γ-氨基丁酸A型受体调节哮喘动物模型气道重构的实验研究

Bicuculline inhibits airway remodeling in a murine model of chronic asthma

目的探讨γ-氨基丁酸A受体(GABAAR)拮抗剂荷包牡丹碱对于小鼠哮喘气道重构模型的干预作用。方法BALB/C小鼠被随机分为4组:空白对照组、哮喘模型组、激素治疗组和荷包牡丹碱干预组。除空白对照组外其他各组小鼠在第1、7、14天通过腹腔注射10μg卵清蛋白(OVA)和100μgAl(OH)3的PBS液致敏,从实验的第15～81天小鼠每天雾化吸入5%OVA溶液1h。从第15天开始,激素治疗组每天雾化吸入0.02%布地奈德溶液0.5h,荷包牡丹碱组每天鼻腔滴入20%荷包牡丹碱悬浊液50μl。于最后一次激发后24h放血处死小鼠,检测BALF中细胞总数、嗜酸性粒细胞和淋巴细胞,肺组织标本切片PAS染色检测粘蛋白糖原,Masson染色检测上皮下胶原沉积面积,免疫组织化学方法检测GABAARβ2和血管内皮生长因子(VEGF)的表达水平。结果(1)荷包牡丹碱干预组BALF中细胞总数、嗜酸性粒细胞和淋巴细胞数分别为(20.50±2.40)×104、(8.30±0.23)×104和(4.20±0.05)×104,与对照组[(2.43±2.10)×104、(0.00±0.00)×104和(0.90±0.05)×104]和激素干预组[(5.50±1.90)×104、(3.60±0.25)×104和(1.90±0.03)×104]比较差异有统计学意义(均为P<0.01),与哮喘模型组[(20.40±1.80)×104、(8.80±0.14)×104和(4.20±0.10)×104]比较差异无统计学意义(P>0.05)。(2)荷包牡丹碱干预组气道基底膜胶原沉积面积为(0.34±0.17)μm2/μm,与对照组[(0.27±0.02)μm2/μm]和激素干预组[(0.30±0.06)μm2/μm]比较差异无统计学意义(均为P>0.05),与哮喘模型组[(0.72±0.08)μm2/μm]比较差异有统计学意义(P<0.05)。(3)荷包牡丹碱干预组PAS阳性细胞百分数为(19.00±2.08)%,与对照组[(0.00±0.0)%]、激素干预组[(30.00±1.66)%]和哮喘模型组[(58.00±1.57)%]比较差异有统计学意义(均为P<0.05)。(4)荷包牡丹碱干预组GABAA受体β2-亚基表达阳性率为(11.30±2.7)%,与对照组[(2.00±0.7)%]和激素干预组[(7.3±1.58)%]比较差异有统计学意义(P<0.01和P<0.05),与哮喘模型组[(12.50±0.31)%]比较差异无统计学意义(P>0.05)。(5)荷包牡丹碱干预组肺血管周围细胞VEGF表达阳性率为(10.60±1.20)%,与对照组[(1.70±0.34)%]和激素干预组[(8.30±0.78)%]比较差异有统计学意义(P<0.01和P<0.05),与哮喘模型组[(18.30±0.80)%]比较差异无统计学意义(P>0.05)。结论气道上皮细胞和气道平滑肌细胞广泛表达GABAARβ2,荷包牡丹碱吸入可以有效抑制粘液分泌和上皮下胶原沉积,其抑制粘液分泌的作用强于吸入激素,但不能减轻气道炎症水平,也不能抑制GABAARβ2和VEGF的表达。

Objective To investigate the effect of bicuculline,a selective GABAA receptor antagonist,on airway remodeling in the murine model of chronic allergen-induced asthma.Methods Forty BALB/C mice were randomized into 4 groups,namely the control group,asthmatic model （induced by ovalbumin sensitization and challenge） group,budesonide inhalation group and bicuculline inhalation group.The mice were sacrificed 24 h after the last ovalbumin inhalation,and the lungs were lavaged with PBS and the total cells,eosinophils and lymphocytes counts were examined.Periodic acid-Schiff （PAS） staining was used for counting mucin-positive goblet cells in the lung tissue,and Masson Trichrome staining was used to evaluate collagen deposition.GABAARβ2 and VEGF were quantified by immunohistochemistry.Results The numbers of the total cells,eosinophils and lymphocytes counts in BALF were significantly greater in the bicuculline group than in the control and budesonide groups （P0.01）,but comparable to those in the asthmatic model group （P0.05）.The airway collagen deposition in the bicuculline group was comparable to that in the control and budesonide group （P0.05）,but was significantly less than that in the asthmatic model group （P0.05）.Significant differences were found in the airway histological mucus index between the bicuculline group and the other 3 groups （P0.05）.The airway GABAARβ2-positive cell percentage in the bicuculline group was significantly greater that those in the control and budesonide （P0.01 and 0.05）,but similar with that in the asthmatic model group （P0.05）.The percentage of pulmonary perivascular VEGF-positive cells in the bicuculline group was significantly greater in the control and budesonide groups （P0.01 and P0.05）,but comparable to that in the asthmatic model group （P0.05）.Conclusion GABAARβ2 is expressed in both the airway epithelium and smooth muscles.Bicuculline inhalation can effectively suppress collagen deposition with a stronger inhibitory effect on mucus hypersecretion than budesonide.