PM2.5通过MAPK-NF-κB信号通路抑制气道上皮细胞人β防御素-1表达致细菌侵袭性增加的机制研究

Based on researches, acute and chronic exposure to PM 2.5 are closely related to chronic obstructive pulmonary disease and other diseases. Preliminary experiments showed that after PM 2.5 exposure, the expression of DEFB1, the coding gene of human β-defensin 1 (HBD-1) in airway epithelial cells decreased, the corresponding expression of HBD-1 decreased, and the invasiveness of bacteria increased. MAPK and NF-κB inhibitors can reverse the decrease of HBD-1 expression and the increase of bacterial invasiveness. Based on the above preliminary results, we propose the following scientific hypothesis: PM 2.5 inhibits the expression of HBD-1 in airway epithelial cells via the MAPK-NF-κB signaling pathway, which leads to the decrease of airway mucosal immune function and the increase of bacterial invasiveness. The aim of this study is to clarify the regulatory relationship among MAPK, NF-κB and HBD-1 by using the methods of chromatin immunoprecipitation, double luciferase reporter gene and siRNA interference in PM 2.5 acute exposure and Pseudomonas aeruginosa invasive cell and animal models in order to elucidate the mechanism of HBD-1 expression and bacterial invasiveness under PM 2.5 exposure. This study will provide a new theoretical basis for the pathogenesis and interventions of PM 2.5 exposure-related pulmonary infections, such as COPD exacerbation and pneumonia.

我们及国内外研究发现，PM2.5急慢性暴露与慢性阻塞性肺病等疾病密切相关。预实验发现，PM2.5暴露后气道上皮细胞人β防御素-1(HBD-1)的编码基因DEFB1表达降低，相应的HBD-1多肽表达下降，细菌侵袭性增加。MAPK和NF-κB抑制剂可以逆转这种HBD-1表达的降低并抑制细菌侵袭性增加。基于上述结果，我们提出科学假设：PM2.5通过MAPK-NF-κB信号通路抑制气道上皮细胞HBD-1表达致气道黏膜免疫功能下降，细菌侵袭性增加。本研究拟通过PM2.5急性暴露和铜绿假单胞菌侵袭的细胞及动物模型，利用染色质免疫共沉淀、双荧光素酶报告基因和siRNA干扰等实验方法，明确MAPK、NF-κB和HBD-1之间的调控关系，以阐明PM2.5暴露下HBD-1表达和细菌侵袭性影响机制。本研究将为与PM2.5暴露相关的慢阻肺急性加重和肺炎等肺部感染的发病机制及干预措施提供新的理论依据。

人β防御素(HBD)是一类在黏膜固有免疫中发挥抗感染重要作用的多肽类物质，在人气道上皮中组成性表达。前期研究显示，细颗粒物(PM2.5)暴露下，气道上皮细胞中持续表达的HBD家族尤其是HBD1显著降低。本研究通过建立PM2.5暴露+铜绿假单胞(PA)感染的细胞及动物模型，研究PM2.5暴露是否增加PA的侵袭性，以及HBD1在这一过程中的抗菌作用和表达变化，并进一步采用人支气管上皮样细胞（HBE）模型及生物信息学技术对PM2.5下调HBD1的机制展开研究。结果显示，在PM2.5 浓度100μg/ml、暴露时间24小时、PA感染复数10、HBD1浓度10000ng/ml为实验条件下，PM2.5暴露会显著降低气道上皮细胞活性，通过降低细胞内HBD1表达来增加PA对细胞及小鼠模型的细菌侵袭性，且这个过程可以被外源性HBD1改善。细胞模型实验发现，MAPK通路抑制剂作用于PA+PM2.5共培养细胞模型时，HBD1表达量较PA+PM2.5组提高(P<0.05)。动物模型中，MAPK和NF-κB通路抑制剂可延长PA+PM2.5组动物的生存时间(P<0.05)。抑制剂组100小时内生存率较PA+PM2.5组提升约30%，小鼠肺内细菌数目均较 PA+PM2.5 组有所减少(P<0.05)。同时，MAPK 和 NF-κB 通路抑制剂的干预会在分泌和细胞内两个水平提高小鼠模型的HBD1表达量(P<0.05)。因此，PM2.5通过激活 MAPK及NF-κB通路，下调人β防御素1表达来增加铜绿假单胞菌对人支气管上皮细胞模型及小鼠模型的细菌侵袭性。

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