丁苯酞对慢性阻塞性肺疾病模型大鼠肺功能及TLR4/MyD88/NF-κB通路的影响

Effect of butylphthalide on pulmonary function and TLR4/MyD88/NF-κB pathway in chronic obstructive pulmonary disease model rats

目的探讨丁苯酞对慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)模型大鼠肺功能及Toll样受体4(Toll-like receptor 4,TLR4)/髓样分化因子88(myeloid differentiation factor 88,MyD88)/核因子-κB(nuclear factor-κB,NF-κB)通路的影响。方法 Wistar大鼠随机分为对照组、模型组及丁苯酞低、中、高剂量组,对照组大鼠进行10 mL/kg生理盐水腹腔注射,模型组及丁苯酞低、中、高剂量组大鼠建立COPD模型,并分别进行10 mL/kg生理盐水及20、40、80 mg/kg丁苯酞腹腔注射。用药4周后,测定肺功能指标每0.3 s用力呼气量(forced expiratory volume in 0.3 second,FEV0.3)占用力肺活量(forced vital capacity,FVC)之比、动态顺应性(dynamic compliance,Cdyn)和呼气阻力(expiratory resistance,Re),酶联免疫(enzyme-linked immunosorbent assay,ELISA)法检测大鼠支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中肿瘤坏死因子-α(tumor necrosis factor-alpha,TNF-α)、白细胞介素(interleukin,IL)-1β、IL-8水平,苏木素-伊红(Hematoxylin and Eosin,HE)染色观察肺组织病理学变化,采用蛋白印迹(western blot,WB)法检测肺组织TLR4、MyD88、p-NF-κB p65、NF-κB p65蛋白水平。结果与对照组相比,模型组大鼠FEV0.3/FVC(70.55±1.63)、Cdyn(0.32±0.02)降低,Re(9.23±0.66)及BALF中TNF-α(213.52±15.44)、IL-1β(102.85±3.18)、IL-8(162.58±9.38)水平升高,肺组织中TLR4(0.87±0.07)、MyD88(0.84±0.03)、p-NF-κB p65/NF-κB p65(0.93±0.06)蛋白水平升高(均P<0.05),并出现明显的COPD病理特征;与模型组相比,随着丁苯酞剂量的升高,COPD大鼠FEV0.3/FVC(74.08±1.57、76.77±1.41、80.68±1.76)、Cdyn(0.37±0.02、0.43±0.02、0.47±0.03)升高,Re(7.31±0.85、4.68±0.82、3.35±0.45)及BALF中TNF-α(183.25±17.59、152.44±14.25、128.67±13.48)、IL-1β(82.49±3.32、58.70±3.64、39.68±3.22)、IL-8(138.90±9.44、102.52±7.65、83.65±7.21)水平降低,肺组织中TLR4(0.76±0.05、0.60±0.07、0.41±0.05)、MyD88(0.77±0.03、0.52±0.04、0.43±0.03)、p-NF-κB p65/NF-κB p65(0.84±0.05、0.62±0.06、0.45±0.03)蛋白水平降低(均P<0.05),且COPD各病理特征有明显改善。结论丁苯酞能改善COPD大鼠肺功能,并抑制TLR4/MyD88/NF-κB通路激活,降低炎症反应。

Objective To investigate the effect of butylphthalide on lung function and Toll-like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)/nuclear factor-κB(NF-κB) pathway in model rats with chronic obstructive pulmonary disease(COPD). Methods Wistar rats were randomly divided into control group,model group and low-,medium-,and high-dose butylphthalide groups. Rats in the control group were intraperitoneally injected with 10 mL/kg normal saline. COPD models were established in model group and low-,medium-,and high-dose butylphthalide groups,10 mL/kg normal saline and 20,40 and 80 mg/kg butylphthalide were injected intraperitoneally respectively. After four weeks of treatment,the ratio of forced expiratory volume in 0.3 second(FEV0.3) to forced vital capacity(FVC),dynamic compliance(Cdyn) and expiratory resistance(Re) were measured,the levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β and IL-8 in bronchoalveolar lavage fluid(BALF) were detected by enzyme-linked immunosorbent assay(ELISA),the pathological changes of lung tissue were observed by hematoxylin eosin(HE) staining,andthe protein levels of TLR4,MyD88,p-NF-κB p65 and NF-κB p65 were detected by Western blot(WB).Results Compared with those in the control group(86.62±1.83,0.53±0.03,1.89±0.48,85.40±9.83,27.64±2.86,58.93±4.46,0.29±0.03,0.36±0.02,0.17±0.03),FEV0.3/FVC(70.55±1.63) and Cdyn(0.32±0.02) in the model group were significantly decreased,Re(9.23±0.66) and the levels of TNF-α(213.52±15.44),IL-1β(102.85±3.18) and IL-8(162.58±9.38) in BALF,protein levels of TLR4(0.87±0.07),MyD88(0.84±0.03),p-NF-κB p65/NF-κB p65(0.93±0.06) in lung tissue were significantly increased(all P<0.05), and there were obvious pathological features of COPD. Compared with those in the model group,FEV0.3/FVC(74.08±1.57,76.77±1.41,80.68±1.76) and Cdyn(0.37±0.02,0.43±0.02,0.47±0.03) in COPD rats were significantly increased with the increase of butylphthalide dose,Re(7.31±0.85,4.68±0.82,3.35±0.45) and the levels of TNF-α (183.25±17.59,152.44±14.25,128.67±13.48),IL-1β(82.49±3.32,58.70±3.64,39.68±3.22) and IL-8(138.90±9.44,102.52±7.65,83.65±7.21) in BALF,protein levels of TLR4(0.76 ±0.05,0.60 ±0.07,0.41 ±0.05),MyD88(0.77 ±0.03,0.52 ±0.04,0.43 ±0.03) and p-NF-κB p65/NF-κB p65(0.84 ±0.05,0.62±0.06,0.45±0.03) in lung tissue were significantly decreased(all P<0.05),and the pathological features of COPD were significantly improved. Conclusion Butylphthalide can improve the lung function of COPD rats,inhibit the activation of TLR4/MyD88/NF-κB pathway and reduce inflammatory reaction.