摘要目的 探讨细颗粒物(PM 2.5)对哮喘小鼠呼吸道炎症的影响及和厚朴酚的干预作用.方法 将50只雌性无特殊病原菌(SPF)级BALB/c小鼠按随机数字表法分为5组,每组10只.A组:正常对照组;B组:哮喘模型组;C组:PM 25低剂量暴露哮喘组;D组:PM 2.5高剂量暴露哮喘组;E组:和厚朴酚组.采用卵清蛋白(OVA)致敏并激发建立哮喘小鼠模型,B～E组第0天和第7天经小鼠腹腔注射100 mg/L OVA和氢氧化铝混合液0.1 mL致敏,第14天至第21天,以10 g/L OVA 9 g/L盐水溶液雾化30 min激发;C组及D组在OVA造模同时,分别于第1天、第8天、第15天及第21天对小鼠分别予低剂量(1 mg/kg)及高剂量(15 mg/kg) PM 2.5气管注入,E组在D组造模的基础上予8μg/kg和厚朴酚连续灌胃,A组小鼠以9 g/L盐水代替OVA同步进行实验.末次激发24h后麻醉并解剖小鼠,左肺进行肺泡灌洗留取支气管肺泡灌洗液(BALF)进行炎性细胞总数及分类计数;取右肺行HE染色、病理学检查;实时荧光定量聚合酶链反应(qPCR)分析技术检测小鼠外周血单个核细胞(PBMCs)中Toll样受体4(TLR4)和核因子κB(NF-кB) mRNA的表达及流式细胞仪检测Th17和Treg细胞的表达.结果 与A组比较,B、C、D组在支气管肺组织病理学上可见到支气管黏膜上皮细胞变形、脱落,基底膜增厚及管腔内分泌物增多,大量炎性细胞浸润,D组较C组更为明显,而E组小鼠呼吸道炎症明显减轻;C组及D组BALF中炎性细胞总数分别为(20.28±11.16)×108/L和(27.38±14.64)×108/L,嗜酸性粒细胞比例为0.177 8±0.064 9和0.229 1±0.098 7,与E组[(8.56±3.28)×108/L、0.041 5±0.013 5]比较均明显增高,差异均有统计学意义(均P＜0.01);C组及D组PBMCs中TLR4 mRNA和NF-κB mRNA的表达分别为2.56±0.49、3.21±0.61和2.42±0.30、2.83±0.32,与E组(1.60±0.28、1.54±0.25)比较均显著增高,差异均有统计学意义(均P＜0.05),且D组较C组增高明显,差异有统计学意义(P＜0.05);C组及D组PBMCs中Th17细胞的表达分别为0.043 9±0.008 9、0.052 2 ±0.011 8,与E组(0.018 3±0.002 3)比较均显著增高,差异均有统计学意义(均P＜0.05),C组及D组PBMCs中Treg细胞的表达分别为0.038 2±0.004 2、0.022 7±0.003 3,与E组(0.0645±0.003 8)比较均显著降低,E组情况较C组及D组明显改善.结论 PM 25暴露可导致哮喘小鼠呼吸道炎症加重,且高剂量PM 2.5暴露对其呼吸道的损伤更为明显,和厚朴酚能减轻PM 2.5暴露对哮喘小鼠呼吸道炎症作用,其作用机制与下调TLR4、NF-κB表达和调节Th17及Treg细胞平衡有关.

abstractsObjective To explore the effect of particulate matter (PM 2.5) on airway inflammation in asthmatic mice and the intervention effect of Honokiol.Methods Fifty female BALB/c mice were divided into 5 groups according random number table,group A:normal control group;group B:asthma model group;group C:PM 2.5 low dose exposure asthma group;group D:PM 2.5 high dose exposure asthma group:group E:Honokiol group.Asthmatic mouse models were established by ovalbumin(OVA) sensitization and challenge.On day 0 and 7,B-E groups were intraperitoneally with injection 100 mg/L OVA and Al (OH)3 for sensitization;on day 14 to 21,10 g/L OVA solution was given 30 min per day to challenge.During challenge phrase,C-D groups were received different doze intratracheal injection of PM 2.5 respectively,every 7 days,total 4 times.On this basis,the mice in group E received Honokiol intragastfic administration.The mice in group A were carried out by using saline instead of OVA.Mice were sacrificed 24 h after the final inhalation challenge,and for the recovered bronchoalveolarlavage fluid(BALF) of the left lung was used for differential inflammatory cell counts,HE staining and pathological examination were performed on the right lung.The expression of Toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB at mRNA level were detected by real-time flurescence quantitative polymerase chain reaction (qPCR).Flow cytometry analysis was performed to measure the levels of Th17 and Treg cells.Results Compared with group A,mice in group B,group C and group D expressed more serious disorsers of bronchial epithelial cells,alveolar wall congestion and edema,increased mucus secretion in the airway and infiltration of inflammatory cells in lung,and those in group D was more obvious than those in group C and group E,significantly reduced respiratory inflammation compared with group E[(8.56 ± 3.28) × 108/L,0.041 5 ± 0.013 5],the total number of inflammatory cell counts in group C and group D were (20.28 ± 11.16) × 108/L and (27.38 ± 14.64) × 108/L,eosinophils proportion were 0.177 8 ±0.064 9 and 0.229 1 ±0.098 7,there were statistically significant differences(all P ＜ 0.05);compared with group E (1.60 ± 0.28,1.54 ± 0.25),the expression of TLR4 mRNA and NF-κB mRNA in group C and group D (2.56 ± 0.49,3.21 ± 0.61;2.42 ± 0.30,2.83 ± 0.32) were significantly higher,and there were statistically significant differences(all P ＜0.05),group D was more higher than those in group C (all P ＜ 0.05);compared with group E(0.018 3 ± 0.002 3),the expression of Th17 in group C and group D (0.043 9 ±0.008 9 and 0.052 2 ±0.011 8) were significantly higher,and there were statistically significant differences(all P ＜0.05);compared with group E(0.064 5 ±0.003 8),the expression of Treg in group C and group D (0.038 2 ± 0.004 2) and (0.022 7 ± 0.003 3) were significantly lower,and there were statistically significant differences(all P ＜ 0.05);and those of group E were improved remarkably.Conclusion PM 2.5 exposure can aggravate airway inflammation in asthmatic mice,and the damage to airway is more obvious when exposed to high dose of PM 2.5,Honokiol can relieve PM 2.5 exposure of asthmatic airway inflammation through down regulation the expression of TLR4 and NF-κB and Th17 and regulating the balance of Th17 and Treg cells.