和厚朴酚对PM2.5暴露哮喘小鼠呼吸道炎症的影响及其机制

摘要

Objective To investigate the protective effect of Honokiol on the airway inflammation induced by particulate matter 2.5(PM2.5)in the asthmatic mice and its mechanism.Methods Fifty male specific pathogen free (SPF)Balb/c mice were randomly divided into 5 groups.Group A:normal control group;group B:asthmatic model group;group C:PM2.5 exposure asthmatic group;group D:TAK -242 group;group E:Honokiol group. Asthmatic mouse models were established by ovalbumin(OVA)sensitization and challenge.On days 0 and 7,the mice in B-E groups were injected intraperitoneally with injection 100 mg/L OVA and aluminum hydroxide for sensitization;on days 14 to 21,10 g/L OVA solution was given 30 min per day to challenge.During challenge phrase,the mice in C -E groups received intratracheal injection of PM2.5,every other day,4 times totally.On this basis,the mice in group D re-ceived TAK-242 intraperitoneal injection,and the mice in group E received honokiol intragastric administration.Group A was given saline instead of OVA.Animals were sacrificed 24 h after the final inhalation challenge,and the bronchoal-veolar lavage fluid(BALF)of the left lung was used for differential inflammatory cell counts.The expressions of Toll-like receptors 4(TLR4)and nuclear factor(NF)-κB at mRNA level were detected by real-time quantitative PCR. Flow cytometry analysis was performed to measure the levels of Th17 and Treg cells.Results Compared with group A,mice in group B and group C expressed more serious disorders of bronchial epithelial cells,alveolar wall congestion and edema,increased mucus secretion in the airway and infiltration of inflammatory cells in the lung,and those in group C were more obvious than those of group B and group E significantly reduced respiratory inflammation;compared with group A[(4.15 ± 1.35)×108/L,0.012 0 ± 0.002 3],the total number of inflammatory cell counts[(16.79 ± 5.62)×108/L and(24.58 ± 13.46)×108/L],eosinophils proportions(0.113 8 ± 0.022 3 and 0.197 8 ± 0.084 9)in group B and group C,were significantly higher,and the differences were statistically significant(all P<0.05);The total number of inflammatory cell counts and eosinophils proportion in group E(8.56 ± 3.28)×108/L and 0.041 5 ± 0.013 5)were significantly lower than those in group C,and the differences were statistically significant(all P <0.05);The expressions of TLR4 mRNA and NF-κB mRNA in group B and C(1.85 ± 0.56,1.82 ± 0.28 and 2.97 ± 0.41,2.83 ± 0.32)were significantly higher,and the differences were statistically significant(all P <0.05);The expressions of TLR4 mRNA and NF-κB mRNA in group E(1.60 ± 0.28,1.54 ± 0.25)was significantly lower than those in group C,and the differences were statistically significant(all P<0.05);the expressions of Th17 in group B and C[(2.89 ± 0.61)% and(4.96 ± 0.27)%]were significantly higher than those of group A[(1.03 ± 0.35)%] (all P<0.05);The expression of Th17 in group E[(1.83 ± 0.23)%]was significantly lower than that of group C,and the differences were statistically significant(P<0.05);the expressions of Treg in group B and C[(4.96 ± 0.35)%and(2.27 ± 0.41)%]were significantly lower than those of group A[(7.37 ± 0.56)%],and the differences were sta-tistically significant(all P<0.05);The expression of Treg in group E was significantly increased[(6.45 ± 0.38)%] compared with that in group C,and the difference were statistically significant(P<0.05);and those of group D and E were improved remarkably.Conclusions Honokiol can relieve PM2.5 exposure of asthmatic airway inflammation through down-regulating the expression of TLR4 and NF-κB and Th17 and regulating the balance of Th17 and Treg cells.%目的 探讨和厚朴酚对细颗粒物(PM2.5)所致哮喘小鼠呼吸道炎症是否具有保护作用及其作用途径.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:PM2.5暴露哮喘组;D组:TAK-242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~E组从首次雾化第1天开始到末次激发气管注入PM2.5激发,隔日1次,共4次;在此基础之上,D组给予TAK-242腹腔注射处理,E组给予和厚朴酚灌胃处理.A组小鼠,以9 g/L盐水代替 OVA同步进行实验.末次激发24 h后麻醉并解剖小鼠,左肺进行肺泡灌洗留取肺泡灌洗液(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PCR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-κB)mRNA的表达,流式细胞仪检测Th17和 Treg细胞的表达.结果 与A组比较,B组及C组小鼠支气管黏膜上皮细胞变形、脱落,上皮增生变厚,肺泡壁充血水肿,管腔内黏液分泌增加,肺部炎性细胞浸润,C组较B组更为明显,而E组小鼠呼吸道炎症明显减轻;B组及C组BALF中炎性细胞总数分别为(16.79 ± 5.62)× 108/L和(24.58 ± 13.46)×108/L,嗜酸性粒细胞比例为0.1138 ± 0.0223和0.1978 ± 0.0849,均显著高于A组[(4.15 ± 1.35)×108/L、0.0120 ± 0.0023],差异均有统计学意义(均P<0.05);E组BALF中炎性细胞总数及嗜酸性粒细胞比例[(8.56 ± 3.28)×108/L)、0.0415 ± 0.0135]与C组比较差异均有统计学意义(均P<0.05);B组及C组PBMCs中TLR4 mRNA和NF-κB mRNA的表达分别为1.85 ± 0.56、1.82 ± 0.28和2.97 ± 0.41、2.83 ± 0.32,均显著高于 A组(1.06 ± 0.19、1.08 ± 0.17),差异均有统计学意义(均 P <0.05);E组PBMCs中TLR4 mRNA和NF-κB mRNA的表达(1.60 ± 0.28、1.54 ± 0.25)与C组比较差异均有统计学意义(均P<0.05);B组及C组PBMCs中Th17细胞的表达水平分别为(2.89 ± 0.61)%、(4.96 ± 0.27)%,均显著高于A组[(1.03 ± 0.35)%],差异均有统计学意义(均 P <0.05);E组 PBMCs中 Th17细胞的表达[(1.83 ± 0.23)%]明显低于C组,差异有统计学意义(P<0.05);B组及C组PBMCs中Treg细胞的表达分别为(4.96 ± 0.35)%、(2.27 ± 0.41)%,均显著低于 A组[(7.37 ± 0.56)%],差异均有统计学意义(均 P <0.05);E组PBMCs中Treg细胞的表达[(6.45 ± 0.38)%]明显高于C组,差异有统计学意义(P<0.05);D、E组情况较B组及C组明显改善.结论 和厚朴酚能减轻PM2.5暴露哮喘小鼠的呼吸道炎症,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.