【作者】 赵金镯；

【导师】 宋伟民；

【作者基本信息】 复旦大学 ， 劳动卫生与环境卫生学， 2008， 博士

【摘要】 大气污染是危害人类健康的主要环境因素之一,其中大气颗粒物因其粒径大小不同,含有多种有害化学组分,对人体健康有复杂的影响。大气颗粒物中空气动力学直径≤2.5μm的细颗粒物,由于其粒径小、表面积大,肺沉积率高,易于穿过内皮到达间质,对人体具有更大的危害性。其所含的炭核、氮、硫、有机物（多环芳烃）、生物化合物（内毒素,细胞片段）和过渡金属（铁,镍,锌,钒）等多种有害的化学成分对人体产生的负面影响,也逐渐成为研究者关注的热点。细颗粒物进入呼吸道,不仅可引起肺损伤,还可引起肺外其它器官如心血管系统、免疫系统及DNA的损伤。细颗粒物与心血管系统疾病的关联性已在流行病学的研究中得到了一定的论证,但在毒理学的研究中尚未得到相关的系统论证,细颗粒物对心血管系统影响的作用机制也尚未阐明。本次研究通过动物实验和体外细胞实验从自主神经功能、炎症反应、纤溶/凝血、内皮功能和心肌缺血及细胞凋亡等方面对细颗粒物致心血管系统的损伤进行了较系统的毒理学研究,探索其可能作用机制,并通过他汀类药物的干预验证氧化损伤、炎症反应、内皮功能损伤和细胞凋亡等通路在细颗粒物致心血管系统损伤中的作用,为探寻其作用机制提供科学依据。本研究在上海市市区采集大气细颗粒物,对不同生理病理状态大鼠（高血压大鼠（SHR）作为疾病模型组,Wistar大鼠（WKY）作为健康模型组）进行气管滴注染毒,对照组气管滴注生理盐水,试验组滴注剂量分别为1.6 mg/kg b.w、8.0 mg/kgb.w和40.0 mg/kg b.w的细颗粒物悬液,每天染毒1次,连续染毒3天。观察暴露于细颗粒物后大鼠自主神经功能的改变,包括血压、心率和心电图,以及对大鼠血液及心肌组织中反应炎症、纤溶/凝血、内皮功能及心肌细胞损伤或凋亡等因子的检测,结果发现,细颗粒物染毒组大鼠血压和心率明显升高,心电图ST-段抬高,T波低平。大鼠血液中高敏C-反应蛋白（hs-CRP）、D-二聚体、内皮素-1（ET-1）在染毒组明显高于对照组,并随染毒剂量增加而增加。血液中心肌酶学指标乳酸脱氢酶（LDH）和肌酸激酶同工酶（CK-MB）也随染毒剂量增加而增加,而血管舒张因子一氧化氮（NO）却明显减少。心肌组织内各炎症相关基因IL-1β、IL-6、TNF-α和MIP-2,内皮功能相关基因ET-1,内皮细胞粘附分子ICAM-1及介导细胞凋亡的促凋亡基因bax的mRNA表达均呈现升高趋势,而抗凋亡基因bcl-2的mRNA表达呈下降趋势。同时,利用免疫组织化学方法对暴露于大气细颗粒物后,心肌组织中bax和bcl-2的蛋白表达进行检测。结果发现,与对照组相比,染毒组的bax蛋白表达明显增加,而bcl-2蛋白表达明显降低,表明心肌组织中抗凋亡基因和促凋亡基因的调节出现紊乱,提示心肌细胞凋亡在细颗粒物所致心血管系统损伤中有着非常重要的作用。同样的方法对细颗粒物暴露的大鼠心肌组织中转化生长因子β₁（TGF-β₁）蛋白的表达强度进行了检测,发现暴露于细颗粒物后,心肌组织中TGF-β₁蛋白的表达明显高于对照组。这提示大鼠心肌组织中除内皮屏障功能受到破坏及血管通透性增加外,细胞外基质合成和分泌增加及降解减少、心血管纤维化病变也可能是细颗粒物致心血管系统损伤的作用机制。实验研究也发现,大气细颗粒物染毒剂量相同时,SHR大鼠血液及心肌组织中介导损伤的炎性细胞因子、内皮功能因子、细胞凋亡等因子的表达均明显高于WKY大鼠,表明大气细颗粒物对SHR大鼠心血管系统的毒性作用高于WKY大鼠,这一基于动物模型的实验结果与人群流行病学的研究具有一致性,后者的结论为患有心肺疾病的人群暴露于高浓度细颗粒物后,因心血管疾病发作而增加的死亡率高于健康人群,属于细颗粒物暴露损伤的易感人群。为了更进一步了解大气细颗粒物对心血管系统的作用机制并探索细颗粒物不同化学成分在心血管系统损伤中的作用,本研究比较了细颗粒物不同成分的毒性大小,分别测定SO₄^2-、NO₃^-和Cl^-三种阴离子和五种金属阳离子对人脐静脉血管内皮细胞（EC-304）的毒性作用,结果发现在染毒浓度分别为0.01 mmol/L、0.05 mmol/L、0.1 mmol/L、0.5 mmol/L、1.0 mmol/L、5.0 mmol/L和10.0mmol/L的离子浓度时,阴离子中NO₃^-离子对细胞活性有抑制作用;五种金属阳离子对细胞活性均有抑制作用,其作用大小依次为Ni²⁺＞Cu²⁺＞Zn²⁺＞Fe³⁺＞Mg²⁺。本次研究还提取了大气细颗粒物有机物、水溶成分和非水溶成分,每种成分分别按浓度100μg/ml、200μg/ml和400μg/ml,设为低、中、高三个浓度组对血管内皮细胞进行24h染毒,观察细颗粒物不同成分的毒性作用,进一步了解氧化应激、炎症反应、内皮功能紊乱和细胞凋亡在心血管系统损伤中的作用。结果发现细颗粒物有机提取物、水溶成分和非水溶成分使细胞存活率显著降低。细胞内活性氧族（ROS）、丙二醛（MDA）和上清液LDH/总LDH的比值均随着染毒浓度的增加而增加,而超氧化物歧化酶（SOD）及血管舒张因子NO含量却明显降低。细胞内与炎症相关基因IL-6、IL-4和TNF-α,内皮功能相关基因ET-1和TGF-β₁,凝血功能相关基因P-selectin,凋亡相关基因fas的mRNA表达随着染毒浓度的增加均明显升高,而内皮型一氧化氮合酶（eNOS）明显降低。同时发现,大气细颗粒物不同成分对血管内皮细胞的氧化损伤、炎症反应和内皮功能的作用大小依次为:水溶成分＞有机提取物＞非水溶成分,但对细胞凋亡和细胞ROS含量的影响却是有机提取物＞水溶成分＞非水溶成分。他汀类药物能改善内皮功能,增加NO的生物活性,抑制炎性反应和调节免疫功能,并有很强的抗氧化功能,本次研究使用阿托伐他汀对细颗粒物有机提取物、水溶成分和非水溶成分致EC-304细胞的毒性进行干预,使用浓度为400μg/ml的细颗粒物有机提取物、水溶成分和非水溶成分分别对EC-304细胞进行染毒,同时加入阿托伐他汀药物进行干预,药物浓度为0μmol/L、0.1μmol/L、1μmol/L、10μmol/L,观察药物对细颗粒物不同成分氧化应激、内皮功能损伤和凋亡等毒性的干预作用。结果显示,加入阿托伐他汀后,细颗粒物不同成分染毒组的细胞内ROS、MDA和LDH较未加药组明显降低,而SOD和NO明显增加。同时,加入药物使细胞IL-6、IL-4、TNF-α、ET-1、P-selectin、fas和TGF-β₁ mRAN表达水平明显下调,使eNOS mRNA表达上调,并呈剂量-反应关系。由于他汀类药物在抗氧化损伤、减少炎症和改善内皮功能方面具有良好的药理作用,本次实验表明他汀类药物能明显减少细颗粒物暴露所致的氧化应激、炎症反应、内皮功能损伤和细胞凋亡,结果提示,细颗粒物对心血管系统的损伤是由氧化应激、炎症反应、内皮功能损伤和细胞凋亡等通路介导的。由于大气细颗粒物对心血管系统的作用机制尚不清楚,本次研究通过动物实验和体外细胞实验研究了大气细颗粒物对心血管系统的毒性作用,并探索其可能作用机制。结果表明,自主神经功能失调、炎症反应、纤溶/凝血功能障碍、内皮功能损伤及心肌缺血和细胞凋亡是大气细颗粒物致心血管系统损伤的可能作用机制,通过他汀类药物的干预实验更进一步验证了这些损伤通路的存在。总之,本实验从分子、细胞和动物体等不同的生物学层次进行了较为系统的细颗粒物致心血管损伤的毒理学机制的研究,取得了与人群流行病学研究相一致的结论,为今后大气细颗粒物污染的防治及有效地保护人类健康等方面提供理论依据。更多还原

【Abstract】 Air pollution is one major pollution in perspective of public health.Air particulate matter（PM）,varying in different aerodynamic diameters and generally composed of a complex of carbon,nitrogen,sulfur,biological chemicals and heavy metals,is one of the most important air pollutants.Among others,fine particles （aerodynamic diameter≤2.5μm）,in comparison with coarse particles,have been considered as a more serious pollutant,due to its high deposition efficiency into human respiratory system as well as cardiovascular system.It is of major concern because of their high propensity to penetrate the epithelium and reach interstitial sites, even transportation to other organs by air exchange in human lungs,resulting in respiratory system diseases,and extrapulmonary injuries,such as in cardiovascular and immunonological system.Epidemiological studies have shown that increases in airborne fine particle concentrations are adversely associated with cardiovascular health,but there is a lack of comprehensive studies on the effects of fine particles on cardiovascular system in toxicological perspectives,as well as the potential toxicity mechanisms.The purpose of this study is to explore the toxicological mechanisms of fine particles on cardiovascular system in respect to autonomic nervous system,inflammation, coagulation,endothelial function and cell apoptosis through animal experiment and cell culture studies.In addition,drug intervention studies by statin were performed to verify the possible toxicological mechanisms of fine particles on vein endothelial cells in vitro.In this study,spontaneously hypertensive（SH） rats were used as a model of human cardiovascular disease and Wistar Kyoto（WKY） rats as a model of healthy human.Ambient fine particles were collected by air sampling in the urban area in Shanghai and prepared into particle solutions in different concentrations.Acute exposure to fine particle solution preparations were performed by intratracheally instillation.The SH model was exposed to a gradient of fine particle concentration in 1.6 mg/kg b.w,8.0 mg/kg b.w and 40.0 mg/kg b.w,respectively,while the control model was exposed to physiological saline.The exposure was performed once a day for three days.In 24h after the last exposure,blood pressure,heart rate and cardiogram were monitored in both types of rats,as well as the biomarkers of inflammation,coagulation,endothelial function and myocardial cells lesion in blood and cardiac muscle tissue.The results showed that blood pressure and heart rate significantly increased both in SH rats and WKY rats after exposure to fine particles with an elevation of ST segment in cardiograms.In blood samples,the levels of blood high sensitive C-response protein（hs-CRP）,D-dimer and endothelin-1（ET-1） increased significantly in SHR and WKY rats in a dose-dependent manner,whereas the level of blood nitrous oxide（NO） was decreased.The serum level of myocardial enzyme MB isoenzyme of creatine kinase（CK-MB） and lactate dehydrogenase（LDH） increased both in SHR and WKY rats in a dose-dependent manner.In the cardiac muscle tissue,the mRNA expression of inflammation-related genes IL-1β,IL-6, TNF-α,MIP-2 significantly increased in rats,as well as the endothelial function-related gene ET-1 and coagulation function-related gene ICAM-1.Moreover, expression of apoptosis bax gene increased significantly in rats while the anti-apoptosis gene bcl-2 mRNA expression decreased.The latter result was consistent with the protein expression levels determined by immunohistochemical staining measurement.It was found that in rats,the protein expression of apoptosis bax gene was up-regulated while the anti-apoptosis bcl-2 gene was down-regulated. These results indicated that exposure to fine particle may cause inflammation, coagulation,endothelial dysfunction and apoptosis in rats.Finally,protein expression of transforming growth factor-beta₁（TGF-β₁） measured by the same staining method showed that the TGF-β₁ protein expression obviously increased in SH rats in comparison with WYK rats,which suggested that in the fine particle toxicity mechanisms,in addition to the endothelial dysfunction and the increase of vascular permeability,the extracellular matrix synthesis and secretion and myocardial fibrosis were potentially the additional ways of cardiovascular toxicity effects.Interestingly, when the same exposure level was performed in both SHR and WKY rats,the levels of hs-CRP,D-dimer,ET-1,LDH and CK-MB were significantly higher in SH rats than in WKY rats,which indicated that model rats with the presence of hypertension diseases could be more susceptible to fine particle exposure than normal ones.This is in agreement with the results from epidemiological studies on human population, which prove that after exposure to fine particles,higher rate of cardiopulmonary morbidity is more likely in people with cardiovascular disorders than in healthy people.The cardiovacular effects of fine particle ion components were analyzed on human umbilical vein endothelial cells（EC-304）.Particle samples were extracted for specific anion samples including SO₄^2-,NO₃^-,Cl^-,and five metal ion samples in doses of 0.01mmol/L,0.05mmol/L,0.1mmol/L,0.5mmol/L,1.0mmol/L,5.0mmol/L and 10.0mmol/L,respectively.After exposure experiment,it was found that only NO₃^-anions could inhibit cell activity in all the above concentration levels,and all metal ions could inhibit cell activity in different inhibition rates:Ni²⁺＞Cu²⁺＞Zn²⁺＞Fe³⁺＞Mg²⁺.Furthermore,the cardiovascular effects of chemical components,such as organic extracts,soluble and insoluble components of fine particles（exposure doses: 100μg/mL,200μg/mL and 400μg/mL,respectively for 3 kinds of components） were analyzed on EC-304 cells.Malondialdehyde（MDA）,LDH,reactive oxygen species （ROS） increased and NO and Superoxide dismutase（SOD） decreased in EC-304 cells for all 3 component extraction exposure.The mRNA expression of IL-4,IL-6,TNF-α, ET-1,P-selectin and fas gene increased in EC-304 exposure cells while mRNA expression of eNOS decreased.Within the 3 different components,the inhibition effects of cell activity,oxidative stress,inflammatory response were more significant in soluble extracts than in organic extracts than in insoluble extracts.However,effects of cell apoptosis and ROS level were higher in organic extracts than in soluble extracts than insoluble extracts.In summary,different components of fine particles could lead to different levels of toxicity to cells.Statin can improve endothelial function,elevate NO biological activity,inhibit inflammation,modulate immune function and reduce oxidation in clinical studies.In this study,atorvastatin was used for intervention studies on the possible toxicological mechanisms of fine particle components on EC-304.EC-304 were exposed to the 400μg/mL organic,soluble and insoluble extracts of fine particles,respectively,followed by an intervention of astorvastatin in concentrations of 0.1μmol/L,1μmol/L and 10μmol/L.The results showed that in comparison with the group of cells with no atorvastatin addition,the levels of ROS,MDA and LDH in EC-304 cells induced by either organic extract,soluble or insoluble components of fine particles,were obviously inhibited after the addition of atorvastatin,while cell activities,SOD and NO levels in EC-304 cells increased significantly.Furthermore,the mRNA expression of IL-4,IL-6,TNF-α,ET-1,P-selectin,TGF-β₁ and fas in cells decreased greatly after treatment with atorvastatin as well,and eNOS increased significantly in a dose-dependent manner.Since atorvastatin is a clinical drug for cure of cardiovascular disorders with the known effects of reducing inflammation and improving endothelial functions,this intervention study consistently suggested that oxidative stress, inflammation,endothelial dysfunction and apoptosis were the potential mechanisms of cardiovascular injuries caused by fine particles.In summary,this study examined the potential biological pathways triggering toxicological effects of fine particles on cardiovascular system.By methods based on animal experiment and cell culture,the results indicated that fine particles increased the risk of cardiovascular disease by increasing autonomic nervous dysfunction, oxidative stress,inflammation,coagulation,endothelial dysfunction and apoptosis. Drug intervention studies further supported the presence of the above mechanisms. These results were in consistence with the main conclusions from epidemiological studies on human population exposed to fine particles.It will contribute in further exploration of the real mechanisms by fine particle on cardiovascular diseases,and will be of importance in understanding the effects of air particulate matter on human health.更多还原