3-甲基腺嘌呤对支气管哮喘小鼠气道炎症及气道高反应和黏液分泌的影响

Effects of 3-methyladenine on airway inflammation,airway hyperresponsiveness and mucus secretion in asthmatic mice

目的探讨3-甲基腺嘌呤对支气管哮喘(哮喘)小鼠气道炎症、气道高反应和黏液分泌的影响及其作用机制。方法采用随机数字表法将24只C57BL/6J雌性小鼠随机分为健康对照组(PBS组)、哮喘组(OVA组)、哮喘+3-甲基腺嘌呤组(OVA+3-MA组)、哮喘+4-苯基丁酸钠组(OVA+4-PBA组),每组6只。OVA组、OVA+3-MA组和OVA+4-PBA组均以卵清白蛋白(OVA)致敏、激发构建哮喘模型,PBS组则用PBS溶液致敏并激发作为对照处理;在激发前2 h,OVA+3-MA组给予3-甲基腺嘌呤腹腔注射,OVA+4-PBA组给予4-苯基丁酸钠腹腔注射。通过气道高反应性Penh值、BALF嗜酸粒细胞和肺组织的病理变化,确定哮喘模型的建立,观察各组小鼠肺组织胶原沉积情况和黏液分泌水平,检测肺组织中LC3B、Beclin1、Muc5ac、Atf6、Chop和Bip等蛋白含量的变化。结果OVA组Penh值和BALF中嗜酸粒细胞明显高于PBS组(均P<0.05);OVA+3-MA组和OVA+4-PBA组在乙酰甲胆碱浓度为6.25、12.50、25.00和50.00 g/L激发时,其Penh值显著低于OVA组(均P<0.05),BALF中嗜酸粒细胞也明显低于OVA组(P<0.05)。肺组织病理检查可见OVA组小鼠气管和肺血管周围表现出明显的炎症细胞浸润,肺泡间隔增厚、结构破坏,平滑肌层增厚,胶原沉积和杯状细胞增生;OVA+3-MA组和OVA+4-PBA组小鼠气管和肺血管周炎症细胞浸润、肺泡间隔增厚、结构破坏,胶原沉积和杯状细胞增生均较OVA组明显减少;PBS组未见明显病理损害、胶原沉积和杯状细胞增生。Western blot检测可见OVA组LC3Ⅱ/Ⅰ、Beclin1、Muc5ac、Atf6、Chop和Bip等蛋白表达量(1.09±0.04 vs 0.34±0.09,0.18±0.01 vs 0.06±0.01,1.90±0.38 vs 0.46±0.11,1.67±0.18 vs 0.41±0.08,2.96±0.45 vs 1.11±0.10,2.07±0.34 vs 0.49±0.17)较PBS组均明显高(P<0.05);OVA+3-MA组和OVA+4-PBA组LC3Ⅱ/Ⅰ、Beclin1、Muc5ac、Atf6、Chop、Bip等蛋白表达量(0.46±0.07 vs 1.09±0.04,0.63±0.03 vs 1.09±0.04;0.11±0.02 vs 0.18±0.01,0.12±0.02 vs 0.18±0.01;0.72±0.22 vs 1.90±0.38,0.57±0.13 vs 1.90±0.38;1.06±0.12 vs 1.67±0.18,1.02±0.12 vs 1.67±0.18;1.67±0.21 vs 2.96±0.45,1.10±0.15 vs 2.96±0.45;1.03±0.11 vs 2.07±0.34,0.97±0.10 vs 2.07±0.34)较OVA组均明显降低(P<0.05)。结论3-MA可以抑制哮喘小鼠的气道炎症,减轻气道高反应和黏液分泌,其机制可能与抑制细胞自噬,从而抑制内质网应激反应有关。

ObjectiveTo investigate the effects of 3-methyladenine on airway inflammation,airway hyperresponsiveness and mucus secretion in asthmatic mice,and to explore its mechanism.MethodsC57BL/6J female mice were randomly divided into normal control group(PBS),OVA group(OVA),OVA with 3-methyladenine group(OVA+3-MA),and OVA with 4-phenylbutyrate group(OVA+4-PBA).OVA group,OVA+3-MA group and OVA+4-PBA groups were all sensitized and challenged with OVA to establish asthmatic models,while PBS group was given PBS as a control.At 2 h before challenge,OVA+3-MA group was intraperitoneally injected with 3-methyladenine,and OVA+4-PBA group was intraperitoneally injected with 4-phenylbutyrate.Airway hyperresponsiveness,eosinophils,and pathological changes of pulmonary tissue(hematoxylin-eosin,HE staining)were measured to confirm the establishment of asthmatic models.Sections of pulmonary tissue were also stained with Masson and PAS.The expression level of LC3B was measured by immunofluorescence and Western blot.The Beclin1,Muc5ac,Atf6,Chop and Bip proteins in lung tissues were detected by Western blot.ResultsThe Penh value,and eosinophils in BALF in OVA group was significantly increased compared with PBS group(P<0.05).The Penh value in OVA+3-MA group and OVA+4-PBA group were significantly decreased compared with the OVA group at the concentration of 6.25 g/L,12.50 g/L,25.00 g/L,and 50.00 g/L of methacholine(all the P<0.05),and the eosinophils were also significantly decreased compared with the OVA group(P<0.05).Pulmonary histology revealed that OVA group showed high levels of inflammatory cell infiltration of bronchi and lung vessels,alveolar septal thickening,structural destruction,smooth muscle thickening,collagen deposition,and goblet cell hyperplasia.The levels of inflammatory cell infiltration of bronchi and lung vessels,alveolar septal thickening,structural destruction,smooth muscle thickening,collagen deposition,and goblet cell hyperplasia in OVA+3-MA group and OVA+4-PBA group were significantly lower than the OVA group,while the PBS group was normal.Compared with PBS group,the expression of LC3Ⅱ/Ⅰ,Beclin1,Muc5ac,Atf6,Chop and Bip proteins in lung tissues in the OVA group were significantly increased(1.09±0.04 vs 0.34±0.09,P<0.05;0.18±0.01 vs 0.06±0.01,P<0.05;1.90±0.38 vs 0.46±0.11,P<0.05;1.67±0.18 vs 0.41±0.08,P<0.05;2.96±0.45 vs 1.11±0.10,P<0.05;2.07±0.34 vs 0.49±0.17,P<0.05,respectively).Compared with the OVA group the expression of LC3Ⅱ/Ⅰ,Beclin1,Muc5ac,Atf6,Chop and Bip proteins in lung tissues in the OVA+3-MA group and OVA+4-PBA group were significantly decreased(0.46±0.07 vs 1.09±0.04,0.63±0.03 vs 1.09±0.04,both P<0.05;0.11±0.02 vs 0.18±0.01,0.12±0.02 vs 0.18±0.01,both P<0.05;0.72±0.22 vs 1.90±0.38,0.57±0.13 vs 1.90±0.38,both P<0.05;1.06±0.12 vs 1.67±0.18,1.02±0.12 vs 1.67±0.18,both P<0.05;1.67±0.21 vs 2.96±0.45,1.10±0.15 vs 2.96±0.45,both P<0.05;1.03±0.11 vs 2.07±0.34,0.97±0.10 vs 2.07±0.34,both P<0.05).Conclusion3-MA was shown to inhibit airway inflammation,airway hyperresponsiveness and mucus secretion in mice with bronchial asthma,and the mechanism may be related to inhibiting autophagy,and then inhibiting endoplasmic reticulum stress.