论文标题:新药SGL-2治疗病毒性心肌炎的药理机制及新药SGL-3对心力衰竭治疗作用的研究
论文作者
论文导师 惠汝太,论文学位 博士,论文专业 遗传学
论文单位 中国协和医科大学,点击次数 240,论文页数 117页File Size10573K
2007-05-01论文网 http://www.lw23.com/lunwen_1322777/
Cardiovascular;; Viral Myocarditis;; Cardiac Remodeling;; Heart Failure;; Pharmacology
第一部分：SGL-2抗柯萨奇病毒的药效学、药理学机制 1．研究背景： 病毒性心肌炎严重危害人类健康，发病率为5％～20％；柯萨奇病毒B3(CVB3)是病毒性心肌炎的主要诱因之一。但至今仍无有效治疗手段。SGL-2的有效成分主要用于治疗流感及其他病毒性疾病。本研究的主要目的是探讨SGL-2在体外及体内的抗CVB3病毒的疗效以及其抗病毒机制。 2．实验方法和实验结果： 首先，建立CVB3感染HeLa细胞和心肌细胞的病毒性心肌炎细胞模型，选择利巴韦林作为阳性对照药物，并测定SGL-2在细胞水平的毒性。通过CPE抑制实验、病毒繁殖抑制实验对SGL-2的抗病毒作用进行初步确定，之后通过XTT染色和空斑形成实验计算和验证其抗病毒能力，结果显示，SGL-2的半数有效剂量IC_(50)分别为7.16±0.8和2.63±0.5μg／ml。半数毒性剂量CC_(50)则是利巴韦林的16倍(SGL-2：1648±219μg／ml，Ribavirin：103±14μg／ml)。病毒感染后不同时间点加入SGL-2，发现SGL-2主要在病毒感染的0-4小时内发挥作用。进一步研究结果显示SGL-2对病毒的吸附和穿入具有明确的抑制作用。 在体内试验中，同样首先测定SGL-2的半数致死剂量(LD_(50))，并根据该数据确定实验中SGL-2的使用剂量。病毒性心肌炎动物模型采用4周龄雄性Balb／C小鼠，腹腔注射0.1 ml CVB3悬液(10 PFU)或生理盐水。通过病理切片和RT-PCR对模型进行鉴定。小鼠接种病毒48小时后，灌胃给药，SGL-2的剂量分别为200mg·kg~(-1)·d(-1)、600 mg·kg~(-1)·d~(-1)和1800 mg·kg~(-1)·d~(-1)，利巴韦林(90 mg·kg~(-1)·d~(-1))作为阳性药物，生理盐水作为阴性对照，同时设置V-C、辅酶Q_(10)对照组和生脉饮对照。实验进行28天以后，通过Kaplan-Meier曲线计算各组动物的生存率，与阴性对照组相比(16％)，SGL-2治疗组(1800mg·kg~(-1)·d~(-1))能够明显增加动物的生存率(64％)；抑制CVB3引起的心脏的病理学改变，减少心肌组织内病毒滴度；此外，对心肌酶含量、干扰素水平和中和抗体方面也有一定的作用。 3．结论： SGL-2对CVB3导致的病毒性心肌炎具有确切的治疗作用，其抗病毒机制很可能与SGL-2抑制病毒粘附和穿入有关。 第二部分：SGL-3对心力衰竭的治疗作用及其机制的初步探讨 1．研究背景： 经过数十年探索，医学界对充血性心力衰竭(CHF)的治疗手段和治疗效果已经有了明显改善，但是心衰患者的发病率和死亡率依然据高不下。SGL-3的主要成分一直用于治疗与心衰相关的症状。本研究的目的就是要验证SGL-3是否有确切的治疗作用，同时揭示SGL-3的作用机制。 2．方法与结果： 在原代培养的乳鼠心肌细胞，SGL-3(10μg／ml)可以抑制Angiotensin-Ⅱ(1μmol／L)刺激引起的心肌细胞肥大，通过H~3掺入实验观察到蛋白合成量下降(减少74％±26％)，通过鬼笔环肽染色观察到细胞表面积减少(下降104％±31％)。体内实验采用20周龄雄性Wistar大鼠，对大鼠进行腹主动脉缩窄手术(AAC)或假手术，20周以后，通过超声心动图检测确定AAC术后大鼠出现心脏功能异常。之后将动物分组、灌胃给药，SGL-3的给药浓度为：150 mg·kg~(-1)·d~(-1)、500 mg·kg~(-1)·d~(-1)和1，500 mg·kg~(-1)·d~(-1)，同时设置对照组：卡托普利(270 mg·kg~(-1)·d~(-1))与倍他乐克(1，800 mg·kg~(-1)·d~(-1))合用组，给药18周。之后，使用多导生理仪和超声检测心脏血流动力学和形态学参数。结果显示，SGL-3能够改善心脏的收缩功能(AAC组EF值为51.8％±8.42％，假手术组EF值为75.6％±7.65％，SGL-3治疗组EF值为69.3％±7.73％，卡托普利与倍他乐克合用组的EF值为56.3％±8.41％)。此外，病理学检查发现SGL-3能够明显抑制AAC引起的心肌细胞肥大、心肌纤维化。通过HPLC和放免法测定，发现肾素—血管紧张素—醛固酮系统和ANP血浆浓度均有一定程度的改善(与模型组相比，p＜0.05)。 3．结论： 研究结果显示SGL-3在体外和体内均可以抑制心肌细胞肥大，并抑制心肌肥厚向心力衰竭发展的进程。其机制可能与SGL-3抑制后负荷增高引起的肾素—血管紧张素—醛固酮系统激活有关。
Part 1:SGL-2 Background: Viral myocarditis is an important human disease that affects about 5% to 20% of the population. Coxsackieviruses B3 (CVB3) is one of the major cause for viral myocarditis. So far, not many effective treatments are available for the disease. The main components of SGL-2 have been traditionally used to control common cold and other virus-induced disorders in China. Thus, the aim of this study was to investigate the antiviral effects of SGL-2 on CVB3 in vivo and in vitro. Methods and Results: The effect of SGL-2 in vitro was evaluated in HeLa cells or in cultured rat neonatal cardiac myocytes infected by CVB3, Ribavirin was chosen as positive control. Our results showed that SGL-2 possesses potent antiviral effects on CVB3 in vitro by XTT and plaque forming assay (IC_(50) was 7.16±0.8 and 2.63±0.5μg/ml, respectively). CC_(50) was 16-fold higher in SGL-2 treated cells than in Ribavirin treated cells (SGL-2:1648±219μg/ml vs. Ribavirin: 103±14μg/ml). Time course studies demonstrated that antiviral effect of SGL-2 was mainly found during 0-4 h of the infection. We further explored the mechanism of SGL-2 in CVB_3 and found that SGL-2 effectively blocked the attachment and penetration of CVB_3 into cells. To confirm the antiviral effect of SGL-2 in vivo, 4- week- old male Balb/C mice (n=125) were used and inoculated intraperitoneally with 0.1 ml of CVB_3 suspension (10 PFU) or saline as control (n=25). At 48h postinoculation, the infected mice were administered intragastrically with SGL-2 either 200 mg.kg~(-1).d~(-1), 600 mg.kg~(-1).d~(-1) or 1800 mg.kg~(-1).d~(-1), with Ribavirin (90 mg.kg~(-1).d~(-1), PO) as positive control and saline as sham treatment group (n=25 in each above group). After 28 days, SGL-2 treatment increased the survival rate from 16% (vehicle) to 64% (SGL-2:1800 mg.kg~(-1).d~(-1)) by the Kaplan-Meier method. In addition, SGL-2 protected the cardiac pathological changes induced by CVB_3, and decreased myocardial virus titer significantly (vehicle: 3.6±1.7 vs. SGL-2: 1.9±0.9,p＜0.05). Conclusions: The results showed that SGL-2 is a very promising potent antiviral agent with a highly significant effect on survival and pathological changes in CVB_3-induced myocarditis. Part 2:SGL-3 Background: With significant improvements in the treatment of congestive heart failure (CHF), the morbidity and mortality of patients with CHF remain unacceptably high. The main component of SGL-3 has been traditionally used to treat heart failure symptoms for thousand years in China, thus, we proposed that SGL-3 may inhibit myocardial hypertrophy and prevent heart failure. Methods and results: To investigate effect of SGL-3 on myocardial remodeling and cardiac function, we established models of cardiac hypertrophy in vivo and in vitro. In cultured rat neonatal cardiac myocytes, SGL-3 inhibited cardiac myocyte hypertrophy (10μg/ml) which was induced by angiotensin-Ⅱ(1μtmol/L), reduced [3H] leucine incorporation by 74%±26% and cell surface area by 104%±31%. In vivo, twenty- week- old male Wistar rats were used and subjected to suprarenal abdominal aorta constriction (AAC) operation (n=48) or sham operation (n=12). Twenty weeks after surgery, heart failure was detected in AAC rats by using echocardiography, then the AAC rats were treated with either SGL-3 (150 mg.kg~(-1).d~(-1), 500 mg.kg~(-1).d~(-1) and 1500 mg.kg~(-1).d~(-1), PO, n=12 in each group) or Captopril (270 mg.kg~(-1).d~(-1), PO) plus Metoprolol (1,800 mg.kg~(-1).d~(-1), PO) (n=12) for 18 weeks respectively. Cardiac hemodynamic and morphological parameters were obtained by using multi-functional physiology recorder and echocardiography. The result revealed that SGL-3 can improve cardiac systolic function more effectively (EF: 51.8%±8.42% for AAC group, 75.6%±7.65% for sham operation group, 69.3%±7.73% for SGL-3 treated group) than Captopril plus Metoprolol (EF: 56.3%±8.41%). Histological examination of cardiac structure showed that the extent of cardiac myocyte hypertrophy was restrained and myocardial fibrosis, circulating renin-aldosterone system and ANP were inhibited in AAC rats treated with SGL-3, compared with vehicle (p＜0.05). Conclusion: These fmdings indicated that XL attenuates cardiac myocyte hypertrophy both in vitro and in vivo and halts the process from hypertrophy to heart failure.
|
