基于NCOA4介导的铁自噬探讨针刺对哮喘小鼠气道炎症的影响

Exploration on the effect of acupuncture on airway inflammation in asthmatic mice based on ferritinophagy mediated by NCOA4

关键词:  哮喘  针刺  铁死亡  铁自噬  核受体共激活因子4

Key words:  asthma  acupuncture  ferroptosis  ferritinophagy  NCOA4

摘要:

[目的] 观察针刺通过核受体共激活因子4（NCOA4）介导的铁自噬通路改善哮喘气道炎症的可能机制。[方法] 将48只小鼠随机分为空白组、模型组、对照组、针刺组，每组12只。采用卵清蛋白致敏/激发法建立哮喘模型。苏木精-伊红（HE）染色观察小鼠气道炎症评分和病理变化；免疫磁珠法分选嗜酸性粒细胞，检测小鼠肺泡灌洗液（BALF）中白细胞介素-4（IL-4）、白细胞介素-13（IL-13）水平；测定BALF中嗜酸粒细胞亚铁离子（Fe²⁺）、还原型谷胱甘肽（GSH）、丙二醛（MDA）含量和活性氧（ROS）水平；蛋白免疫印迹（Western blot）法检测NCOA4、抗铁蛋白重链1（FTH1）、谷胱甘肽过氧化物酶4（GPX4）蛋白表达水平；反转录聚合酶链反应（RT-PCR）检测NCOA4、FTH1、GPX4 mRNA表达水平。[结果] 与空白组比较，模型组小鼠肺组织破坏明显，气道炎症评分升高，BALF中IL-4、IL- 13含量升高（P < 0.01），Fe²⁺、MDA含量和ROS水平降低，GSH含量升高（P < 0.01），NCOA4蛋白和mRNA表达降低，FTH1、GPX4蛋白和mRNA表达升高（P < 0.01）。与模型组比较，对照组小鼠病理损伤有所改善，气道炎症评分降低，BALF中IL-4、IL-13含量降低（P < 0.01），Fe²⁺、MDA含量和ROS水平升高，GSH含量降低（P < 0.01），NCOA4蛋白和mRNA表达升高，FTH1、GPX4蛋白和mRNA表达降低（P < 0.01）。与对照组比较，针刺组小鼠病理损伤明显减轻，气道炎症评分下降，BALF中IL-4、IL-13含量降低（P < 0.01），Fe²⁺、MDA含量和ROS水平升高，GSH含量降低（P < 0.01），NCOA4蛋白和mRNA表达升高，FTH1、GPX4蛋白和mRNA表达降低（P < 0.01）。[结论] 针刺调控NCOA4-FTH1通路介导的铁自噬，诱导嗜酸性粒细胞（EOS）铁死亡，改善哮喘气道炎症。

Abstract:

[Objective]To observe the possible mechanism by which acupuncture improves airway inflammation in asthma through the ferritinophagy pathway mediated by nuclear receptor coactivator 4(NCOA4).[Methods]Forty-eight mice were randomly divided into the blank group, the model group, the control group and the acupuncture group, with 12 mice in each group.The asthma model was established by ovalbumin sensitization / activation method. The airway inflammation score and patho - logical changes of mice were observed by HE staining; sorting of eosinophils by immunomagnetic bead method, to detect the levels of interleukin - 4(IL - 4)and interleukin-13(IL-13)in the alveolar lavage fluid(BALF)of mice; determine the contents of Fe²⁺, glutathione(GSH), malondial- dehyde(MDA)and reactive oxygen species(ROS)levels in eosinophil; the protein expression levels of NCOA4, antiferritin heavy chain 1(FTH1), and glutathione peroxidase 4(GPX4)were detected by Western blotting; the expression levels of NCOA4, FTH1 and GPX4 mRNA were detected by RT-PCR.[Results]Compared with the blank group, the lung tissue of mice in the model group was significantly damaged; the airway inflammation score increased; the contents of IL - 4 and IL - 13 in BALF increased(P < 0.01);the contents of Fe²⁺, MDA and the level of ROS decreased; the content of GSH increased(P < 0.01);the expressions of NCOA4 proteins and mRNA decreased; the expressions of FTH1, GPX4 protein and mRNA increased(P < 0.01). Compared with the model group, the pathological injury of mice in the control group was improved, the airway inflammation score was decreased; the contents of IL-4 and IL-13 in BALF were decreased(P < 0.01);the contents of Fe²⁺, MDA and ROS levels were increased; the content of GSH was decreased (P < 0.01);the expressions of NCOA4 proteins and mRNA were increased; the expressions of FTH1, GPX4 protein and mRNA decreased(P < 0.01). Compared with the control group, the pathological injury of mice in the acupuncture group was significantly alleviated; the airway inflammation score decreased; the contents of IL-4 and IL-13 in BALF decreased(P < 0.01);the contents of Fe²⁺, MDA and the level of ROS increased, the content of GSH decreased(P < 0.01);the expressions of NCOA4 proteins and mRNA increased; the expressions of FTH1, GPX4 protein and mRNA decreased(P < 0.01).[Conclusion]Acupuncture regulates ferritinophagy mediated by the NCOA4 -FTH1 pathway, induces ferroptosis of eosinophils and improves airway inflammation in asthma.