中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2013年
28期
2230-2234
,共5页
孟莹%余常辉%李婷%李维%蔡绍曦%李旭
孟瑩%餘常輝%李婷%李維%蔡紹晞%李旭
맹형%여상휘%리정%리유%채소희%리욱
Toll样受体4%脂多糖类%吸烟%肺疾病,慢性阻塞性%NF-κB
Toll樣受體4%脂多糖類%吸煙%肺疾病,慢性阻塞性%NF-κB
Toll양수체4%지다당류%흡연%폐질병,만성조새성%NF-κB
Toll-like receptor 4%Lipopolysaccharides%Smoking%Pulmonary disease,chronic obstructive%NF-kappa B
目的 探讨Toll样受体(TLR)-4受体在单纯烟熏法和脂多糖(LPS)联合烟熏所致肺损伤大鼠模型中的表达及意义.方法 8周龄SD雄性大鼠15只,按随机数字表法随机均分为3组:(1)对照组:常规饲养；(2)烟熏组:置于自制120 L有机玻璃熏箱内,2次/d,第1、14天气管内滴注注射用水；(3)联合组:烟熏同前法,第1、14天气管内滴注脂多糖(1 mg/kg).4周后观察动物的一般情况,测量大鼠肺功能,检测动脉血气和肺组织病理学；免疫组化检测和Western印迹法检测各组肺组织中TLR-4、核因子(NF)-κB蛋白表达及p-Iκ-Kα/β、Iκ-Kα/β、IκB-α蛋白表达.实时定量PCR检测NF-κB信号通路下游炎症因子肿瘤坏死因子(TNF)-α和白细胞介素(IL)-6 mRNA表达.结果 烟熏组和联合组大鼠消瘦,伴有间歇咳嗽和气促.对照组、烟熏组、联合组最大呼气流量分别为(13.5±2.0)、(12.3±0.9)、(10.6±1.4) ml/s,50％容量的呼气流速分别为(1.01±0.08)、(0.91 ±0.10)、(0.77±0.14) ml/s,联合组均显著低于其他2组(均P＜0.05)；3组累积容积分别为(1735±798)、(4358±1501)、(10 077±1866) ml,二氧化碳分压分别为(39.6±1.4)、(52.8±2.0)、(51.0±5.0)mm Hg(1 mm Hg=00.133 kPa),对照组均显著低于其他2组(均P＜0.05).HE染色结果:烟熏组及联合组有明显的慢性气道炎症及肺气肿.联合组单位面积内平均肺泡数(1.56±0.26)低于对照组(3.07±0.78) (P ＜0.05),烟熏组及联合组肺组织平均内衬间隔和肺泡破坏指数分别为(84±13)、(86±10) μm和0.228±0.047、0.294±0.060,均显著高于对照组的(65±6) μm,m和0.036±0.012(均P＜0.05)；免疫组化结果:烟熏组、联合组细胞胞质及细胞膜上TLR-4蛋白表达及细胞核中NF-κB蛋白表达明显高于对照组.Western印迹法检测烟熏组、联合组TLR-4蛋白表达分别为1.12±0.11、1.36±0.07,核蛋白中NF-κB表达分别为0.59 0.06、1.04±0.08,均显著高于对照组的0.68±0.03和0.21±0.08(均P＜0.05)；烟熏组和联合组胞质蛋白Iκ-Kα/β蛋白磷酸化水平均显著高于对照组,IκB-α蛋白表达均显著低于对照组,而TNF-α(3.95±0.29和5.33±0.26)、IL-6 mRNA(5.04±0.28和7.23±0.39)表达均显著高于对照组(1.00±0.37、1.00±0.25)(均P＜0.05).结论 脂多糖联合烟熏与单纯烟熏均可通过NF-κB信号通路诱导大鼠肺部出现类似人类慢性阻塞性肺疾病的损伤,TLR-4参与其中.
目的 探討Toll樣受體(TLR)-4受體在單純煙熏法和脂多糖(LPS)聯閤煙熏所緻肺損傷大鼠模型中的錶達及意義.方法 8週齡SD雄性大鼠15隻,按隨機數字錶法隨機均分為3組:(1)對照組:常規飼養；(2)煙熏組:置于自製120 L有機玻璃熏箱內,2次/d,第1、14天氣管內滴註註射用水；(3)聯閤組:煙熏同前法,第1、14天氣管內滴註脂多糖(1 mg/kg).4週後觀察動物的一般情況,測量大鼠肺功能,檢測動脈血氣和肺組織病理學；免疫組化檢測和Western印跡法檢測各組肺組織中TLR-4、覈因子(NF)-κB蛋白錶達及p-Iκ-Kα/β、Iκ-Kα/β、IκB-α蛋白錶達.實時定量PCR檢測NF-κB信號通路下遊炎癥因子腫瘤壞死因子(TNF)-α和白細胞介素(IL)-6 mRNA錶達.結果 煙熏組和聯閤組大鼠消瘦,伴有間歇咳嗽和氣促.對照組、煙熏組、聯閤組最大呼氣流量分彆為(13.5±2.0)、(12.3±0.9)、(10.6±1.4) ml/s,50％容量的呼氣流速分彆為(1.01±0.08)、(0.91 ±0.10)、(0.77±0.14) ml/s,聯閤組均顯著低于其他2組(均P＜0.05)；3組纍積容積分彆為(1735±798)、(4358±1501)、(10 077±1866) ml,二氧化碳分壓分彆為(39.6±1.4)、(52.8±2.0)、(51.0±5.0)mm Hg(1 mm Hg=00.133 kPa),對照組均顯著低于其他2組(均P＜0.05).HE染色結果:煙熏組及聯閤組有明顯的慢性氣道炎癥及肺氣腫.聯閤組單位麵積內平均肺泡數(1.56±0.26)低于對照組(3.07±0.78) (P ＜0.05),煙熏組及聯閤組肺組織平均內襯間隔和肺泡破壞指數分彆為(84±13)、(86±10) μm和0.228±0.047、0.294±0.060,均顯著高于對照組的(65±6) μm,m和0.036±0.012(均P＜0.05)；免疫組化結果:煙熏組、聯閤組細胞胞質及細胞膜上TLR-4蛋白錶達及細胞覈中NF-κB蛋白錶達明顯高于對照組.Western印跡法檢測煙熏組、聯閤組TLR-4蛋白錶達分彆為1.12±0.11、1.36±0.07,覈蛋白中NF-κB錶達分彆為0.59 0.06、1.04±0.08,均顯著高于對照組的0.68±0.03和0.21±0.08(均P＜0.05)；煙熏組和聯閤組胞質蛋白Iκ-Kα/β蛋白燐痠化水平均顯著高于對照組,IκB-α蛋白錶達均顯著低于對照組,而TNF-α(3.95±0.29和5.33±0.26)、IL-6 mRNA(5.04±0.28和7.23±0.39)錶達均顯著高于對照組(1.00±0.37、1.00±0.25)(均P＜0.05).結論 脂多糖聯閤煙熏與單純煙熏均可通過NF-κB信號通路誘導大鼠肺部齣現類似人類慢性阻塞性肺疾病的損傷,TLR-4參與其中.
목적 탐토Toll양수체(TLR)-4수체재단순연훈법화지다당(LPS)연합연훈소치폐손상대서모형중적표체급의의.방법 8주령SD웅성대서15지,안수궤수자표법수궤균분위3조:(1)대조조:상규사양；(2)연훈조:치우자제120 L유궤파리훈상내,2차/d,제1、14천기관내적주주사용수；(3)연합조:연훈동전법,제1、14천기관내적주지다당(1 mg/kg).4주후관찰동물적일반정황,측량대서폐공능,검측동맥혈기화폐조직병이학；면역조화검측화Western인적법검측각조폐조직중TLR-4、핵인자(NF)-κB단백표체급p-Iκ-Kα/β、Iκ-Kα/β、IκB-α단백표체.실시정량PCR검측NF-κB신호통로하유염증인자종류배사인자(TNF)-α화백세포개소(IL)-6 mRNA표체.결과 연훈조화연합조대서소수,반유간헐해수화기촉.대조조、연훈조、연합조최대호기류량분별위(13.5±2.0)、(12.3±0.9)、(10.6±1.4) ml/s,50％용량적호기류속분별위(1.01±0.08)、(0.91 ±0.10)、(0.77±0.14) ml/s,연합조균현저저우기타2조(균P＜0.05)；3조루적용적분별위(1735±798)、(4358±1501)、(10 077±1866) ml,이양화탄분압분별위(39.6±1.4)、(52.8±2.0)、(51.0±5.0)mm Hg(1 mm Hg=00.133 kPa),대조조균현저저우기타2조(균P＜0.05).HE염색결과:연훈조급연합조유명현적만성기도염증급폐기종.연합조단위면적내평균폐포수(1.56±0.26)저우대조조(3.07±0.78) (P ＜0.05),연훈조급연합조폐조직평균내츤간격화폐포파배지수분별위(84±13)、(86±10) μm화0.228±0.047、0.294±0.060,균현저고우대조조적(65±6) μm,m화0.036±0.012(균P＜0.05)；면역조화결과:연훈조、연합조세포포질급세포막상TLR-4단백표체급세포핵중NF-κB단백표체명현고우대조조.Western인적법검측연훈조、연합조TLR-4단백표체분별위1.12±0.11、1.36±0.07,핵단백중NF-κB표체분별위0.59 0.06、1.04±0.08,균현저고우대조조적0.68±0.03화0.21±0.08(균P＜0.05)；연훈조화연합조포질단백Iκ-Kα/β단백린산화수평균현저고우대조조,IκB-α단백표체균현저저우대조조,이TNF-α(3.95±0.29화5.33±0.26)、IL-6 mRNA(5.04±0.28화7.23±0.39)표체균현저고우대조조(1.00±0.37、1.00±0.25)(균P＜0.05).결론 지다당연합연훈여단순연훈균가통과NF-κB신호통로유도대서폐부출현유사인류만성조새성폐질병적손상,TLR-4삼여기중.
Objective To explore the expression and effect of Toll-like receptor 4 (TLR-4) in the lung tissue of rats established by passive smoking or intratracheal instillation of lipopolysaccharide (LPS).Methods Eight-week-old male Sprague-Dawley rats (n =15) were randomly divided into 3 groups,including:(1) group A:conventional breeding； (2) group B:the rats were placed into a 120-L organic glass box with twice-daily exposure to cigarette smoking plus an intratracheal instillation of water at Day 1 and 14; (3) Group C:exposure to cigarette smoking the same as group B plus intratracheal instillation of lipopolysaccharide(1 mg/kg) at Day 1 and 14.Four weeks later,general status,arterial blood gas,pulmonary function and histopathology were analyzed.The expressions of TLR-4 and nuclear factor kappa-B (NF-κB) were determined by immunohistochemistry.Western blot was used to measure the protein contents of TLR-4,NF-κB,p-Iκ-Kα/β,Iκ-Kα/β,IκB-α.And real-time polymerase chain reaction (PCR) was employed to detect the mRNA levels of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6).Results Rats in Groups B and C were marantic with intermittent cough and dyspnea.Peak expiratory flow (PEF) and 50％ expiratory flow-volume (EP50) were much lower in Group C ((10.6 ± 1.4),(0.77 ± 0.14) ml/s) than that in Groups A ((13.5 ±2.0),(1.01 ±0.08) and B (12.3 ±0.9),(0.91 ±0.10) ml/s) (all P ＜ 0.05).Accumulated volume (AV) and carbon dioxide pressure (PCO2) were much higher in Groups B ((4358±1501) ml,(52.77 ±1.97) mm Hg) (1 mm Hg=0.133 kPa) and C ((10 077 ± 1866) ml,(51.03 ±4.96) mm Hg) than that in Group A ((1735 ±798) ml,(39.57 ±1.43) mm Hg) (all P ＜ 0.05).Hematoxylin and eosin stain showed chronic bronchitis and emphysema in Groups B and C.Besides,quantitative analysis demonstrated that in unit area,mean lining interval (MLI) and destruction index (DI) in Group B ((84 ± 13) μm,0.228 ±0.047) and Group C ((86 ± 10) μm,0.294 ±0.060) significantly increased versus Group A ((65 ± 6) μm,0.036 ± 0.012) (all P ＜ 0.05).Immunohistochemical staining indicated that the expression of TLR-4 in cytoplasm and cytomembrane and NF-κB in nucleus markedly increased in Groups B and C versus Group A.Relative expressions of TLR-4 and NF-κB assayed by Western blot increased in Group B (0.68 ± 0.03,0.21 ± 0.08) and Group C (1.12 ± 0.11,0.59 ± 0.06) than that in Group A (1.36 ± 0.07,1.04 ± 0.08).Compared with Group A,the expression levels of TLR-4,NF-κB and IκB-α and the phosphorylation levels of Iκ-Kα/β in Group B and C significantly increased (all P ＜ 0.05).The mRNA levels of TNF-α and IL-6 increased in Group B (3.95 ± 0.29,5.04 ± 0.28) and C (5.33 ± 0.26,7.23 ± 0.39) versus that in Group C (1.00 ± 0.37,1.00 ± 0.25) (all P ＜ 0.05).Conclusions Both passive smoking and intratracheal instillation of LPS may cause lung injury analogous to chronic obstructive pulmonary disease via NF-κB signaling pathway.And TLR4 plays an important role in this process.
