| Objective:1. To observe the changes of RhoA and ROCKl expressions in rat myocardial tissues with pressure overload and to explore the relationship between the myocardial fibrosis and RhoA/ROCK signaling pathway.2. To evaluate the intervention of tanshinone ⅡA (Tan ⅡA) on the expression of RhoA and ROCK1protein and the content of promote fibrosis substance myocardial transforming growth factor-β1(TGF-β1), connective tissue growth factor (CTGF), nuclear factor-xB p65(NF-κB p65) and serum tumor necrosis factor-a (TNF-a), and to explore the effects and mechanisms on myocardial fibrosis with pressure overload.Methods:1. Sixty Sprague-Dawley (SD) rats were randomly allocated into two groups: sham-operated (Sham group, n=8) and abdominal aortic banding (n=52). All abdominal aorta binding rats were induced by suprarenal abdominal aorta constriction. After4weeks,32abdominal aorta binding rats were divided randomly into4groups (each, n=8):Model group, Tan Ⅱ A of low dose group (L-Tan Ⅱ A group) Tanshinone Ⅱ A sodium injection (10mg/kg), qd i.p, Tan Ⅱ A of high dose group (H-Tan ⅡA group) Tanshinone ⅡA sodium injection (20mg/kg), qd i.p, positive captopril group (captopril group) captopril (100mg/kg), qd p.o and age matched SD sham operated group (n=8) as control. Animals of the model and Sham groups were given with natria chloride (4ml/kg), qd i.p. Rats were given continuously for4weeks in every goup.2.8weeks after operation, the animals were sacrificed and accurately weight body quality (BW), heart weight (HW), left ventricular weight (LVW). Cardiac mass Index (HWI)=(HW/BW) and left ventricular mass index (LVWI)=(LVW/BW). Cardiac hypertrophy was indicated by HWI and LVWI.3. Myocardial histological and morphological changes were determined by HE staining and Masson staining, in concurrent evaluation of myocardial hydroxyproline (HYP) content by ELISA, as well as RhoA and ROCK1content by immunohistochemistry staining and Western Blot and immunohistochemistry staining for myocardial TGF-β1, CTGF, NF-κB p65content and ELISA for serum TNF-α content.Results:1. Cardiac Mass Index (HWI) and Left Ventricular Mass Index (LVWI)Compared with those in Sham group, HWI and LVWI were significantly increased in Model group (P<0.01); Compared with those in Model group, HWI and LVWI were decreased in the L-Tan Ⅱ A group (P<0.05), while these indexes were significantly decreased in the H-Tan ⅡA group and Captopril group (P<0.01).2. Alterations of myocardial histology and morphologyThe HE staining show:the sham rats, myocardial fibers arranged in neat rows, stripes obvious and nucleus clear, cell swelling and myocardial interstitial and microvascular normal; the Model rats, myocardial fibers significantly enlarged and disorganized, some myocardial cell edema, stripes fuzzy, some myocardial fracture, the muscle bundles gap was increased, myocardial nuclear pyknosis, interstitial inflammatory cells infiltration. While these changes were improved to some extent by L-Tan ⅡA, H-TanⅡA and Captopril groups. The Masson staining show:collagen fibers were blue, while myocardial cells were red. Compared with Sham group, collagen deposition was increased significantly in Model group, while L-Tan ⅡA group, H-Tan ⅡA group and Captopril group have alleviated in some extent.3. Myocardial hydroxyproline (HYP) contentCompared with this in Sham group, myocardial HYP content was increased significantly in Model group (P<0.01); Compared with this in Model group, myocardial HYP content was deceased in the L-Tan ⅡA group (P<0.05), while this index was decreased significantly in the H-Tan ⅡA group and Captopril group (P<0.01).4. The expression of myocardial TGF-01Compared with this in Sham group, myocardial TGF-β1expression was increased significantly in Model group (P<0.01); Compared with this in Model group, myocardial TGF-β1expression was deceased in the L-Tan ⅡA group (P<0.05), while this index was decreased significantly in the H-Tan ⅡA group and Captopril group (P<0.01).5. The expression of myocardial CTGFCompared with this in Sham group, myocardial CTGF expression was increased significantly in Model group (P<0.01); Compared with this in Model group, myocardial CTGF expression was deceased in the L-Tan ⅡA group (P<0.05), while this index was decreased significantly in the H-Tan ⅡA group and Captopril group (P<0.01).6. The expression of myocardial NF-κB p65Compared with this in Sham group, myocardial NF-κB p65expression was increased significantly in Model group (P<0.01); Compared with this in Model group, myocardial NF-κB p65expression was deceased in the L-Tan ⅡA group (P<0.05), while this index was decreased significantly in the H-Tan ⅡA group and Captopril group (P<0.01).7. The expression of serum TNF-aCompared with this in Sham group, serum TNF-a expression was increased significantly in Model group (P<0.01); Compared with this in Model group, serum TNF-aexpression was deceased in the L-Tan ⅡA group(P<0.05), while this index was decreased significantly in the H-Tan ⅡA group and Captopril group (P<0.01).8. The expression of myocardial RhoA and ROCK1Western Blot analysis shows that compared with those in Sham group, myocardial RhoA and ROCK1protein expressions were increased significantly in Model group (P<0.01); Compared with those in Model group, myocardial RhoA and ROCK1protein expressions were deceased in the L-Tan ⅡA group (P<0.05), while this index was decreased significantly in the H-Tan ⅡA group and Captopril group (P<0.01). Immunohistochemical staining has the same trend with Western Blot.Conclusion:1. RhoA/ROCK signaling pathway may be closely linked with the occurrence and development of myocardial fibrosis.2. The fibrogenic cytokines (TGF-β1and CTGF) and proinflammatory cytokines (NF-κB and TNF-a) may play an important role in the occurrence and development of myocardial fibrosis.3. Tan ⅡA may reduce myocardial fibrosia by inhibiting the the activity of RhoA/ROCK signaling pathway, down-regulating the fibrogenic cytokines and proinflammatory cytokines, reduce myocardial fibrosis. |
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