| BackgroudA direct relation has been found between the decrease of cardiac mortality and the regression of cardiac hypertrophy. Pressure overload-induced myocardial remodeling is characterized by cardiac hypertrophy and fibrosis, which increases myocardial stiffness and inducts to diastolic dysfunction even to death. However, a convincing and potent antifibrogenic therapeutic modality by targeting myocardial local inflammation activation is still unavailable. Cardiac fibrosis is a hallmark of the cardiomyopathic processes associated with metabolic disturbances such as diabetesand obesity. Cardiac fibrosis is characterized by net accumulation of extracellular matrix in the myocardium and is an integral component of most cardiac pathologic conditions. Fibrotic remodeling of the myocardium is the end-result of most types of cardiac injury.Sophora alopecuroides Linn.(compound Kudouzi injection) has been used mainly for the treatment of inflammation, fever, pain, and edema. Sophocarpine (Figure1), the most abundant active ingredient in Kudouzi, is a tetracyclic quinolizidine alkaloid. Of note, sophocarpine injection (called Kangke injection) has been shown to have obviously therapeutic effects for viral myocarditis in clinical trials. Importantly, the administration of sophocarpine significantly improved cardiac function and reduced infarct size in ischemia reperfusion injury (I/R) rat heart in vivo. Furthermore, sophocarpine ameliorated the contents of inflammatory mediators, neutrophil infiltration, and myeloperoxidase activity. Recently, studies have shown that sophocarpine could inhibit the levels of IL-6and TNF-a in murine macrophages and prevent cachexia-related symptoms induced by colon26adenocarcinoma in mice. Chen et al.(2005) have also suggested that sophocarpine could improve the end-systolic pressure and the end-diastolic volume, and play a pivotal role in maintaining healthy cardiac function.ObjectiveIn light of previous studies, we hypothesized that sophocarpine could protect heart against pressure overloadinduced cardiac fibrosis. To verify our hypothesis, we used a rat model of cardiac hypertrophy to investigate the cardioprotective properties of sophocarpine on hypertensive ventricular remodeling and the potential mechanisms involved.MethodsSixty male rats were randomly divided into four groups:Sham group (n=9), Model group (n=11), Sophocarpine(10mg/kg) group (n=9), Sophocarpine(20mg/kg) group (n=9)and Sophocarpine(40mg/kg) group (n=10). The test substance, sophocarpine, was isolated from the dry seeds of Kudouzi. Adult male Sprague-Dawley rats were subjected to a suprarenal abdominal aorta constriction (AC) or sham to induce sustained pressure overload. Six weeks later, rats were randomly assigned to receive sophocapine (10,20, and40mg/kg, gavage) or vehicle treatment for an additional6weeks. Six weeks after treatment, cardiac dysfunction, cardiac coefficient, cardiac fibrosis, hydroxyproline concentration, and inflammation mediators were examined. The levels of pro-inflammatory cytokines, MMP-2, and MMP-9were measured using a commercially available ELISA kits. The collagen content was quantitated spectrophotometrically at560nm against a hydroxyproline standard curve. Assessment of cardiac fibrosis wasobservedbyMasson’s trichrome staining. The p-IKBa expression was examined by western blot analysis.Results1. Effects of sophocarpine on hemodynamics and cardiac coefficientFollowing6weeks after cardiac hypertrophy, all animal’s body weights were same (data not shown). Whereas, the left ventricular weight/body weight in model animals was higher than that of sham animals, indicating cardiac hypertrophy (P<0.01). In contrast, sophocarpine (20and40mg/kg) obviously ameliorated these parameters (P<0.05, P<0.01),(Table1). Table1also depicted the hemodynamic values recorded in experimented animals. After AC, LVSP was decreased, while LVEDP was increased when compared with sham group (P<0.01). Early treatment with sophocarpine (20and40mg/kg) significantly improved LVSP and LVEDP (P<0.05, P<0.01).2. Effects of sophocarpine on the expression of pro-inflammatory cy tokinesIn the current study, two representative pro-inflammatory cytokines, i.e. IL-6and IL-1β were measured. In model animals, circulating IL-6(102.29±16.46vs.65.98±10.88, pg/mL) and IL-1β (126.08±25.03vs.82.84±12.34, pg/mL) were higher than that in sham-operated animals (P<0.01). However, sophocarpine treatment significantly decreased the levels of two pro-inflammatory cytokines (IL-6,87.36±7.97and83.49±13.34at dose of20and40mg/kg, respectively; IL-1β,97.02±7.18and85.00±17.21at dose of20and40mg/kg, respectively. P<0.05and P<0.01, pg/mL), suggesting an anti-inflammatory effect of sophocarpine treatment.3. Effects of sophocarpine on collagen content levelBoth in experimental and clinical conditions, markers of collagen turnover were regarded as a reliable tool for monitoring from a distance cardiac tissue repair and fibrosis. We then tested the effects of sophocarpine on collagen content level in animals. At6weeks following cardiac hypertrophy, the collagen content was greater in AC groups as compared with sham group. However, chronic continuous treatment with sophocarpine (20and40mg/kg) significantly ameliorated this parameter (P<0.05, P<0.01).4. Effects of sophocarpine on cardiac fibrosisThe effects of sophocarpine on the extent of injury were examined in Masson’s trichrome stained sections from vehicle-and sophocarpine-treated hearts after ligation or sham surgery. A significant increase in fibrosis (collagen staining in blue color) was evident in AC rats compared with sham-operated animals. However, sophocarpine markedly attenuated those pathological changes. 5. Effects of sophocarpine on MMP-2and MMP-9contentAs shown in Fig.5, the content of MMP-2and MMP-9in serum were also elevated in model group (34.10±5.49and21.01±4.17, respectively, ng/mL) when compared with sham-operated group (18.14±3.71and11.83±1.76, respectively, ng/mL) at the end of the6th week. Treatment with sophocarpine reduced these two matrix metalloproteinases (MMP-2,30.19±3.98and27.83±4.45at dose of20and40mg/kg, respectively; MMP-9,17.60±2.41and16.56±1.06at dose of20and40mg/kg, respectively, ng/mL).6. Effects of sophocarpine on p-IKBa expressionSix weeks later, the expression of p-IKBa was examined by western blot, and the result indicated that this parameter was significantly higher in model group than that in the sham group (2.15±0.38vs.1.03±0.08, P<0.01), while sophocarpine dramatically ameliorated this change (1.72±0.24, P<0.01), indicating that test substance might block IKBa phosphorylation.Conclusion1. Sophocarpine (20and40mg/kg) significantly decreased left ventricular weight/body weight. The beneficial effects were associated with amelioration of left ventricular systolic pressure (LVSP) and left ventricular enddiastolic pressure (LVEDP).2. These findings indicated that sophocarpine potentially had antifibrotic effects. The mechanism might be due to modulation of the balance between pro-inflammatory cytokine expression and collagen content level as well as MMPs expression via the NF-Kb signaling pathway. |
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