PPARγ信号通路对香烟烟雾所致COPD模型小鼠肺泡巨噬细胞的影响

Effect of peroxisome proliferator-activated receptor γ signal pathway on alveolar macrophages in cigarette smoke-induced chronic obstructive pulmonary disease mouse model

目的观察过氧化物酶增殖物激活受体γ(PPARγ)对香烟烟雾诱导的小鼠慢性阻塞性肺疾病(COPD)模型体质量、肺功能、炎症因子、凋亡指数、PPARγ相关蛋白表达水平等的影响。方法选择24只SPF级C57BL/6小鼠,按随机数字表法分为正常对照组、模型组、PPARγ激动剂组、PPARγ抑制剂组,每组6只。采用香烟熏吸法复制COPD小鼠模型。制模后PPARγ激动剂组给予生理盐水灌胃同时腹腔注射PPARγ激动剂吡格列酮(给药量为10 mg·kg^(-1)·d^(-1));PPARγ抑制剂组给予生理盐水灌胃同时腹腔注射PPARγ抑制剂GW9662(给药量为1 mg·kg^(-1)·d^(-1)),模型组和正常对照组给予生理盐水灌胃(雄鼠每次0.25 mL,雌鼠每次0.2 mL)及腹腔注射(雄鼠每次0.2 mL,雌鼠每次0.15 mL),均每日1次,每周6次,连续给药8周。给药后观察各组小鼠体质量、肺功能、肺组织病理变化;采用酶联免疫吸附试验(ELISA)检测各组肺组织匀浆中肿瘤坏死因子-α(TNF-α)、白细胞介素-4(IL-4)水平;采用原位末端缺刻标记试验(TUNEL)检测肺组织凋亡细胞凋亡情况;采用免疫组化法检测肺组织巨噬细胞诱导型一氧化氮合酶(iNOS)、CD163,PPARγ的蛋含量。结果与正常对照组相比,模型组体质量明显减轻(g:21.05±0.84比27.12±1.13,P<0.05),最终潮气量(TV)、每分钟呼气量(MV)、呼气峰流速(PEF)、50%潮气量呼气流量(EF50)、PPARγ的蛋白表达水平均明显降低〔TV(mL):0.28±0.03比0.39±0.02,MV(mL/min):120.35±13.01比179.32±17.70,PEF(mL/s):5.72±0.38比9.99±0.67,EF50(mL/s):0.27±0.05比0.49±0.04,PPARγ(A值):12.69±0.85比18.85±0.39,均P<0.01〕;TNF-α、IL-4、肺组织细胞凋亡指数、小鼠肺泡巨噬细胞iNOS、CD163的蛋白含量均明显升高〔TNF-α(ng/L):642.16±146.03比325.99±84.63,IL-4(ng/L):431.39±50.17比343.24±48.87,细胞凋亡指数:(61.68±4.37)%比(41.34±1.69)%,iNOS(A值):17.06±1.16比8.58±0.84,CD163(A值):15.11±0.44比9.04±0.39,均P<0.01〕;光镜下可见小鼠肺泡壁断裂融合,泡腔增大,支气管管壁厚度增加,周围纤维组织增生。与模型组比较,PPARγ激动剂组体质量、TV、MV、PEF、EF50均明显升高〔体质量(g):25.35±0.70比21.05±0.84,TV(mL):0.34±0.01比0.28±0.03,MV(mL/min):151.24±9.37比,120.35±13.01,PEF(mL/s):6.84±0.59比5.72±0.38,EF50(mL/s):0.36±0.05比0.27±0.05,均P<0.05〕,肺组织细胞凋亡指数、iNOS、CD163均明显下降〔肺组织凋亡指数:(38.78±3.65)%比(61.68±4.37)%,iNOS(A值):9.07±0.78比17.06±1.16,CD163(A值):9.36±0.35比15.11±0.44,均P<0.01〕,PPARγ水平明显升高(A值:19.14±0.36比12.69±0.85);而PPARγ激动剂组和PPARγ抑制剂组TNF-α、IL-4均明显降低〔TNF-α(ng/L):286.57±44.66、354.93±88.40比642.16±146.03,IL-4(ng/L):237.05±32.05、285.58±50.47比431.39±50.17,均P<0.01〕。光镜下可见PPARγ激动剂组肺组织病理学改变均有所改善,PPARγ抑制剂组则无明显变化。结论PPARγ通路参与了COPD的病理生理过程;PPARγ信号通路激动剂可改善COPD模型小鼠的一般情况、肺功能、炎症因子、肺组织凋亡指数,抑制肺泡巨噬细胞的极化。

Objective To observe the effects of peroxisome proliferator-activated receptor γ (PPAR γ )on body mass,pulmonary function,inflammatory factors,apoptosis index,and expression of PPAR γ related proteins in cigarette smoke-induced chronic obstructive pulmonary disease(COPD)model in mice.Methods A total of 24 SPF C57BL/6 mice were randomly divided into normal control group,model group,PPAR γ agonist group and PPAR γ inhibitor group,with 6 mice in each group.The COPD model mice were reproduced by cigarette smoking.After modeling,PPAR γ agonist group was given normal saline intragastric administration and intraperitoneal injection of PPAR γ agonist pioglitazone(10 mg·kg^(-1)·d^(-1));PPAR γ inhibitor group received normal saline intragastric administration and intraperitoneal injection of PPAR γ inhibitor group GW9662(1 mg·kg^(-1)·d^(-1));the model group and the normal control group were given normal saline intragastric administration(0.25 mL every time for male mice and 0.2 mL for female mice)and intraperitoneal injection(0.2 mL for male mice and 0.15 mL for female mice).All drugs were given once a day,6 times a week for 8 weeks.After 8 weeks of administration,the body mass,lung function,and pathophysiological changes of lung tissue were observed,and the levels of tumor necrosis factor-α(TNF-α)and interleukin-4(IL-4)in lung homogenate were detected by enzyme linked immunosorbent assay(ELISA).The apoptosis of lung tissue was detected by TdT-mediated dUTP-nick end labeling(TUNEL),and the protein content of macrophage inducible nitric oxide synthase(iNOS)and CD163,PPAR γ in lung tissue was detected by immunohistochemistry.Results Compared with the normal control group,the body weight of the model group was lighter(g:21.05±0.84 vs.27.12±1.13,P<0.05),final tidal volume(TV),minute volume(MV),peak expiratory flow(PEF),expiratory flow at 50%expired volume(EF50)and the protein expression levels of PPAR γ were significantly decreased[TV(mL):0.28±0.03 vs.0.39±0.02,MV(mL/min):120.35±13.01 vs.179.32±17.70,PEF(mL/s):5.72±0.38 vs.9.99±0.67,EF50(mL/s):0.27±0.05 vs.0.49±0.04,PPAR γ (A value):12.69±0.85 vs.18.85±0.39,all P<0.01],TNF-α,IL-4,apoptosis index of lung tissue and protein content of iNOS and CD163 in mouse alveolar macrophages were significantly increased[TNF-α(ng/L):642.16±146.03 vs.325.99±84.63,IL-4(ng/L):431.39±50.17 vs.343.24±48.87,apoptosis index:(61.68±4.37)%vs.(41.34±1.69)%,iNOS(A value):17.06±1.16 vs.8.58±0.84,CD163(A value):15.11±0.44 vs.9.04±0.39,all P<0.01].Under light microscope,the alveolar wall of mice was broken and fused,the alveolar cavity enlarged,the thickness of bronchial wall increased and the surrounding fibrous tissue proliferated.Compared with the model group,the body mass,TV,MV,PEF and EF50 in the PPAR γ agonist group were significantly increased[body mass(g):25.35±0.70 vs.21.05±0.84,TV(mL):0.34±0.01 vs.0.28±0.03,MV(mL/min):151.24±9.37 vs.120.35±13.01,PEF(mL/s):6.84±0.59 vs.5.72±0.38,EF50(mL/s):0.36±0.05 vs.0.27±0.05,all P<0.05],apoptosis index,iNOS and CD163 in lung tissue decreased significantly[apoptosis index:(38.78±3.65)%vs.(61.68±4.37)%,iNOS(A value):9.07±0.78 vs.17.06±1.16,CD163(A value):9.36±0.35 vs.15.11±0.44,all P<0.01],the level of PPAR γ increased significantly(A value:19.14±0.36 vs.12.69±0.85).However,TNF-αand IL-4 decreased significantly in PPAR γ agonist group and PPAR γ inhibitor group[TNF-α(ng/L):286.57±44.66.354.93±88.40 vs.642.16±146.03,IL-4(ng/L):237.05±32.05,285.58±50.47 vs.431.39±50.17,both P<0.01].Under light microscope,the pathophysiological changes of lung tissue were improved in PPAR γ agonist group,but there was no significant change in PPAR γ inhibitor group.Conclusions The PPAR γ pathway participates in the pathophysiological process of COPD.PPAR γ signal pathway agonist improves the general condition,pulmonary function,inflammatory factors,and lung apoptosis index of COPD model mice and inhibits the polarization of alveolar macrophages.