孤儿核受体Nur77调控气道平滑肌增殖参与哮喘气道重塑的作用及机制

Increased proliferation of airway smooth muscle cells plays a pivotal role in asthmatic airway remodeling，but its regulatory mechanism, especially the key transcription factors has not been elucidated. Orphan nuclear receptor Nur77 is known as a transcription factor for the regulation of cell proliferation, apoptosis and differentiation. Its role in asthmatic airway smooth muscle remodeling remains unclear. Our previous study found that asthma inflammation medium can promote the expression of Nur77 in airway smooth muscle cells, and silence its expression can significantly suppress the airway smooth muscle cell proliferation induced by PDGF which means Nur77 play an important role in airway remodeling in asthma.In the present study, a house dust mite induced chronic asthma mice will be used to detect the expression of Nur77 in the airway smooth muscle. Then wildtype and Nur77-/- mice will be used to evaluate the effects of Nur77 on airway smooth muscle remodeling in the HDM-induced mice by detecting airway resistance, airway smooth muscle mass and other asthmatic markers. Primary airway smooth muscle cells seperated from WT or Nur77-/- mice will be used to assess the role of Nur77 on the HDM sentisized T lymphocytes induced proliferation of airway smooth muscle cells. Based on these, the mechanisms of how Nur77 was regulated by methylation, how the expression and location of Nur77 were affected by PI3K/Akt and MAPK/ERK1/2 signal pathway, and the potential target genes regulated by Nur77 will be detected using realtime PCR, western blot, CHIP, EMSA, et al. This study will provide a new insight into the prevention and treatment of asthma.

气道平滑肌细胞持续增殖是引起哮喘气道结构重塑的关键环节，但其调控机制尚未阐明。孤儿核受体Nur77是一种重要的核转录因子，可以在炎症等因素刺激下早期快速表达入核，参与细胞增殖。我们前期研究发现哮喘相关炎症因子可促进气道平滑肌细胞表达Nur77，而下调其表达可显著抑制PDGF诱导的平滑肌细胞增殖，推测Nur77可能在哮喘气道重塑中发挥重要作用。本研究拟采用Western blot、免疫组化等检测慢性哮喘模型气道Nur77表达情况；比较模型组Nur77-/-小鼠气道阻力及气道平滑肌面积变化，评价Nur77对哮喘气道结构重塑的作用；然后观察调控Nur77表达对原代支气管平滑肌细胞增殖的影响；并进一步通过启动子甲基化分析明确Nur77基因表达异常机制，PI3K、ERK1/2信号通路调控Nur77活化入核机制，生物信息学预测及验证Nur77下游的关键靶基因，为制定哮喘诊治新策略提供靶点和参考。

目的：研究Nur77调控气道平滑肌增殖参与哮喘气道重塑的作用及机制。方法：制备哮喘模型，检测BALF中Th1/Th2细胞因子水平， Masson trichrome染色观察细胞外基质和纤维化情况，SMA染色观察平滑肌厚度。Rt-PCR检测IGFBP-3、Nur77和miR-34/449转录情况，Western blot检测IGFBP-3、Nur77蛋白表达变化。Western blot检测bFGF、collagen I、collagen III、α-SMA水平，检测自噬相关蛋白LC3-II/LC3-I 、Beclin-1和P62表达情况，同时检测c-JNK磷酸化和Akt磷酸化状态，免疫荧光检测IGFBP-3和Nur77的分布，免疫共沉淀检测IGFBP-3和Nur77的相互作用，MTT法检测细胞增殖变化。荧光素报告实验检测IGFBP-3和miR-34/449之间的相互作用，体外添加IGFBP-3及下调Nur77后检测对Nur77的核输出和细胞增殖的影响。采集哮喘及正常人外周血，检测miR--34/449水平及自噬相关蛋白表达水平。结果：哮喘组小鼠肺组织炎症细胞浸润明显，以EOS为主，BALF中IL-5、IL-6、TNF-α水平、肺组织TGF-β、bFGF、collagen I、 collagen III、α-SMA水平升高，同时自噬相关蛋白LC3-II/LC3-I、Beclin-1的表达升高。哮喘组小鼠肺组织miR34/449表达低于正常组，miR-34/449 mimics治疗组miR-34/449表达水平较正常组及哮喘组明显升高 (P＜0.05)。miR-34/449 mimics治疗组与哮喘组相比，小鼠肺组织炎症细胞浸润明显减轻，BALF中IL-5、IL-6、TNF-α、肺组织TGF-β、bFGF、collagen I、collagen III、α-SMA水平下降，同时自噬相关蛋白LC3-II/LC3-I、Beclin-1表达下降 (P＜0.05)。IGFBP-3可影响Nur77亚细胞定位，IGFBP-3表达上调时促进Nur77出核，Nur77可与Bcl-2相互作用。Beclin-1与Bcl-2结合后，抑制下游自噬发生。Nur77表达增高，竞争性与Bcl-2结合，解除了对Bcl-2的束缚，进而促进了Bcl-2对beclin-1的抑制，最终抑制了自噬的发生。

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