【作者】 佘晓俊；

【导师】 刘洪涛； 吴铭权；

【作者基本信息】 中国人民解放军军事医学科学院 ， 劳动卫生与环境卫生学， 2011， 博士

【摘要】 目的:交通噪声、建筑噪声、社会生活噪声,不但干扰正常的工作和休息,而且影响身体健康。WHO报告指出心血管疾病是目前死亡率最高的疾病。流行病学调查显示噪声影响心血管系统,造成血压升高、心电图异常,甚至增加缺血性心脏病的危险性,对健康产生极大危害。但该方面的实验室研究较少,其致病机制尚未明确。心脏局部肾素-血管紧张素系统(renin-angiotensin system,RAS)与多种心脏疾病相关,如心肌梗塞、心脏肥大、心脏重塑、心衰等。当心脏局部RAS兴奋,其关键效应分子血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)水平升高,与其受体(AngⅡreceptor, AT)结合,促发下游信号转导过程,引起心肌收缩力增强,心肌蛋白质合成加速,心肌结构改变等。另外,AngⅡ可激活尼克酰胺腺嘌呤二核苷酸磷酸氧化酶(nicotinamide adeine dincleotide phosphate oxidase, NADPH氧化酶),该酶是心血管系统产生活性氧(reactive oxygen species, ROS)的主要酶体。并且研究发现ROS可能参与了噪声损伤机体的过程。那么心脏RAS是否在噪声暴露时,通过影响来源于NADPH氧化酶的ROS的产生,并启动下游信号转导分子,从而致心肌损伤的作用尚不清楚。因此本研究以心脏局部RAS为切入点,观察噪声暴露及给予AT1受体阻断剂(AngⅡtypeⅠreceptor blocker,ARB)-替米沙坦(Telmisartan)干预后心脏的变化,如心电图的改变、心脏氧化/抗氧化指标的变化;心脏RAS主要成分的变化;PKC介导的NADPH氧化酶/ROS信号通路的改变,来探讨局部RAS在噪声暴露致心肌损伤中的作用及其信号转导途径,为进一步认识噪声的心血管危害及预防措施研究提供理论和实验依据。方法:健康成年雄性SD大鼠,耳廓反射灵敏,体质量140-150g,暴露于100dB白噪声,6h/d,连续12周。采用电镜观察心肌超微结构,采用生化法观察心肌组织MDA、T-AOC来反映心肌氧化损伤,放射免疫法检测循环、心肌AngⅡ水平的变化,荧光定量RT-PCR法检测心肌AT1A受体、血管紧张素原(angiotensinogen)mRNA表达水平变化,以探讨长期噪声暴露对心肌的损伤,及对心脏RAS的影响。在该模型的基础上,噪声暴露同时给予替米沙坦灌胃,采用电生理、形态学、生化学、ELISA、RT-PCR、Western blotting等技术方法,观察心肌形态结构、氧化/抗氧化能力、心脏RAS、NADPH氧化酶等指标的变化,以期明确通过阻断AT1受体,阻止AngⅡ作用的发挥,是否能减轻噪声对心肌的损伤,以及噪声暴露引起心肌氧化损伤是否与NADPH氧化酶相关,阐明噪声损伤心肌的机制。结果:1、100dB,6h/d,12wk,白噪声暴露可轻度损伤大鼠听力,表现为ABR阈值升高、波潜伏期延长。2、长期噪声暴露可引起心电图改变,噪声暴露组心率(HR)(291.22 bpm)较正常组(379.63 bpm)明显降低(p<0.01);噪声暴露组心电图P波时间、P-R间期、QRS间期延长,P波电压增大;替米沙坦干预组HR(312.61 bpm)较噪声暴露组有升高趋势,但无统计学差异(p>0.05);替米沙坦干预组P波电压下降,较噪声暴露组明显降低(p<0.01)。3、长期噪声暴露后,大鼠心肌超微结构损伤,表现为线粒体聚集、肿胀、嵴消失甚至呈空泡状;心肌纤维排列紊乱,甚至溶解消失。替米沙坦干预可部分减轻噪声对心肌超微结构的损伤,体现为线粒体、心肌纤维损伤程度减轻。4、噪声暴露组心肌组织超氧阴离子(O2–)、MDA浓度较正常对照组明显升高(p<0.01,p<0.05),噪声暴露组SOD活性较正常对照组有降低趋势,差异不显著(p>0.05),噪声暴露组T-AOC较正常对照组明显降低(p<0.01)。替米沙坦干预组心肌组织O2–、MDA浓度较噪声暴露组明显降低(p<0.01),较正常对照组无明显差异(p>0.05);替米沙坦干预组心肌组织SOD活性较噪声暴露组明显升高(p<0.01),较正常对照组无明显差异(p>0.05);替米沙坦干预组心肌组织T-AOC较噪声暴露组有升高趋势,差异不显著(p>0.05),较正常对照组无明显差异(p>0.05)。说明噪声暴露可引起心肌组织ROS升高、脂质过氧化损伤,可降低心肌组织抗氧化能力,引起心肌氧化损伤;替米沙坦干预可以改善心肌氧化/抗氧化状态,增强心肌抗氧化能力、减轻氧化损伤。5、长期噪声暴露可引起循环、心脏AngⅡ水平较正常对照组明显升高(p<0.05,p<0.01)。噪声暴露组心肌AGT mRNA表达较正常对照组有升高趋势,但无统计学意义(p>0.05)。噪声暴露组心肌AT1A mRNA表达较正常对照组明显增加(p<0.01)。替米沙坦干预组AngⅡ浓度和AGT mRNA表达水平较噪声暴露组、正常对照组明显降低(p<0.01);替米沙坦干预组AT1A mRNA表达水平较噪声暴露组明显降低(p<0.01),较正常对照组有降低趋势,但差异不显著(p>0.05)。说明噪声暴露可兴奋心脏RAS,替米沙坦干预可抑制心脏RAS,降低心肌AngⅡ水平、抑制AGT、AT1A mRNA表达。6、噪声暴露组、替米沙坦干预组心肌蛋白激酶C(PKC)mRNA表达较正常对照组明显增加(p<0.01);替米沙坦干预组PKC mRNA表达较噪声暴露组有降低趋势,但无统计学差异(p>0.05)。噪声暴露组NADPH氧化酶亚基p47phox、gp91phox、p22phox mRNA表达较正常对照组明显增加(p<0.05),p47phox蛋白表达较正常对照组明显增加(p<0.01)。替米沙坦干预组NADPH氧化酶亚基p47phox mRNA和蛋白表达较噪声暴露组有降低趋势,但无统计学意义(p>0.05);替米沙坦干预组NADPH氧化酶亚基gp91phox、p22phox mRNA表达较噪声暴露组明显降低(p<0.05)。结论:1、长期噪声暴露后,大鼠心电图出现异常;大鼠心肌线粒体损伤严重,心肌纤维排列紊乱,甚至溶解消失。说明长期接触噪声不仅影响心脏功能,还影响心肌形态结构。2、噪声暴露可引起心肌氧化/抗氧化失衡,导致氧化损伤;给予替米沙坦干预可以提高心肌抗氧化能力,减轻氧化损伤,说明ROS增多是噪声损伤心肌的机理之一,且通过AT1受体介导。3、噪声暴露可引起心脏AngⅡ水平升高,AGT、AT1A mRNA基因表达增多;给予替米沙坦可以抑制心脏RAS,降低AngⅡ水平,抑制AGT、AT1A mRNA基因表达,说明噪声暴露可以兴奋心脏RAS,给予替米沙坦干预可以抑制心脏RAS过度兴奋。4、噪声暴露可使心肌PKC表达增多,NADPH氧化酶表达增多;给予替米沙坦可以一定程度抑制PKC、NADPH氧化酶亚基表达,说明PKC/NADPH氧化酶途径可能介导AngⅡ损伤心肌过程。综上,噪声损伤心肌可能机制为:噪声暴露引发心脏RAS兴奋,高水平的AngⅡ与其受体结合,激活PKC,促使P47phox激活,从而促进了NADPH氧化酶的组装,组装好的NADPH氧化酶具有了催化活性,通过催化电子传递过程产生ROS,最终导致心肌氧化损伤。这些研究结果为进一步阐明噪声导致心肌损伤的机制提供了启示,并为研究噪声导致心肌损伤的防护措施提供新的实验依据及思路。更多还原

【Abstract】 OBJECTIVE: Noise pollution has become an increasingly serious problem. Traffic noise、construction noise、community noise have not only disturbed people’s normal life, but also affected their health. The World Health Organization has reported cardiovascular disease as the highest cause of mortality. Epidemiological studies suggested that noise affects the cardiovascular system. Noise can increase the blood pressure and electrocardiogram abnormality rate, enve increase the relative risk of ischemic heart disease, which may affect people’s health seriously. However, few laboratory-based studied have been done in this area. The exact mechanism of noise-induced cardiovascular disease has not been fully explored. Local cardiac renin-angiotensin system (RAS) is related to a variety of cardiovascular diseases such as myocardial infarction, cardiac hypertrophy, cardiac remolding and heart failure. Studies have suggested that when the local RAS is stimulated, angiotensin II (Ang II) is increased, binds with Ang II receptor (AT), and therefore triggers signal transduction. This causes the enhancement of cardiomyocytes contractility, acceleration of protein synthesis and the rearrangement of cardiomyocytes, eventually leading to cardiomyocyte apoptosis, necrosis, hypertrophy, and remodeling. Research has demonstrated that Ang II can stimulate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the primary enzyme to produce reactive oxygen species (ROS) in the cardiovascular system. Studies have also shown that noise exposure can produce ROS in the blood and cochlea. It has not yet been determined whether cardiac RAS mediated any of the effects of noise on heart, or whether ROS derived by RAS mediated the effects of noise on heart. Against this background, we plan to investigate the alteration of cardiac RAS, electrocardiogram, the index of oxidation/antioxidation, the NADPH oxidase subunits and PKC in rat hearts damaged by noise treated with Telmisartan. Based on these changes, this research aims to explore the role of the cardiac RAS in myocardial injury caused by noise exposure and its path of signal transduction, and therefore provide more theoretical and experimental basis for understanding the influence of noise exposure on the cardiovascular system and eventual implications for prevention.METHODS: First, healthy male rats, weighted 140-150g, were exposed to 100dB white niose, 6h/d, 12 wk, building the animal model of heart damaged by noise, according to cardiomyocytes ultrastructure and the oxidative indicies of MDA content and T-AOC in myocardium. And then, radio-immunity method was used to detect the content of Ang II in plasm and myocardium; fluorescent quantitation method of RT-PCR was used to detect the expression of AGT and AT1A mRNA in myocardium. Second, on the basis of this model, ARB was administrated before noise exposure to observe the role of the Ang II, the indicies including myocardiac ultrastructure,electrocardiogram,oxidant/antioxidant, cardiac RAS, expression of NADPH oxidase and PKC. ARB was applied to block the action of Ang II, to explore whether it can reduce noise-induced myocardium impairment and whether oxidative damage caused by noise would be relate to NADPH oxidase, through which to clarify the underlying mechanisms of noise-induced myocardium damaged.RESULTS:1. The hearing of rat was damaged by long term noise exposure with white noise of 100dB, 6h/d, 12 wk, showing the thresholds of ABR increased and peak latency extended.2. Electrocardiogram was changed by long term noise exposure. The HR of noise -exposure group(291.22 bpm) was significantly decreased(p<0.01), comparing with that of the normal control group(379.63 bpm). The duration of P wave, the interval of P-R, and the interval of QRS were extended of the noise-exposure group. The voltage of P wave was raised of the noise-exposure group. The HR of Telmisartan intervention group(291.22 bpm) had the tendency of accelation, but had no significance(p>0.05), comparing with the noise-exposure group. The voltage of P wave was decreased significantly comparing with that of the noise-exposure group.3. Long term noise exposure damaged the myocardium, showing mitochondrial accumulating, swelling, cristae disappearing, even vacuoles happen. The damage of mitochondrial and myocardium were relieved by Telmisartan intervention.4. The O2–and MDA content of myocardium of noise-exposure group were significantly raised(p<0.01, p<0.05), comaring with the normal control group. The SOD activity of noise-exposure group was unsignificantly changed(p>0.05), comparing with the normal control group. The T-AOC of noise-exposure group was significantly decreased(p<0.01), comparing with that of the normal control group. Telmisartan intervention may raise the SOD activity and T-AOC in the myocardium, decrease the O2–and MDA content of myocardium after noise exposure, Which indicating noise exposure can increase ROS generating, damaged antioxidant capability of myocardium, and then lead to myocardium oxidative damaged. Telmisartan can raise the antioxidant capability of myocardium, reduce the oxidative damaged.5. Long term noise exposure can raise the AngⅡcontent of plasma and myocardium(p<0.05,p<0.01), comparing with the normal control group. The expression of AGT mRNA of noise-exposure group had the tendency of augmentation, comparing with the normal control group. The expression of AT1A mRNA of noise-exposure group was significantly raised(p<0.01), comparing with the normal control group. The AngⅡcontent , the expression of AGT and AT1A mRNA of myocardium of the telmisartan intervention group were decreased significantly(p<0.01), comaring with the noise-exposure group. 6. Noise can cause increased expression of PKC and NADPH oxidase subunits: p47phox, p22phox, gp91phox. Telmisartan was shown to inhibit the expression of PKC and NADPH oxidase subunits.CONCLUSION:1. Heart damaged by noise may be related to oxidative stress injury.2. Noise exposure can stimulate local cardiac RAS.3. Heart damaged by noise may be related to overexpression of PKC and NADPH oxidase.4. Heart damaged by noise may be mediated by Ang II-AT1 pathway.5 Telmisartan can reduce heart injury by blocking the action of Ang II. One of the signal transduction process of noise-induced heart damaged is composed of the following steps: noise exposure induction of overexcitation of cardiac RAS, which increas Ang II levels, binds with AT1A, activates of PKC, increases of P47 expression, and augments NADPH oxidase assembly. Assembled NADPH oxidase has enzyme activity, which then catalyzes electron transport to produce ROS. ROS should be the signal messenger, activating signal pathways, and causing heart injury.更多还原