【作者】 吴俊标；

【导师】 周玖瑶；

【作者基本信息】 广州中医药大学 ， 中药学， 2012， 硕士

【摘要】 目的：环维黄杨星D (Cyclovirobuxinum D, Cvb-D)为黄杨科植物小叶黄杨及其同属植物中提取精制所得生物碱,可行气活血,通络止痛,对与心肌肥厚发生发展密切相关的因子都有明显调节作用,但对心肌肥厚疗效尚未见报道。本研究利用异丙肾上腺素(ISO)诱导小鼠心肌肥厚、左旋甲状腺素(L-thy)诱导大鼠心肌肥厚两个模型,从组织病理形态学、神经体液因子、心肌能量代谢以及相关信号通路探讨Cvb-D对心肌肥厚的药效及其作用机制,为其临床应用提供实验依据,拓展中医药防治心血管疾病的新领域。方法：取KM小鼠皮下注射ISO1.0mg/kg,每d两次,连续14d,制备小鼠心肌肥厚模型,给予低、中、高剂量环维黄杨星D(6.0、9.0、12.0mg/kg·d),灌胃给药14d。以小鼠心脏重量指数(HW=全心重量/体重)、左心室重量指数(LIWI=左心室重量/体重)、病理形态学(HE染色)、心肌能量代谢变化(游离脂肪酸FFA、乳酸LD)和凋亡调控基因bax、bcl-2蛋白阳性表达量(免疫组织化学)为指标考察Cvb-D对小鼠心肌肥厚的保护作用。取SD大鼠腹腔注射L-thy1.0mg/kg-d,连续14d,制备大鼠心肌肥厚模型,给予低、中、高剂量环维黄杨星D(6.0、12.0、24.0mg/kg·d),灌胃给药14d。以大鼠心脏重量指数、左心室重量指数、病理形态学、心肌NO系统(NO、NOS)、抗氧化损伤(SOD、MDA含量)为指标考察Cvb-D对大鼠心肌肥厚的疗效。以凋亡调控基因bax、bcl-2蛋白阳性表达量、原癌基因c-fos、c-jun mRNA和丝裂原活化蛋白激酶MAPK通路中p38a, p381βmRNA的表达变化(实时荧光相对定量RT-PCR)为指标,初步探讨Cvb-D对大鼠心肌肥厚的保护作用机理。结果：1环维黄杨星D (Cvb-D)对心肌肥厚的药效研究与模型对照组比较,Cvb-D高剂量组小鼠心脏重量指数(HW)和左心室重量指数(LHWI)明显降低(P<0.01或P<0.05)；Cvb-D低、中、高剂量组大鼠心脏重量指数(HW)和左心室重量指数(LHWI)明显降低(P<0.01或P<0.05)：Cvb-D中、高剂量组大鼠和小鼠心肌细胞肥厚扩大、细胞间隙紊乱现象有明显的改善；Cvb-D低、中、高剂量组小鼠血清游离脂肪酸(FFA)和乳酸(LD)含量显著下降(P<0.01,或P<0.05);Cvb-D中、高剂量组大鼠心肌组织SOD活性显著升高；Cvb-D低、中、高剂量组大鼠心肌组织MDA含量明显降低(P<0.05)；Cvb-D低、中、高剂量组大鼠心肌组织NO含量显著上升(P<0.01或P<0.05)；Cvb-D中、高剂量组大鼠心肌组织NOS的含量显著升高(P<0.01或P<0.05)2环维黄杨星D (Cvb-D)对心肌肥厚的作用机理与模型对照组比较,Cvb-D低、中、高剂量组小鼠和大鼠心肌组织bax蛋白的表达显著下调,且使bcl-2蛋白的表达明显上调,bax、bcl-2累积光密度IOD比值显著降低(P<0.05或P<0.01),减少心肌细胞的凋亡；Cvb-D中、高剂量组大鼠心肌c-fos mRNA显著降低(P<0.01)；Cvb-D中、高剂量组大鼠心肌c-jun mRNA显著下调(P<0.05或P<0.01)；Cvb-D中、高剂量组大鼠心肌p38a、p38βmRNA显著下调(P<0.01)结论：1Cvb-D抑制心肌肥厚,改善心肌肥厚病理形态学,阻止左室心肌肥厚的发生发展。其机制与其能通过影响心肌一氧化氮系统,抗氧化损伤进而改善心功能、阻止氧自由基对心肌细胞的损伤作用有关。2Cvb-D通过减少心肌肥厚心肌组织游离脂肪酸(FFA)、乳酸(LD)的生成,改善心肌能量代谢,减轻心脏负荷,改善心肌肥厚预后。3Cvb-D对ISO、L-thy诱导的心肌肥厚细胞凋亡具有较好的抑制作用,其机制可能与其能够下调控制凋亡的bax基因和上调bcl-2基因的表达有关。4环维黄杨星D对心肌肥厚具有良好的保护作用,其分子机制与其能够调节原癌基因c-fos、c-jun mRNA的表达、影响p38MAPK信号通路有关。更多还原

【Abstract】 Objectives:Cyclovirobuxine D (Cvb-D), an alkaloid extracted from Buxusn microphylla Sieb.et Zucc.var.sinica Rehd.et Wils and its congeneric plants, can promote Qi and blood flowing,dredge collaterals and relieve pain.Cyclovirobuxine D plays an important role in the regulation of neuroendocrine factors,which are linked closely with myocardial hypertrophy.But there were no reports about the influence of Cvb-D on myocardial hypertrophy.In this study, the model of myocardial hypertrophy of mouse and rat were induced by isoproterenol (ISO) and L-thyroxine (L-thy), respectively. Cvb-D’s efficacy and its underlying mechanism of effect on cardiac hypertrophy were studied, in the aspects of pathomophology, NO system, antioxidant effect, myocardial energy metabolism and related signal pathways, aiming to provide new ideas and experimental bases for the clinical application of Cvb-D and expand a new research filed for prevention and treatment of cardiovascular by TCM.Methods:The Kunming mice were subcutaneous injected of ISO (1.0mg/kg), b.i.d.,for14consecutive days,to induce the mouse model of cardiac hypertrophy. The treatment groups were oral administrated Cvb-D (6.0,9.0,12.0mg/kg·d) for14days. The protective effects of Cvb-D on myocardial hypertrophy in mice were investigated with the heart weight index(HW=heart weight/body weight), left heart weight index(LHWI=left ventricular weight/body weight), the myocardial histopathological morphology(HE staining), the energy metabolism(The content of free fatty acid and lactic acid) and apoptosis regulation genes of bax and bcl-2(Immunohistochemistry) as indicators.The Sprague Dawley rats were intraperitoneal injected of L-thy (1.0mg/kg·d,) for14consecutive days to induce the rat model of cardiac hypertrophy. The treatment groups were oral administration of Cvb-D (6.0,12.0,24.0mg/kg·d) for14days. The protective effects of Cvb-D on myocardial hypertrophy in rats were investigated on the base of the heart weight index(HW=heart weight/body weight), left heart weight index(LHWI=left ventricular weight/body weight), the myocardial histopathological morphology(HE staining), NO system of myocardial (The content of NO and NOS), anti-oxidative damage(The content of SOD and MDA). Through the positive expression changes of bax and bcl-2proteins, changes of c-fos, c-jun,p38α and p38β mRNA(Real time QPCR) to explore the mechanism of Cyclovirobuxinum D on preventing cardiac hypertrophy preliminarilyResults:1The effects of Cyclovirobuxinum D on cardiac hypertrophyCompared with model group, high dose of Cvb-D could significantly reduce the weight index of the rat’s heart and the mass index of the left ventricular in mice (P<0.Olor P<0.05); high-, mid-, low-dose of Cvb-D all could significantly reduce the weight index of the rat’s heart and the mass index of the left ventricular in rats (P<0.01or P<0.05); high-, mid-dose of Cvb-D could improve the hypertrophy of myocardial cells, intercellular space disorder phenomenon in mice and rats; high-, mid-, low-dose of Cvb-D all could significantly reduce the content of free fatty acid (FFA) and lactic acid (LD) in serum(P><0.05or P<0.01);high-, mid-dose of Cvb-D could also improve the activity of SOD in myocardial organization, reduce the MDA’s content (P<0.05orP<0.0I) and significantly increase the content of NO and NOS (P<0.05or P<0.01).2The mechanism of Cyclovirobuxinum D on preventing cardiac hypertrophyCompared with model group, high-, mid-, low-dose of Cvb-D all could significantly reduce the expression of bax’s protein of myocardial tissue in mice and rats, upregulate the protein of bcl-2(P<0.05or P<0.01), and reduce the apoptosis of cardiac cells. High-, mid-dose of Cvb-D were able to significantly reduce the expression of mRNA of c-fos and c-jun in myocardial tissue(P<0.05orP<0.01);High-, mid-dose of Cvb-D were able to significantly down-regulated the expression of mRNA of p38a and p38p in myocardial tissue(P<0.01).Conclusions:1. The Cvb-D could inhibit cardiac hypertrophy, improving the morphology of left ventricular hypertrophy,and preventing the development of left ventricular hypertrophy. Its mechanism may be related to its effects on the myocardial nitric oxide system, anti-oxidative damage and thus improve the cardiac function and prevent injury related to oxygen free radicals on myocardial cells.2. The Cvb-D could ameliorate the mycardial energy metabolism and relieve the dardiac stress,by reducing the generation of free fatty acids(FFA) and lactic acid(LD) in cardiac tissue of hypertrophic cadiac.3. The Cvb-D showed a better inhibition effect of apoptosis of cardiomyocytes induced by ISO, L-thy, its mechanism may be related to down-regulating the bax gene which control apoptosis and up-regulating the bcl-2gene expression.4. The Cvb-D showed a great protective effect on cardiac hypertrophy. Its molecular mechanism could involve the regulation of the mRNA expression of proto-oncogene of c-fos and c-jun, and influence of the p38in MAPK signaling pathway.更多还原