echocardiogram: In comparison with control group, the LVEF and FS of model group were decreased obviously (P<0.01) and the LVIDd, LVIDs, LVEDV and LVESV of model group was increased markedly (P<0.01). The LVEDV and LVESV of model group was increased by tow- and eight-fold , respectively, compared to the control group. Comparing with model group, the LVEF and FS of the treatment with puerarin groups were increased (P<0.01). The LVEF and FS of early puerarin-treated group were more increased than later puerarin-treated group (P<0.05, P<0.01, respectively). However, comparing with model group, the LVIDd, LVEDV, LVIDs, LVESV of later puerarin-treated group were decreased (P<0.05, P<0.05, P<0.01, P<0.01, respectively). The LVIDs, LVESV of early puerarin-treated group were decreased in comparison with later puerarin-treated group(P<0.01, P<0.05, respectively). Furthermore, nonsignificant was the difference in the LVIDd and LVEDV in early puerarin-treated group, later puerarin-treated group and control group.②Myocardial fibrosis: It was observed that excessive collagen deposited in LV myocardial interstitium of the model rats after VG collagen staining. In comparison with control group, LV weight/ body weight ratio, CVF and ratio of hydroxyproline/ total protein in LV myocardial tissue of model group were significantly increased (P<0.01). LV weight/ body weight ratio, CVF and ratio of hydroxyproline/total protein in LV myocardial tissue of early puerarin-treated group were 2.3825±0.0755 mg/g, 0.1286±0.0097, 0.4873±0.0267μg/mg, respectively. In the same time, those of later puerarin-treated group were 2.4863±0.1131 mg/g, 0.1674±0.0158, 0.5304±0.0334μg/mg, respectively. Comparing with model group, LV weight/ body weight ratio, CVF and ratio of hydroxyproline/total protein in LV myocardial tissue of the treatment with puerarin groups were decreased (P<0.01). LV weight/ body weight ratio, CVF and ratio of hydroxyproline/total protein in LV myocardial tissue of early puerarin-treated group were lower than later puerarin-treated group (P<0.05, P<0.01, P<0.05, respectively).③Profibrotic cytokines: In comparison with control group, the CTGF mRNA and TGF-β1 mRNA in LV myocardial tissue of model group were upregulated noticeably (P<0.01), and those were increased by three- and two-fold respectively. In this study, puerarin downregulated the overexpression of CTGF gene and TGF-β1 gene in LV myocardial tissue induced by ISO in rats. Comparing with model group, the CTGF mRNA in LV myocardial tissue of early puerarin-treated group and later puerarin-treated group were significantly downregulated (P<0.01), and the TGF-β1 mRNA in LV myocardial tissue of early puerarin-treated group and later puerarin-treated group were downregulated (P<0.01, P<0.05, respectively). The CTGF mRNA and TGF-β1 mRNA in LV myocardial tissue of early puerarin-treated group were lower than those of later puerarin-treated group (P<0.05, P<0.01, respectively). The proteins of CTGF and TGF-β1 in LV myocardial tissue of model group were markedly increased compared to control group (P<0.01), and those were increased by sevene- and three-fold respectively. When compared with model group, the CTGF protein in LV myocardial tissue of early puerarin-treated group and later puerarin-treated group were markedly decreased (P<0.01), and the TGF-β1 protein in LV myocardial tissue of early puerarin-treated group and later puerarin-treated group were decreased (P<0.01, P<0.05, respectively). The CTGF protein and TGF-β1 protein in LV myocardial tissue of early puerarin-treated group were lower than those of later puerarin-treated group (P<0.05, P<0.01, respectively).④correlation analyze: CTGF protein was correlated with ratio of hydroxyproline/ total protein and CVF in LV of rats of every group (P<0.05).Conclusions: These data demonstrated that peurarin reduced the excessive collagen deposition in cardiac interstitial, and improved LV systolic dysfunction, and reduced LV dilatation of rats with chronic myocardial fibrosis induced by ISO. One of the mechanisms of the effects is related to downregulate the overexpression of CTGF and TGF-β1, two profibrotic cytokins, in LV myocardial tissue. Furthermore, the effects of early treatment with puerarin are better than those of later treatment with puerarin. |
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