高温联合PM_(2.5)暴露致COPD大鼠肺损伤的机制研究

Mechanism of lung injury caused by high temperature and PM_(2.5) in COPD rats

[目的]研究高温及大气PM_(2.5)暴露致慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)大鼠肺组织损伤的作用机制。[方法] 84只7周龄健康SPF级雄性Wistar大鼠随机分成对照组和COPD模型组,利用卷烟烟雾和气管内滴注脂多糖的方法建立COPD大鼠模型。对照组和COPD模型组行PM_(2.5)气管滴注(0、3.2、12.8 mg/mL),然后暴露于高温(40℃)8 h,每天1次,持续3 d,同时选择20℃作为常温对照温度。末次染毒24 h后,检测两组大鼠的最大吸气流量(peak inspiratory flow,PIF)和最大呼气流量(peak expiratory flow,PEF)等肺功能指标;HE染色制作肺组织病理切片;利用试剂盒测定COPD大鼠肺组织超氧化物歧化酶(superoxide dismutase,SOD)、诱导性一氧化氮合酶(inducible nitric oxide synthase,iNOS)、丙二醛(malonyldialdehyde,MDA)的含量;采用链霉素抗生物素蛋白-过氧化物酶连接法测定8-羟基脱氧鸟苷(8-hydroxydeoxyguanosine,8-OHdG)表达水平;采用蛋白质免疫印迹法分析血红素氧合酶(heme oxygenase,HO-1)的表达水平。采用析因分析法分析温度、PM_(2.5)和COPD模型对肺功能的影响,以及温度、PM_(2.5)对氧化应激指标的影响。[结果]高温状态下,对照组和COPD组大鼠经不同剂量PM_(2.5)染毒,PIF和PEF均低于常温状态相应染毒剂量组(均P <0.05);对于PIF,高温、PM_(2.5)及COPD三者存在交互作用(F=7.585,P <0.05)。病理切片显示,COPD组大鼠肺损伤明显,尤其在高温和PM_(2.5)暴露下变化较对照组更为明显。高温状态COPD组大鼠肺组织中iNOS含量在各PM_(2.5)染毒剂量下均高于相应的常温状态COPD组(均P <0.05);高温和PM_(2.5)对MDA(F=1.779,P=0.183)、iNOS(F=0.128,P=0.880)及SOD(F=1.792,P=0.175)的影响不存在交互作用。此外,高温状态COPD组大鼠肺组织8-OHd G蛋白表达在0 mg/mL和3.2 mg/mL PM_(2.5)染毒剂量组时高于相应剂量下的常温状态组(P <0.05);而相同PM_(2.5)剂量下,高温状态COPD组大鼠HO-1蛋白表达在0、12.8 mg/mL PM_(2.5)染毒剂量下低于常温状态组(P <0.05)。[结论]高温状态下,COPD大鼠肺功能更易受PM_(2.5)的影响,氧化/抗氧化系统失衡可能是高温和PM_(2.5)联合暴露致COPD大鼠肺组织损伤的主要机制。

[ Objective ] To explore the possible mechanism of lung injury caused by high temperature and PM25 in chronic obstructive groups. A COPD rat model was established by cigarette smoke exposure and intralraeheal instillation of lipopolysacharide.The rats were instilled with PM2.5 （0, 3.2, and 12.8 mg/mL） separately and then exposed 1o high temperature at 40℃ for 8 h, once a day for three consecutive days; 20℃ was selected as the normal control temperature. Lung function indicators [peak inspiralory flow （PIF） and peak expiralory flow （PEF）] were measured 24 h after the last exposure, Lung tissue samples were collected for pathologic analysis by HE staining. The levels of superoxide dismutase （SOD）, inducible nitric oxide synthase （iNOS）, and malonyldialdehyde （MDA） in lung homogenate of COPD rats were detected by conrresponding test kits. The expression levels of 8-hydroxydeoxyguanosine （8-OHdG） and heine oxygenase （HO-1） were determined by streptomycin anti-biotin-peroxidase and Western blot respectively. Factorial analysis was used to analyze the effects of temperature, PM2.5, and COPD on lung function, and also the effects of temperature and PM2.5 on oxidative stress indicators. [ Results ] Under high temperature stimulation, the PIF and PEF of the control and COPD rats treated with different PM2.5 doses were lower than the rats treated with corresponding exposure dose at normal temperature （Ps〈0.05）; high temperature, PM2.5, and COPD had interactions on PIF （F=7.585, P〈0.05）. The injury in lung tissues of the COPD rats was obvious, especially under high temperature and PM2.5 exposure, which was more obvious than that in the control group. The content of iNOS in lung tissues of the COPD group under high temperature was higher than that of the COPD group under normal temperature treated with various doses of PM25 （Ps 〈 0.05）. There was no interaction between high temperature and PM2.5 on MDA （F=1.779, P=0.183）, iNOS （F=0.128, P=0.880）, and SOD （F= 1.792, P=0.175）. In addition, the expression of 8-0HdG protein in lung tissues of the COPD group under high temperature was higher than that of the COPD group under normal temperature lreated with 0 and 3.2 mg/mL PM2.5 （P 〈 0.05）; the expression of HO- 1 protein in the COPD rats under high temperature was lower than that in the rats under normal temperature at 0 and 12.8 mg/mL PMzs （P〈0.05）. [ Conclusion ] COPD rats＇ lung function is more susceptible to PM2.5 under high temperature, and the imbalance of oxidative/ antioxidant system may be the main mechanism of lung injury in COPD rats with high temperature and PM2.5 co-exposure.