生物燃料烟雾与慢性阻塞性肺疾病关系的研究
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摘要
研究背景:慢性阻塞性肺疾病(COPD)是一种具有气流受限特征的可以预防和治疗的疾病,气流受限不完全可逆、呈进行性发展,与肺部对香烟烟雾等有害气体或有害颗粒的异常炎症反应有关。它是一种全球性患病率较高的疾病,而且呈上升趋势。有针对性的干预能降低COPD的患病率。全球疾病负担评估表明,使用生物燃料造成的空气污染是一项威胁全球公共卫生的重要问题,是发展中国家农村地区最大的死亡负担,它可造成每年200万以上的死亡和4%的全球疾病负担。生物燃料烟雾是发展中国家室内空气污染的主要来源,越来越多的证据表明,在发展中国家,使用生物燃料取暖和做饭与COPD密切相关,是广大发展中国家COPD的一个重要危险因素。然而,在各种人群中,生物燃料烟雾暴露与COPD的相关程度,生物燃料烟雾暴露量与COPD的危险度关系以及生物燃料烟雾暴露与香烟烟雾暴露的相互影响尚不清楚。
COPD是一种全身性疾病,对全身各个系统都产生影响。但是,COPD的临床研究却受到了很多因素的限制。首先临床上要获得COPD病人全身各个系统标本相当困难,只有血标本的获取相对容易;其次,外周血中一些指标的变化不一定与肺部及其他系统的改变相平行。因此,许多学者致力于COPD动物模型的建立。现已有多种方法可复制出COPD动物模型,但是尚没有建立生物燃料烟雾暴露的COPD动物模型,因此对生物燃料烟雾暴露所致COPD的研究尚少。
COPD是一种全身性的疾病,存在多系统的氧化应激,但是生物燃料烟雾暴露所致COPD的全身氧化应激状况如何,生物燃料烟雾和香烟烟雾暴露所致全身氧化应激有何异同尚不清楚。
研究目的:本课题分三部分:第一部分采用循证医学的方法研究生物燃料烟雾暴露与COPD的关系,以明确生物燃料导致COPD的危险度,以及种族、性别及吸烟对生物燃料烟雾导致COPD的危险度的影响。第二部分观察生物燃料烟雾对大鼠肺脏病理组织学的影响,并与香烟烟雾对大鼠肺脏病理组织学改变相比较。第三部分比较生物燃料烟雾暴露和香烟烟雾暴露大鼠全身氧化应激状况的异同,以进一步探讨COPD的发病机制。
第一部分:生物燃料烟雾暴露与慢性阻塞性肺疾病的循证医学研究
方法:我们通过电子检索Medline,Embase,LILACS数据库,并且查阅相关文献的参考文献,共鉴定出15个流行病学研究(4个病例-对照研究和11个横断面研究)符合纳入的研究标准。用标准化的数据提取方案来提取资料并进行统计学分析。结果:和不暴露者相比,生物燃料烟雾暴露者发展为COPD的OR值为2.44(95%CI为1.90-3.33)。对于不同性别、不同种族、不同吸烟状态,生物燃料烟雾暴露是COPD的危险因素(女性OR: 2.73, 95% CI: 2.28-3.28;男性OR4.30, 95% CI: 1.85-10.01;亚洲人:OR: 2.31, 95% CI: 1.41-3.78;非亚洲人OR: 2.56, 95% CI: 1.71-3.83;吸烟者OR=4.39, 95%CI:1.40-4.66,不吸烟者OR=2.55, 95%CI: 2.06-3.15)。生物燃料烟雾暴露者发展为慢性支气管炎的OR值为2.56(95%CI为1.71-3.83),发展为通过肺功能诊断的COPD的OR值为2.65(95% CI: 1.75-4.03)。生物燃料烟雾暴露量越大,发展为COPD的危险性越大。小结:生物燃料烟雾暴露是COPD的危险因素,吸烟和生物燃料烟雾暴露存在协同作用。生物燃料烟雾暴露量越大,发展为COPD的危险性越大。
第二部分:生物燃料烟雾暴露对大鼠肺脏病理组织学的影响
方法:30只雌性SD大鼠,随机分为正常对照组、生物燃料烟雾暴露组、香烟烟雾暴露组,每组10只。每组动物每天置于烟室内两次,每次45min,分别向烟室内充新鲜空气,生物燃料烟雾和香烟烟雾,共100天。熏烟时同时测量烟室中O2、SO2、CO和NO的浓度。100天后,处死动物,HE染色检查大鼠肺脏病理组织学。ELISA法检测血浆IL-8含量,同时进行全血细胞计数和分类,肺泡灌洗液中白细胞计数和分类,右心室重量/室间隔+左心室重量。结果:生物燃料烟雾暴露大鼠熏烟期间箱内O2、CO、SO2和NO的浓度分别为(20.31±0.21)%、(981.72±429.59) mg/m3、(3.44±3.36) mg/m3和(2.59±1.71) mg/m3,香烟烟雾暴露大鼠熏烟期间箱内O2、CO、SO2和NO的浓度分别为(20.28±0.15)%、(745.56±683.24) mg/m3、(12.64±11.44) mg/m3和(3.37±0.52) mg/m3。病理切片HE染色镜下见对照大鼠小气道黏膜上皮完整,纤毛未见黏连脱落,管壁规整未见增厚,未见炎性细胞浸润,管腔内未见炎性渗出物,肺泡腔未见病理性扩大。生物燃料烟雾暴露和香烟烟雾暴露大鼠肺脏病理组织学无明显区别,都可见小气道上皮呈锯齿状增生增厚,支气管壁增厚,见炎性细胞浸润。管腔内见大量炎性渗出物,管壁结缔组织增生,可见淋巴小结。肺泡结构紊乱,肺泡壁断裂,肺泡腔扩大,部分融合成肺大疱。生物燃料烟雾暴露组和香烟烟雾暴露组支气管炎症评分分别为32.66±13.84和32.25±12.51,高于对照组11.11±9.84;平均肺泡间隔分别为130.62±33.64μm、119.03±26.20μm高于对照组89.84±17.34um;而平均肺泡数低于对照组,分别为169.21±76.33个/mm2、173.86±68.33个/mm2和280.26±103.95个/mm2。生物燃料组、香烟烟雾暴露组大鼠血浆中IL-8的浓度分别为240.79±161.28 pg/ml、188.14±20.85 pg/ml高于对照组107.91±35.84 pg/ml。生物燃料烟雾组、香烟烟雾暴露组大鼠BALF白细胞计数及中性白细胞比例高于对照组。而生物燃料烟雾暴露组大鼠全血白细胞和红细胞计数以及血红蛋白含量高于对照组和吸烟组。生物燃料组、香烟烟雾暴露可使大鼠体重减轻,右心室重量/左心室+室间隔重量增加。小结:生物燃料烟雾暴露和香烟烟雾一样都可使大鼠产生慢性支气管炎和肺气肿, BALF中白细胞计数及中性分类增高,血浆IL-8增加,同时,两种烟雾所致大鼠肺脏病理组织学无明显差异。生物燃料烟雾暴露可使大鼠全血白细胞和红细胞计数以及血红蛋白含量上升。
第三部分:生物燃料烟雾和香烟烟雾暴露大鼠全身氧化应激状况比较的研究
方法:使用试剂盒测定血浆T-SOD活力;肺脏、心脏总抗氧化能力;血浆、肺脏、心脏、肝脏MDA和GSH含量;血浆、肺脏和心脏iNOS活力,心脏和肺脏GST活力等氧化/抗氧化指标。应用荧光定量PCR的方法检测肺组织GCLC-mRNA、GSTM1-mRNA、GSTP-mRNA和SLPI-mRNA以及心脏组织GCLC-mRNA、GSTM1-mRNA和GSTP- mRNA的表达。免疫组化分析肺脏组织GCLC的表达。Western Blot检测肺组织γ-GCSc(GCLC)蛋白表达。
结果:香烟烟雾暴露和生物燃料烟雾暴露大鼠血浆中T-SOD活力分别为(218.65±65.60)u/ml,(223.91±56.86) u/ml,低于对照组(283.11±41.71) u/ml。香烟烟雾暴露组大鼠肺和心脏的总抗氧化能力低于对照组和生物燃料暴露组;生物燃料暴露组大鼠肺和心脏的总抗氧化能力与对照组无区别。生物燃料烟雾暴露组肺脏MDA的浓度高于对照组,而吸烟组肺脏MDA的浓度低于对照组;生物燃料烟雾暴露组和吸烟组心脏MDA的浓度低于对照组;血浆和肝组织中MDA浓度三组无明显差异。生物燃料烟雾暴露组血浆iNOS活力低于吸烟组和对照组,肺脏iNOS活力与对照组和吸烟组无明显差异,心脏iNOS活力高于对照组和吸烟组。生物燃料组、吸烟组和对照组血浆、肺脏、心脏、肝脏组织匀浆中GSH浓度没有明显的差异。在肺脏组织中,香烟组GST酶的活力为(28.43±8.80) RFU/min/ mgpro,生物燃料组GST酶的活力为(15.44±11.34) RFU/min/ mgpro,对照组GST酶的活力为(20.54±10.12) RFU/min/ mgpro。心脏组织中香烟组GST酶的活力为(4.35±1.94) RFU/min/mgpro,低于生物燃料组(6.27±1.31)RFU/min/mgpro和对照组(6.76±1.26) RFU/min/ mgpro。吸烟组大鼠肺组织GCLC-mRNA的表达高于对照组和生物燃料组,GSTP1-mRNA的表达低于对照组和生物燃料烟雾暴露组,差异具有统计学意义。肺脏GSTM1mRNA和SLPImRNA的表达三组间没有差别。生物燃料组大鼠心脏GSTM1-mRNA的表达高于对照组,差异具有统计学意义。吸烟组心脏GSTM1-mRNA的表达高于对照组,差异无统计学意义。心脏GSTP-mRNA和GCLC-mRNA的表达三组间无差别。免疫组化显示对照组支气管上皮和肺泡上皮GCLC呈弱阳性表达。生物燃料组和香烟组大鼠支气管上皮和肺泡上皮GCLC表达增强,且均为胞浆表达;而且肺间质纤维化越明显的地方,GCLC的表达越强。免疫组化定量分析和Western-blot分析显示香烟组和生物燃料组肺脏γ-GCSc蛋白的表达强于对照组。小结:本研究显示,香烟烟雾和生物燃料烟雾暴露都可导致大鼠出现全身性氧化应激。两种烟雾刺激所引起的全身氧化应激是不同的。香烟烟雾暴露使大鼠血浆SOD活力降低,心脏和肺脏的总抗氧化力下降,心脏GST活力下降。肺组织中GCLC-mRNA表达增加,GSTP-mRNA表达下降,心肌组织中GSTM1-mRNA表达增强。肺组织GCLC蛋白表达增强,在肺纤维化越明显的部位GCLC表达越高。生物燃料烟雾暴露使大鼠血浆SOD和INOS活力降低,心脏INOS活力增强;肺脏MDA水平升高,心脏MDA降低;肺脏GST活力下降。心肌组织中GSTM1-mRNA表达增强。肺组织GCLC蛋白表达增强,在肺纤维化越明显的部位GCLC表达越高。
结论:生物燃料烟雾暴露是COPD的危险因素,吸烟和生物燃料烟雾暴露存在协同作用。生物燃料烟雾暴露量越大,发展为COPD的危险性越大。生物燃料烟雾和香烟烟雾暴露一样可使大鼠产生慢性支气管炎和肺气肿,两种原因所致肺气肿病理组织学上无明显区别。香烟烟雾和生物燃料烟雾暴露可导致机体出现全身性的氧化抗氧化失衡,导致全身的氧化应激。两种烟雾刺激所引起的全身氧化应激存在一定的差异。
Background: Chronic obstructive pulmonary disease (COPD) is a preventable and treatable disease with some significant extrapulmonary effects that may contribute to the severity in individual patients. Its pulmonary component is characterized by airflow limitation that is not fully reversible. The airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lung to noxious particles or gases. COPD is a global disease with a high and increasing prevalence. Evidence showed that some kind of intervention can reduce the prevalence of COPD. It has been estimated that indoor air pollution resulting from the combustion of solid fuels may be one of the leading contributors to the global burden of disease.Exposure to indoor air pollution may be responsible for nearly 2 million excess deaths in developing countries and for some 4% of the global burden of disease. There is increasing evidence for a possible association between COPD and the use of biomass fuels for cooking and heating in developing countries.However,the extent of the association between biomass smoke and COPD among different Population, the duration of biomass smoke on COPD and the relation between biomass smoke and cigarette smoke is not fully known.
Indoor air pollution is a major global public health threat requiring greatly increased efforts in the areas of research and policy-making.COPD is a system disease.However,the clinical research of COPD were restricted,because it is very differcult to obtain the sample of each system.We only can get the sample of blood.However,the vary of the biomarker in blood is not parallelled with lung and the other organs. So, several kinds of COPD animal model developed.However,there is no COPD animal model caused by biomass smoke.Exposure to biomass smoke is a risk factor for COPD.And COPD is a system disease with system oxidative stress, however there is no information about system oxidative stress caused by biomass smoke and about the difference of oxidative stress between biomass smoke and cigarette smoke.
Objective: To get the information about the extent of the association between biomass smoke and COPD among different Population, the duration of biomass smoke on COPD and the relationship between biomass smoke and cigarette smoke. To compare the pathological morphology caused by biomass smoke and cigarette smoke.To compare the system oxidative stress caused by exposure to biomass smoke and cigarette smoke.
Part I Risk of chronic obstructive pulmonary disease from exposure to biomass smoke: Result from meta-analysis
Methods:We searched the Medline, Embase, LILACS and reviewed citations in relevant articles to identify 15 epidemiologic (11 cross-sectional and 4 case-control) studies that met inclusion criteria. Data were extracted and analyzed independently by two investigators using a standardized protocol.Results: Overall, people exposed to biomass smoke have odds ratio (OR) of 2.44 (95 percent confidence interval (95% CI) 1.9-3.33) for developing COPD, compared with those not exposed to biomass smoke. Biomass smoke exposure was consistently a risk factor for developing COPD in women (OR: 2.73, 95% CI: 2.28-3.28),in men (OR: 4.30, 95% CI: 1.85-10.01), in Asian population (OR: 2.31, 95% CI: 1.41-3.78) ,in Non-Asian population (OR: 2.56, 95% CI: 1.71-3.83),in the phenotype of chronic bronchitis (OR: 2.56, 95% CI: 1.77-3.70),in the phenotype of COPD (OR: 2.65, 95% CI: 1.75-4.03),and in cigarettes smoker (OR=4.39, 95%CI:1.40-4.66) and in cigarettes nonsmoker (OR=2.55, 95%CI: 2.06-3.15). Conclusions: Exposure to biomass smoke is a risk factor for COPD. There is interaction between cigarette and biomass smoke.
Part II The influence of biomass smoke on rat lung pathological morphology
Methods: Sprague Dawley Rat were divided into three groups: control group(C group) rats were exposed to fresh air; biomass smoke group(BS group) were exposed to biomass smoke; cigarette smoke(CS) group were exposed to cigarette smoke. All rats were exposed to the smoke/fresh air 45 minutes a time and twice a day for 100 days. During the exposure time, the concentration of O2、SO2、CO and NO were detected. On the end of day 100, animals were sacrificed. Lung tissues were obtained for histological detections using H&E staining. Sera were detected for il-8. BALF were obtained for cell analysis.
Result: During the exposure time, the concentration of O2、SO2、CO and NO in the biomass smoke group were (20.31±0.21)%、(981.72±429.59) mg/m3、(3.44±3.36) mg/m3 and (2.59±1.71) mg/m3 respectively and the concentration of O2、SO2、CO and NO in the cigarette group were (20.28±0.15)%、(745.56±683.24) mg/m3、(12.64±11.44) mg/m3 and (3.37±0.52) mg/m3 respectively. The pathological morphology showed that airway epithelia were intact, ciliary arrangements were regular and the structures of alveoli were normal in C group; Characteristic pathological changes of bronchitis and emphysema were observed in BS group and CS group.The pathological score of airway abnormities was higher in CS group(32.66±13.84) and BS group(32.25±12.51) than that in C group (11.11±9.84). The MLI in BS group (130.62±33.64μm) and CS (119.03±26.20μm) were longer than that in C group( 89.84±17.34um ). The MAN in BS group(169.21±76.33/mm2) and CS group (173.86±68.33/mm2 )were smaller than that in C group (280.26±103.95/mm2 ).The serum concentration of IL-8 was higher in CS group(188.14±20.85 pg/ ml) and BS group (240.79±161.28 pg/ml) than that in C group(107.91±35.84 pg/ml).The total inflammatory cells count and the proportion of neutrophils of BALF in BS group and CS group was higher than in C group. The total white blood cell、red blood cell and Hemoglobin in BS group were higher than that in CS group and c group.Conclusion: Exposure to biomass smoke and cigarette smoke will lead to emphysema. The pathological morphology induced by biomass smoke and cigarette smoke were similar.
PART III The study of system oxidative stress in the rat exposure to biomass smoke and cigarette smoke
Method: The activities of superoxide dismutase (SOD) in sera, the total ability of antioxidant in lung tissue homogenate and in heart tissue homogenate,the levels of glutathione (GSH), malondialdehyde (MDA)in sera、lung tissue homogenate、heart tissue homogenate and liver homogenate, the activities of iNOS in in sera、lung tissue homogenate、heart tissue homogenate and the activities of GST in lung tissue homogenate and heart tissue homogenate were measured .The expressions of GCLC-mRNA、GSTM1-mRNA、GSTP-mRNA and SLPI-mRNA in the lung tissue and the GCLC-mRNA、GSTM1-mRNA and GSTP-mRNA in heart tissue were respectively measured with real-time-PCR methods. The expressions ofγ-GCSc(GCLC)protein were measured with immunohistochemisty and Western-blot. Result: The activities of serum superoxide dismutase (SOD) in BS group(223.91±56.86 u/ml) and CS group (218.65±65.60u/ml) were lower than that in C group(283.11±41.71 u/ml). The total ability of antioxidant in lung tissue homogenate and heart homogenate in CS group were lower than that in BS group and C group. The levels of MDA in heart tissue homogenate in BS and CS group were lower than that in C group. The levels of MDA in lung tissue homogenate in BS group were higher than that in C group and CS group. The levels of MDA in liver tissue homogenate and sera were similar in the three groups. The activities of iNOS in lung tissue homogenate in BS group were lower than that in CS group and C group. The activities of iNOS in heart tissue homogenate in BS group and CS group were lower than that in C group. There is no difference of activities of iNOS in sera in the three group. There is no difference of the levels of glutathione (GSH) in sera、lung tissue homogenate、heart tissue homogenate and liver homogenate between the three group. The activities of GST in lung tissue homogenate in CS group (28.43±8.80 RFU/min/ mgpro) were higher than that in BS group(15.44±11.34 RFU/min/ mgpro) and C group(20.54±10.12)RFU/min/ mgpro). The activities of GST in heart tissue homogenate in CS group (4.35±1.94) RFU/min/ mgpro were lower than that in BS group(6.27±1.31 RFU/min/mgpro) and C group(6.76±1.26RFU/min/mgpro).For lung,the expressions of GCLC-mRNA in CS group were stronger than that in BS group and C group. The expressions of GSTP1-mRNA in BS group were weaker than that in CS group and C group.For heart, The expressions of GSTM1-mRNA in BS group and CS group were stronger than that in C group;There is no difference of the expressions of GSTP-mRNA and GCLC-mRNA in BS、CS group and C group.
The immunohistochemisty showed that a few GCLC were expressed in endochylema of alveolar and airway epithelial cells in control group.The GCLC were expressed in BS group and CS group were stronger than that in C group. The more the pulmonary fibrosis is, the stronger of the GCLC expressed. Western-blot analysis also showed that the GCLC were expressed stronger in CS group and BS group than in C group. Conclusion: Exposure to biomass smoke and cigarette smoke will lead to system oxidative stress.There is some difference of system oxidative stress between the two kind of exposure.
COPD是一种全身性疾病,对全身各个系统都产生影响。但是,COPD的临床研究却受到了很多因素的限制。首先临床上要获得COPD病人全身各个系统标本相当困难,只有血标本的获取相对容易;其次,外周血中一些指标的变化不一定与肺部及其他系统的改变相平行。因此,许多学者致力于COPD动物模型的建立。现已有多种方法可复制出COPD动物模型,但是尚没有建立生物燃料烟雾暴露的COPD动物模型,因此对生物燃料烟雾暴露所致COPD的研究尚少。
COPD是一种全身性的疾病,存在多系统的氧化应激,但是生物燃料烟雾暴露所致COPD的全身氧化应激状况如何,生物燃料烟雾和香烟烟雾暴露所致全身氧化应激有何异同尚不清楚。
研究目的:本课题分三部分:第一部分采用循证医学的方法研究生物燃料烟雾暴露与COPD的关系,以明确生物燃料导致COPD的危险度,以及种族、性别及吸烟对生物燃料烟雾导致COPD的危险度的影响。第二部分观察生物燃料烟雾对大鼠肺脏病理组织学的影响,并与香烟烟雾对大鼠肺脏病理组织学改变相比较。第三部分比较生物燃料烟雾暴露和香烟烟雾暴露大鼠全身氧化应激状况的异同,以进一步探讨COPD的发病机制。
第一部分:生物燃料烟雾暴露与慢性阻塞性肺疾病的循证医学研究
方法:我们通过电子检索Medline,Embase,LILACS数据库,并且查阅相关文献的参考文献,共鉴定出15个流行病学研究(4个病例-对照研究和11个横断面研究)符合纳入的研究标准。用标准化的数据提取方案来提取资料并进行统计学分析。结果:和不暴露者相比,生物燃料烟雾暴露者发展为COPD的OR值为2.44(95%CI为1.90-3.33)。对于不同性别、不同种族、不同吸烟状态,生物燃料烟雾暴露是COPD的危险因素(女性OR: 2.73, 95% CI: 2.28-3.28;男性OR4.30, 95% CI: 1.85-10.01;亚洲人:OR: 2.31, 95% CI: 1.41-3.78;非亚洲人OR: 2.56, 95% CI: 1.71-3.83;吸烟者OR=4.39, 95%CI:1.40-4.66,不吸烟者OR=2.55, 95%CI: 2.06-3.15)。生物燃料烟雾暴露者发展为慢性支气管炎的OR值为2.56(95%CI为1.71-3.83),发展为通过肺功能诊断的COPD的OR值为2.65(95% CI: 1.75-4.03)。生物燃料烟雾暴露量越大,发展为COPD的危险性越大。小结:生物燃料烟雾暴露是COPD的危险因素,吸烟和生物燃料烟雾暴露存在协同作用。生物燃料烟雾暴露量越大,发展为COPD的危险性越大。
第二部分:生物燃料烟雾暴露对大鼠肺脏病理组织学的影响
方法:30只雌性SD大鼠,随机分为正常对照组、生物燃料烟雾暴露组、香烟烟雾暴露组,每组10只。每组动物每天置于烟室内两次,每次45min,分别向烟室内充新鲜空气,生物燃料烟雾和香烟烟雾,共100天。熏烟时同时测量烟室中O2、SO2、CO和NO的浓度。100天后,处死动物,HE染色检查大鼠肺脏病理组织学。ELISA法检测血浆IL-8含量,同时进行全血细胞计数和分类,肺泡灌洗液中白细胞计数和分类,右心室重量/室间隔+左心室重量。结果:生物燃料烟雾暴露大鼠熏烟期间箱内O2、CO、SO2和NO的浓度分别为(20.31±0.21)%、(981.72±429.59) mg/m3、(3.44±3.36) mg/m3和(2.59±1.71) mg/m3,香烟烟雾暴露大鼠熏烟期间箱内O2、CO、SO2和NO的浓度分别为(20.28±0.15)%、(745.56±683.24) mg/m3、(12.64±11.44) mg/m3和(3.37±0.52) mg/m3。病理切片HE染色镜下见对照大鼠小气道黏膜上皮完整,纤毛未见黏连脱落,管壁规整未见增厚,未见炎性细胞浸润,管腔内未见炎性渗出物,肺泡腔未见病理性扩大。生物燃料烟雾暴露和香烟烟雾暴露大鼠肺脏病理组织学无明显区别,都可见小气道上皮呈锯齿状增生增厚,支气管壁增厚,见炎性细胞浸润。管腔内见大量炎性渗出物,管壁结缔组织增生,可见淋巴小结。肺泡结构紊乱,肺泡壁断裂,肺泡腔扩大,部分融合成肺大疱。生物燃料烟雾暴露组和香烟烟雾暴露组支气管炎症评分分别为32.66±13.84和32.25±12.51,高于对照组11.11±9.84;平均肺泡间隔分别为130.62±33.64μm、119.03±26.20μm高于对照组89.84±17.34um;而平均肺泡数低于对照组,分别为169.21±76.33个/mm2、173.86±68.33个/mm2和280.26±103.95个/mm2。生物燃料组、香烟烟雾暴露组大鼠血浆中IL-8的浓度分别为240.79±161.28 pg/ml、188.14±20.85 pg/ml高于对照组107.91±35.84 pg/ml。生物燃料烟雾组、香烟烟雾暴露组大鼠BALF白细胞计数及中性白细胞比例高于对照组。而生物燃料烟雾暴露组大鼠全血白细胞和红细胞计数以及血红蛋白含量高于对照组和吸烟组。生物燃料组、香烟烟雾暴露可使大鼠体重减轻,右心室重量/左心室+室间隔重量增加。小结:生物燃料烟雾暴露和香烟烟雾一样都可使大鼠产生慢性支气管炎和肺气肿, BALF中白细胞计数及中性分类增高,血浆IL-8增加,同时,两种烟雾所致大鼠肺脏病理组织学无明显差异。生物燃料烟雾暴露可使大鼠全血白细胞和红细胞计数以及血红蛋白含量上升。
第三部分:生物燃料烟雾和香烟烟雾暴露大鼠全身氧化应激状况比较的研究
方法:使用试剂盒测定血浆T-SOD活力;肺脏、心脏总抗氧化能力;血浆、肺脏、心脏、肝脏MDA和GSH含量;血浆、肺脏和心脏iNOS活力,心脏和肺脏GST活力等氧化/抗氧化指标。应用荧光定量PCR的方法检测肺组织GCLC-mRNA、GSTM1-mRNA、GSTP-mRNA和SLPI-mRNA以及心脏组织GCLC-mRNA、GSTM1-mRNA和GSTP- mRNA的表达。免疫组化分析肺脏组织GCLC的表达。Western Blot检测肺组织γ-GCSc(GCLC)蛋白表达。
结果:香烟烟雾暴露和生物燃料烟雾暴露大鼠血浆中T-SOD活力分别为(218.65±65.60)u/ml,(223.91±56.86) u/ml,低于对照组(283.11±41.71) u/ml。香烟烟雾暴露组大鼠肺和心脏的总抗氧化能力低于对照组和生物燃料暴露组;生物燃料暴露组大鼠肺和心脏的总抗氧化能力与对照组无区别。生物燃料烟雾暴露组肺脏MDA的浓度高于对照组,而吸烟组肺脏MDA的浓度低于对照组;生物燃料烟雾暴露组和吸烟组心脏MDA的浓度低于对照组;血浆和肝组织中MDA浓度三组无明显差异。生物燃料烟雾暴露组血浆iNOS活力低于吸烟组和对照组,肺脏iNOS活力与对照组和吸烟组无明显差异,心脏iNOS活力高于对照组和吸烟组。生物燃料组、吸烟组和对照组血浆、肺脏、心脏、肝脏组织匀浆中GSH浓度没有明显的差异。在肺脏组织中,香烟组GST酶的活力为(28.43±8.80) RFU/min/ mgpro,生物燃料组GST酶的活力为(15.44±11.34) RFU/min/ mgpro,对照组GST酶的活力为(20.54±10.12) RFU/min/ mgpro。心脏组织中香烟组GST酶的活力为(4.35±1.94) RFU/min/mgpro,低于生物燃料组(6.27±1.31)RFU/min/mgpro和对照组(6.76±1.26) RFU/min/ mgpro。吸烟组大鼠肺组织GCLC-mRNA的表达高于对照组和生物燃料组,GSTP1-mRNA的表达低于对照组和生物燃料烟雾暴露组,差异具有统计学意义。肺脏GSTM1mRNA和SLPImRNA的表达三组间没有差别。生物燃料组大鼠心脏GSTM1-mRNA的表达高于对照组,差异具有统计学意义。吸烟组心脏GSTM1-mRNA的表达高于对照组,差异无统计学意义。心脏GSTP-mRNA和GCLC-mRNA的表达三组间无差别。免疫组化显示对照组支气管上皮和肺泡上皮GCLC呈弱阳性表达。生物燃料组和香烟组大鼠支气管上皮和肺泡上皮GCLC表达增强,且均为胞浆表达;而且肺间质纤维化越明显的地方,GCLC的表达越强。免疫组化定量分析和Western-blot分析显示香烟组和生物燃料组肺脏γ-GCSc蛋白的表达强于对照组。小结:本研究显示,香烟烟雾和生物燃料烟雾暴露都可导致大鼠出现全身性氧化应激。两种烟雾刺激所引起的全身氧化应激是不同的。香烟烟雾暴露使大鼠血浆SOD活力降低,心脏和肺脏的总抗氧化力下降,心脏GST活力下降。肺组织中GCLC-mRNA表达增加,GSTP-mRNA表达下降,心肌组织中GSTM1-mRNA表达增强。肺组织GCLC蛋白表达增强,在肺纤维化越明显的部位GCLC表达越高。生物燃料烟雾暴露使大鼠血浆SOD和INOS活力降低,心脏INOS活力增强;肺脏MDA水平升高,心脏MDA降低;肺脏GST活力下降。心肌组织中GSTM1-mRNA表达增强。肺组织GCLC蛋白表达增强,在肺纤维化越明显的部位GCLC表达越高。
结论:生物燃料烟雾暴露是COPD的危险因素,吸烟和生物燃料烟雾暴露存在协同作用。生物燃料烟雾暴露量越大,发展为COPD的危险性越大。生物燃料烟雾和香烟烟雾暴露一样可使大鼠产生慢性支气管炎和肺气肿,两种原因所致肺气肿病理组织学上无明显区别。香烟烟雾和生物燃料烟雾暴露可导致机体出现全身性的氧化抗氧化失衡,导致全身的氧化应激。两种烟雾刺激所引起的全身氧化应激存在一定的差异。
Background: Chronic obstructive pulmonary disease (COPD) is a preventable and treatable disease with some significant extrapulmonary effects that may contribute to the severity in individual patients. Its pulmonary component is characterized by airflow limitation that is not fully reversible. The airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lung to noxious particles or gases. COPD is a global disease with a high and increasing prevalence. Evidence showed that some kind of intervention can reduce the prevalence of COPD. It has been estimated that indoor air pollution resulting from the combustion of solid fuels may be one of the leading contributors to the global burden of disease.Exposure to indoor air pollution may be responsible for nearly 2 million excess deaths in developing countries and for some 4% of the global burden of disease. There is increasing evidence for a possible association between COPD and the use of biomass fuels for cooking and heating in developing countries.However,the extent of the association between biomass smoke and COPD among different Population, the duration of biomass smoke on COPD and the relation between biomass smoke and cigarette smoke is not fully known.
Indoor air pollution is a major global public health threat requiring greatly increased efforts in the areas of research and policy-making.COPD is a system disease.However,the clinical research of COPD were restricted,because it is very differcult to obtain the sample of each system.We only can get the sample of blood.However,the vary of the biomarker in blood is not parallelled with lung and the other organs. So, several kinds of COPD animal model developed.However,there is no COPD animal model caused by biomass smoke.Exposure to biomass smoke is a risk factor for COPD.And COPD is a system disease with system oxidative stress, however there is no information about system oxidative stress caused by biomass smoke and about the difference of oxidative stress between biomass smoke and cigarette smoke.
Objective: To get the information about the extent of the association between biomass smoke and COPD among different Population, the duration of biomass smoke on COPD and the relationship between biomass smoke and cigarette smoke. To compare the pathological morphology caused by biomass smoke and cigarette smoke.To compare the system oxidative stress caused by exposure to biomass smoke and cigarette smoke.
Part I Risk of chronic obstructive pulmonary disease from exposure to biomass smoke: Result from meta-analysis
Methods:We searched the Medline, Embase, LILACS and reviewed citations in relevant articles to identify 15 epidemiologic (11 cross-sectional and 4 case-control) studies that met inclusion criteria. Data were extracted and analyzed independently by two investigators using a standardized protocol.Results: Overall, people exposed to biomass smoke have odds ratio (OR) of 2.44 (95 percent confidence interval (95% CI) 1.9-3.33) for developing COPD, compared with those not exposed to biomass smoke. Biomass smoke exposure was consistently a risk factor for developing COPD in women (OR: 2.73, 95% CI: 2.28-3.28),in men (OR: 4.30, 95% CI: 1.85-10.01), in Asian population (OR: 2.31, 95% CI: 1.41-3.78) ,in Non-Asian population (OR: 2.56, 95% CI: 1.71-3.83),in the phenotype of chronic bronchitis (OR: 2.56, 95% CI: 1.77-3.70),in the phenotype of COPD (OR: 2.65, 95% CI: 1.75-4.03),and in cigarettes smoker (OR=4.39, 95%CI:1.40-4.66) and in cigarettes nonsmoker (OR=2.55, 95%CI: 2.06-3.15). Conclusions: Exposure to biomass smoke is a risk factor for COPD. There is interaction between cigarette and biomass smoke.
Part II The influence of biomass smoke on rat lung pathological morphology
Methods: Sprague Dawley Rat were divided into three groups: control group(C group) rats were exposed to fresh air; biomass smoke group(BS group) were exposed to biomass smoke; cigarette smoke(CS) group were exposed to cigarette smoke. All rats were exposed to the smoke/fresh air 45 minutes a time and twice a day for 100 days. During the exposure time, the concentration of O2、SO2、CO and NO were detected. On the end of day 100, animals were sacrificed. Lung tissues were obtained for histological detections using H&E staining. Sera were detected for il-8. BALF were obtained for cell analysis.
Result: During the exposure time, the concentration of O2、SO2、CO and NO in the biomass smoke group were (20.31±0.21)%、(981.72±429.59) mg/m3、(3.44±3.36) mg/m3 and (2.59±1.71) mg/m3 respectively and the concentration of O2、SO2、CO and NO in the cigarette group were (20.28±0.15)%、(745.56±683.24) mg/m3、(12.64±11.44) mg/m3 and (3.37±0.52) mg/m3 respectively. The pathological morphology showed that airway epithelia were intact, ciliary arrangements were regular and the structures of alveoli were normal in C group; Characteristic pathological changes of bronchitis and emphysema were observed in BS group and CS group.The pathological score of airway abnormities was higher in CS group(32.66±13.84) and BS group(32.25±12.51) than that in C group (11.11±9.84). The MLI in BS group (130.62±33.64μm) and CS (119.03±26.20μm) were longer than that in C group( 89.84±17.34um ). The MAN in BS group(169.21±76.33/mm2) and CS group (173.86±68.33/mm2 )were smaller than that in C group (280.26±103.95/mm2 ).The serum concentration of IL-8 was higher in CS group(188.14±20.85 pg/ ml) and BS group (240.79±161.28 pg/ml) than that in C group(107.91±35.84 pg/ml).The total inflammatory cells count and the proportion of neutrophils of BALF in BS group and CS group was higher than in C group. The total white blood cell、red blood cell and Hemoglobin in BS group were higher than that in CS group and c group.Conclusion: Exposure to biomass smoke and cigarette smoke will lead to emphysema. The pathological morphology induced by biomass smoke and cigarette smoke were similar.
PART III The study of system oxidative stress in the rat exposure to biomass smoke and cigarette smoke
Method: The activities of superoxide dismutase (SOD) in sera, the total ability of antioxidant in lung tissue homogenate and in heart tissue homogenate,the levels of glutathione (GSH), malondialdehyde (MDA)in sera、lung tissue homogenate、heart tissue homogenate and liver homogenate, the activities of iNOS in in sera、lung tissue homogenate、heart tissue homogenate and the activities of GST in lung tissue homogenate and heart tissue homogenate were measured .The expressions of GCLC-mRNA、GSTM1-mRNA、GSTP-mRNA and SLPI-mRNA in the lung tissue and the GCLC-mRNA、GSTM1-mRNA and GSTP-mRNA in heart tissue were respectively measured with real-time-PCR methods. The expressions ofγ-GCSc(GCLC)protein were measured with immunohistochemisty and Western-blot. Result: The activities of serum superoxide dismutase (SOD) in BS group(223.91±56.86 u/ml) and CS group (218.65±65.60u/ml) were lower than that in C group(283.11±41.71 u/ml). The total ability of antioxidant in lung tissue homogenate and heart homogenate in CS group were lower than that in BS group and C group. The levels of MDA in heart tissue homogenate in BS and CS group were lower than that in C group. The levels of MDA in lung tissue homogenate in BS group were higher than that in C group and CS group. The levels of MDA in liver tissue homogenate and sera were similar in the three groups. The activities of iNOS in lung tissue homogenate in BS group were lower than that in CS group and C group. The activities of iNOS in heart tissue homogenate in BS group and CS group were lower than that in C group. There is no difference of activities of iNOS in sera in the three group. There is no difference of the levels of glutathione (GSH) in sera、lung tissue homogenate、heart tissue homogenate and liver homogenate between the three group. The activities of GST in lung tissue homogenate in CS group (28.43±8.80 RFU/min/ mgpro) were higher than that in BS group(15.44±11.34 RFU/min/ mgpro) and C group(20.54±10.12)RFU/min/ mgpro). The activities of GST in heart tissue homogenate in CS group (4.35±1.94) RFU/min/ mgpro were lower than that in BS group(6.27±1.31 RFU/min/mgpro) and C group(6.76±1.26RFU/min/mgpro).For lung,the expressions of GCLC-mRNA in CS group were stronger than that in BS group and C group. The expressions of GSTP1-mRNA in BS group were weaker than that in CS group and C group.For heart, The expressions of GSTM1-mRNA in BS group and CS group were stronger than that in C group;There is no difference of the expressions of GSTP-mRNA and GCLC-mRNA in BS、CS group and C group.
The immunohistochemisty showed that a few GCLC were expressed in endochylema of alveolar and airway epithelial cells in control group.The GCLC were expressed in BS group and CS group were stronger than that in C group. The more the pulmonary fibrosis is, the stronger of the GCLC expressed. Western-blot analysis also showed that the GCLC were expressed stronger in CS group and BS group than in C group. Conclusion: Exposure to biomass smoke and cigarette smoke will lead to system oxidative stress.There is some difference of system oxidative stress between the two kind of exposure.
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