柴胡皂苷d对哮喘小鼠气道炎症及TLR4/NF-κB通路的影响

Effects of saikosaponin-d on airway inflammation and TLR4/NF-κB pathway in mice with asthma

目的探讨柴胡皂苷d(SSd)对哮喘小鼠气道炎症及Toll样受体4(TLR4)/核转录因子-κB(NF-κB)通路的影响。方法健康BALB/c雌性小鼠40只,随机取6只作为对照组,除对照组外均在第1、13天腹腔注射20μg卵蛋白(OVA)和1 mg氢氧化铝[Al(OH)3]混悬液致敏,第14~30天用2%OVA 0.9%氯化钠溶液雾化吸入激发造模。造模成功后,分为模型组、阿奇霉素(AZM)组、低中高剂量SSd组(n=6),AZM组及低中高剂量SSd组分别经腹腔注射给予AZM 250 mg/kg,SSd 1.0、1.5、2.0 mg/kg,1次/d,连续14 d,对照组和模型组给予等量0.9%氯化钠溶液。所有小鼠均在最后一次给药后2 h处死,收集支气管肺泡灌洗液(BALF),检测各组小鼠免疫细胞数量。HE染色观察各组小鼠右侧肺脏组织病理学变化;采用实时荧光定量PCR法(qRT-PCR)法检测小鼠肺脏组织中TLR4、NF-κB、肿瘤坏死因子-α(TNF-α)及白介素-1β(IL-1β)mRNA的水平;免疫印迹法(WB)检测肺脏组织中TLR4、NF-κB、TNF-α、IL-1β蛋白水平。结果与对照组比较,模型组小鼠出现呼吸急促、大小便失禁现象,气道组织出现出血、肺泡间隙炎性细胞聚集、肺泡璧增厚及肺泡水肿,BALF中中性粒细胞、巨噬细胞、淋巴细胞、嗜酸性粒细胞及肺组织中TLR4、NF-κB、TNF-α、IL-1βmRNA和蛋白水平均升高(P<0.05);与模型组相比,AZM组、低中高剂量SSd组小鼠,饮食、饮水逐渐恢复,炎性细胞浸润和毛细血管充血、出血程度逐渐减轻,BALF中中性粒细胞、巨噬细胞、淋巴细胞、嗜酸性粒细胞及肺组织中TLR4、NF-κB、TNF-α、IL-1βmRNA和蛋白水平均降低(P<0.05);与AZM组相比,高剂量SSd组差异无统计学意义(P>0.05)。结论SSd可能通过抑制TLR4/NF-κB通路表达,降低哮喘小鼠气道炎症水平,达到改善哮喘的目的,可能作为潜在的治疗哮喘的药物。

Objective To investigate the effects of saikosaponin-d(SSd)on airway inflammation and Toll-like receptor 4(TLR4)/nuclear transcription factor-κB(NF-κB)pathway in mice with asthma.Methods The six of 40 healthy female BALB/c mice were randomly enrolled as control group,and the other 34 mice were sensitized by intraperitoneal injection of 20μg ovalbumin(OVA)and 1mg aluminum hydroxide[Al(OH)3]suspension on the 1st and 13th day,and were given aerosol inhalation of 2%OVA normal saline solution from the 14th to 30th day to establish the animal models with asthma.After successful modeling,the mice were divided into model group,azithromycin(AZM)group,and low,medium and high dose SSd groups,the mice in AZM group and low,medium and high dose SSd groups were given AZM 250mg/kg,SSd 1.0,1.5,2.0mg/kg intraperitoneally,once a day,for 14 days.However the mice in control group and model group were given the same amount of normal saline.All the mice were sacrificed at 2h after the last administration,and bronchoalveolar lavage fluid(BALF)was collected to detect the number of immune cells.The histopathological changes of the right lung were observed by HE staining,and the mRNA levels of TLR4,NF-κB,tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in lung tissue of mice were detected by real-time fluorescence quantitative PCR(qRT-PCR),and the protein levels of TLR4,NF-κB,TNF-αand IL-1βwere detected by Western Blot.Results Compared with those in control group,the mice in the model group had shortness of breath and incontinence,there were hemorrhage,inflammatory cell aggregation,alveolar wall thickening and alveolar edema in airway tissue,the neutrophils,macrophages,lymphocytes and eosinophils in BALF,and the mRNA and protein levels of TLR4,NF-κB,TNF-αand IL-1βin lung tissues were significantly increased(P<0.05).Compared with those in model group,the diet and drinking water of mice in AZM group,low,medium and high dose SSd groups were gradually recovered,the degree of inflammatory cell infiltration,capillary congestion and bleeding as well as the numbers of neutrophils,macrophages,lymphocytes,eosinophils in BALF,and the mRNA and protein levels of TLR4,NF-κB,TNF-αand IL-1βin lung tissues were significantly decreased(P<0.05),however,there were no significant differences between AZM group and high-dose SSd group(P>0.05).Conclusion SSd may reduce the levels of airway inflammation in mice with asthma by inhibiting the expression of TLR4/NF-κB pathway,so as to improve asthma,which may be regarded as a potential drug in treatment of asthma.