特异性组蛋白去乙酰化酶6抑制剂调控HSP90乙酰化治疗支气管哮喘小鼠气道炎症

Specific histone deacetylase 6 inhibitor regulates heat shock protein 90 acetylation to treat airway inflammation in mice with bronchial asthma

目的探讨特异性组蛋白去乙酰化酶6(HDAC6)抑制剂Tubastatin A Hcl对支气管哮喘(哮喘)小鼠气道炎症的作用及Tubastatin A Hcl调控HDAC6/热休克蛋白90(HSP90)/κB抑制因子激酶(IKK)/核因子κB(NF-κB)信号通路治疗哮喘小鼠气道炎症的分子机制。方法本研究为实验性研究。6~8周SPF级雌性BALB/C小鼠随机分为4组:正常组、哮喘组、地塞米松组、Tubastatin A Hcl组,每组6只。哮喘小鼠模型的构建使用卵清蛋白致敏和卵清蛋白激发的方式。测定各组小鼠气道阻力以评估气道反应性。采用HE、AB-PAS和Masson染色观察各组小鼠气道炎症细胞浸润、黏液分泌、气道上皮杯状细胞增生以及气道周围胶原沉积情况。免疫组织化学染色检测各组小鼠肺组织中磷酸化IKK和磷酸化NF-κB的表达分布。蛋白质印迹法检测各组小鼠肺组织中HDAC6、磷酸化IKK、磷酸化NF-κB、IKK和NF-κB的表达水平。免疫沉淀技术检测各组小鼠肺组织中HSP90乙酰化水平变化情况。结果(1)哮喘小鼠模型构建及气道炎症水平检测:与正常组相比,哮喘组小鼠气道高反应性增高;与哮喘组相比,地塞米松组和Tubastatin A Hcl组小鼠气道高反应性降低;HE、AB-PAS和Masson染色结果显示,与正常组相比,哮喘组小鼠气道周围出现大量炎性细胞浸润,气道内可见黏液分泌,气道上皮杯状细胞增生明显,上皮下胶原纤维沉积增加。与哮喘组相比,地塞米松组和Tubastatin A Hcl组小鼠气道炎症、气道上皮杯状细胞增生以及气道周围胶原沉积水平降低。(2)小鼠肺组织中HDAC6表达水平及HSP90乙酰化水平:与正常组相比,哮喘组小鼠肺组织中HDAC6表达水平升高;Tubastatin A Hcl干预后小鼠肺组织中HDAC6表达水平降低。与正常组相比,哮喘组小鼠肺组织中HSP90乙酰化水平降低;Tubastatin A Hcl干预后小鼠肺组织中HSP90乙酰化水平升高。(3)小鼠肺组织中IKK和NF-κB磷酸化水平:与正常组相比,哮喘组小鼠肺组织中IKK和NF-κB磷酸化水平升高;Tubastatin A Hcl干预后小鼠肺组织中IKK和NF-κB磷酸化水平均降低。结论特异性HDAC6抑制剂Tubastatin A Hcl能够有效缓解哮喘小鼠的气道炎症,其机制可能与Tubastatin A Hcl上调HSP90乙酰化水平,进而抑制IKK/NF-κB信号通路有关。

Objective To investigate the effect of specific histone deacetylase 6(HDAC6)inhibitor Tubastatin A Hcl on airway inflammation in asthmatic mice and to explore the molecular mechanism of Tubastatin A Hcl regulating HDAC6/heat shock protein 90(HSP90)/inhibitor of kappa B kinase(IKK)/nuclear factor kappa B(NF-κB)signaling pathway in the treatment of airway inflammation in asthmatic mice.Methods This study was an experimental study.6-8-week SPF female BALB/C mice were randomly divided into 4 groups:normal group,asthma group,dexamethasone group,and Tubastatin A Hcl group,with 6 mice in each group.Ovalbumin(OVA)sensitization and ova challenge were used to establish the mouse model of asthma.Airway resistance was measured to assess airway responsiveness.HE,AB-PAS,and Masson staining were used to observe airway inflammatory cell infiltration,mucus secretion,airway epithelial goblet cell proliferation,and collagen deposition around the airway in each group.Immunohistochemical staining was adopted to detect the expression and distribution of p-IKK and p-NF-κB in lung tissue.Western blot was used to detect the expression levels of HDAC6,p-IKK,p-NF-κB,IKK,and NF-κB in lung tissue of mice in each group.Immunoprecipitation was adopted to detect the levels of HSP90 acetylation in lung tissues.Results(1)Establishment of asthma mouse model and detection of airway inflammation:compared with the normal group,the airway hyperresponsiveness of asthma group mice was significantly increased;compared with the asthma group,the airway hyperresponsiveness of dexamethasone group and Tubastatin A Hcl group was significantly decreased.HE,AB-PAS,and Masson staining showed that compared with the normal group,the asthma group had a large number of inflammatory cells infiltrating around the airway,mucus secretion in the airway,obvious proliferation of airway epithelial goblet cells,and increased deposition of subepithelium collagen fibers.Compared with the asthma group,the levels of airway inflammation,airway epithelial goblet cell proliferation,and collagen deposition around the airway were significantly decreased in the dexamethasone group and Tubastatin A Hcl group.(2)HDAC6 expression level and HSP90 acetylation level in the lung tissue of mice:compared with the normal group,the asthma group showed increased expression level of HDAC6,which was decreased after Tubastatin A Hcl intervention,and decreased acetylation level of HSP90 in the lung tissue,which was increased after Tubastatin A Hcl intervention.(3)IKK and NF-κB phosphorylation levels in the lung tissue of mice:compared with normal group,the asthma group showed increased levels of both IKK and NF-κB phosphorylation in the lung tissue,which were decreased after Tubastatin A Hcl intervention.Conclusions Specific HDAC6 inhibitor Tubastatin A Hcl can effectively alleviate airway inflammation in asthmatic mice,and the mechanism may be related to the up-regulation of HSP90 acetylation level by Tubastatin A Hcl,thereby inhibiting IKK/NF-κB signaling pathway.