原花青素抑制病毒性心肌炎大鼠心肌纤维化的机制

Mechanism of proanthocyanidins inhibiting myocardial fibrosis in rats with viral myocarditis

目的探究原花青素对病毒性心肌炎大鼠心肌纤维化的抑制效应机制,观察转化因子-β(TGF-β)/Smads信号通路的变化,研究其抑制病毒性心肌炎心肌纤维化的机制。方法选择60只雄性SD大鼠随机分为空白组(腹腔注射生理盐水)、模型组、阳性对照组(腹腔注射利巴韦林药液36 mg/kg)及原花青素高、中、低剂量组(400、200、100 mg/ml原花青素灌胃)各10只,除空白组外,均采用柯萨奇B3病毒法制作病毒性心肌炎大鼠模型,采用酶联免疫吸附试验法(ELISA)测定大鼠血心肌酶谱、心肌肌钙蛋白I(cTnI)含量及I型前胶原羧基端前肽(PICP)、I型胶原交联羧基末端肽(ICTP)水平;免疫组化染色光镜下观察各组大鼠心肌组织纤维化程度;测定各组全心质量指数(HMI)与左心室质量指数(LVMI);蛋白印迹法(WB)测定各组大鼠心室组织TGF-β/Smads信号通路相关蛋白TGF-β1及Ⅰ型、Ⅲ胶原纤维蛋白表达水平;实时荧光定量聚合酶链反应(RT-PCR)测定心肌组织TGF-β1 mRNA、Smad2 mRNA、Smad3 mRNA水平。结果与空白组比较,模型组、阳性对照组肌红蛋白(MB)、肌酸激酶同工酶(CK-MB)、cTnI、PICP、ICTP、HMI、LVMI、TGF-β1、I型胶原纤维蛋白、Ⅲ型胶原纤维蛋白及TGF-β1 mRNA、Smad2 mRNA、Smad3 mRNA均上升(P<0.05),与模型组比较,阳性对照组、原花青素低、中、高剂量组MB、CK-MB、cTnI、PICP、ICTP、HMI、LVMI、TGF-β1、I型胶原纤维蛋白、Ⅲ型胶原纤维蛋白及TGF-β1 mRNA、Smad2 mRNA、Smad3 mRNA均降低(P<0.05),与阳性对照组比较,原花青素低、中、高剂量组MB、CK-MB、cTnI、PICP、ICTP、HMI、LVMI、TGF-β1、I型胶原纤维蛋白、Ⅲ型胶原纤维蛋白及TGF-β1 mRNA、Smad2 mRNA、Smad3 mRNA均下降(P<0.05),原花青素各剂量组各指标比较差异无统计学意义。结论原花青素可减轻病毒性心肌炎大鼠心肌纤维化病变程度,其作用可能与抑制TGF-β/Smads信号通过转导,阻止胶原合成及沉积有关。

OBJECTIVE To investigate the inhibitory effect of proanthocyanidins on myocardial fibrosis in rats with viral myocarditis, observe the changes of transforming factor β(TGF-β)/Smads signaling pathway, and to study its mechanism of inhibiting myocardial fibrosis in viral myocarditis. METHODS Totally 60 male SD rats were randomly divided into blank group(intraperitoneal injection of normal saline), model group, positive control group(intraperitoneal injection of ribavirin solution 36 mg/kg), high, medium and low dose of proanthocyanidin groups(gavage with 400, 200 and 100 mg/ml of proanthocyanidin), with 10 rats in each group. Except for the blank group, all rat models of viral myocarditis were made by the Coxsackievirus B3 virus. Enzyme-linked immunosorbent assay(ELISA) was used to determine the blood myocardial enzymes spectrum, cardiac troponin I(cTnI), carboxyterminal propeptide of typeⅠ procollagen(PICP) and type I collagen carboxy-terminal cross-linked peptide(ICTP). The degree of myocardial tissue fibrosis in each group of rats was observed under the immumohistochemical staining light microscope. The whole heart weight index(HMI) and left ventricular mass index(LVMI) of each group were measured. The Western blotting(WB) method was used to determine the expression levels of TGF-β/Smads signaling pathway-related protein TGF-β1 and type Ⅰ and Ⅲ collagen fibrin protein in ventricular tissues of each group. Real-time fluorescence quantitative polymerase chain reaction(RT-PCR) was used to determine the levels of TGF-β1 mRNA, Smad2 mRNA and Smad3 mRNA in myocardial tissues. RESULTS Compared with those in the blank group, MB, CK-MB, cTnI, PICP, ICTP, HMI, LVMI, TGF-β1, type I collagen fibrin protein, type III collagen fibrin protein, TGF-β1 mRNA, Smad2 mRNA and Smad3 mRNA were all increased in the model group and the positive control group(P<0.05). Compared with those in the model group, MB, CK-MB, cTnI, PICP, ICTP, HMI, LVMI, TGF-β1, type I collagen fibrin protein, type III collagen fibrin protein, TGF-β1 mRNA, Smad2 mRNA and Smad3 mRNA in the positive control group, low, medium and high dose proanthocyanidins groups were all decreased(P<0.05). Compared with those in the positive control group, MB, CK-MB, cTnI, PICP, ICTP, HMI, LVMI, TGF-β1, type I collagen fibrin protein, type III collagen fibrin protein, TGF-β1 mRNA, Smad2 mRNA and Smad3 mRNA in the low, medium and high dose proanthocyanidins groups were all decreased(P<0.05), and there was no significant difference among the 3 proanthocyanidins groups(P>0.05). CONCLUSION Proanthocyanidins can reduce the degree of myocardial fibrosis in rats with viral myocarditis, and its effect may be related to the inhibiting of TGF-β/Smads signal transduction and preventing of collagen synthesis and deposition.