鹿茸多肽介导TGF-β/samds对心梗后心肌缺血损伤大鼠保护作用及机制研究

Protective effect and mechanism of TGF-β/samds mediation by antler polyskin on post-infarction myocardial ischemic injury in rats

目的观察鹿茸多肽(velvet antler peptide,VAP)对冠状动脉左前降肢结扎后心肌梗死缺血及纤维化损伤大鼠的保护作用,阐明VAP介导TGF-β/samds信号通路的作用机制。方法60只大鼠随机分为6组:假手术组(SHAM group)、模型组(MI group)、卡托普利阳性药对照组(KTPL group,30 mg/kg)、鹿茸多肽低、中、高剂量组(VAP group,100、200、300 mg/kg)。大鼠采用冠状动脉左前降肢结扎法复制大鼠心肌损伤模型,制备模型3 d后大鼠经口服(灌胃)给药,灌胃1 mL/d,连续28 d。末次给药2 h后,麻醉大鼠经腹主动脉无菌采血并离心取血清。心电图分析其心肌损伤程度。HE染色法观察各组大鼠心脏切片病理形态学改变;ELISA法检测各组大鼠血清中肌酸激酶同工酶(CK-MB)、心肌肌钙蛋白(cTn)水平;Western blot法检测各组大鼠心肌组织CollagenⅠ、CollagenⅢ、TGF-β1、Smad7、Smad4、Smad2/3、p-Smad2/3相关蛋白水平。结果心电图分析表明,通过ST段抬高、T波倒置、Q波形成的变化提示心肌损伤。HE染色发现,与SHAM组比较,MI组大鼠心肌纤维排列紊乱、横向条纹消失,细胞肿胀破裂、坏死,细胞核变形移位;与MI组相比,KTPL组和VAP组大鼠心肌病理学形态明显改善。ELASA法检测,与SHAM组相比,MI组大鼠的CK-MB、cTnT和cTnI含量被明显诱导(P<0.01);与MI组相比,KTPL组和VAP组大鼠心肌组织中CK-MB、cTnT和cTnI含量明显降低(P<0.01)。Western blot法检测,与SHAM组相比,MI组大鼠通过上调TGF-β1、Smad7、Smad4、Smad2/3、p-Smad2/3、CollagenⅠ、CollagenⅢ蛋白表达水平及下调Smad7表达水平导致心肌损伤(P<0.01);与MI组比较,KTPL组和VAP组大鼠显著下调TGF-β1、Smad2/3、p-Smad2/3、Smad4、Collagen I、CollagenⅢ蛋白表达水平及升高Smad7蛋白表达量,改善纤维化(P<0.01)。结论心肌缺血梗死大鼠能激活TGF-β/smads信号转导,通过卡托普利及鹿茸多肽给药后可抑制TGF-β1、smads蛋白改善心肌梗死后心肌纤维化。鹿茸多肽(VAP)可能通过调控TGF-β/smad信号通路保护心肌梗死后心肌缺血及纤维化损伤。

Objective To observe the protective effects of velvet antler peptide(VAP) on myocardial infarction(MI) ischemia and fibrosis injury in rats after coronary artery ligation and the mechanism of TGF-β/Smad signaling pathway mediation by VAP. Methods 60 SPF male Wistar rats were randomly divided into six groups: Sham operation;MI;positive drug control(captopril, 30 mg/kg);low-, medium-, and high-dose(100, 200, and 300 mg/kg) VAP groups. The left anterior descending limb of the coronary artery was ligated to provide a model of myocardial injury in rats. Three days after the model was made, the rats were orally administered with 1 mL/d VAP for 28 days. Two hours after the last administration, we collected blood aseptically from the abdominal aorta of anesthetized rats and centrifuged the samples to obtain VAP serum. Electrocardiogram was used to analyze the degree of myocardial injury. HE staining was used to observe pathological changes in the myocardial tissue of rats. Serum creatine kinase isoenzyme(CK-MB) and cardiac troponin(cTn) levels were measured by enzyme linked immunosorbent assay ELISA. The levels of TGF-β1, Smad2/3, p-Smad2/3, Smad4, Smad7, Collagen I and Collagen Ⅲ proteins in myocardial tissue were detected by Western blot. Results HE staining in the MI group showed that myocardial fibers group were disordered;transverse stripes had disappeared;cells were swollen, cracked, and necrotic;and nuclei were deformed and displaced compared with the findings in the Sham group. The pathological morphology of myocardium in KTPL group and VAP group was significantly improved compared with that in the MI group. ELISA showed significantly higher CK-MB, cTnT(cardiac troponin T), and cTnI(cardiac troponin I) levels in the MI group compared with the findings in the Sham group(P<0.01). There were significantly lower CK-MB, cTnT, and cTnI contents in the myocardial tissue of positive drug control group KTPL and VAP groups compared with the findings in the MI group(P<0.01). The Western blot showed that myocardial injury was caused by the up-regulation of TGF-β1, Smad2/3, p-Smad2/3, Smad4, Collagen I and Collagen Ⅲ and down-regulation of Smad7 protein expression in the MI group(P<0.01). Compared with the findings in the Sham group. The expression of TGF-β1, Smad2/3, p-Smad2/3, Smad4, Collagen I and Collagen Ⅲ were significantly down-regulated and Smad7 was up-regulated in the KTPL group and VAP group, which improved fibrosis(P<0.01). Conclusions MI rats activate TGF-β/Smad signal transduction, and captopril and VAP inhibit TGF-β1 and Smad proteins to improve myocardial fibrosis after MI. VAP may protect against myocardial ischemia and fibrosis after MI by regulating the TGF-β/Smad signaling pathway.