百草枯致肺损伤潜在治疗靶点：MUC5B的功能与机制研究

Paraquat poisoning can cause acute lung injury and death are remains a challenge in toxication field of emergency medicine. Our previous study found that Muc5b protein level was increased in the paraquat poisoning patient plasma samples as well as paraquat-induced mouse model; Muc5b-/- mice had significantly lower damage in lung and 100% survival rate compared to the wild type mice and Muc5b transgenic mice(CCSP-Tg and SPC-Tg); Muc5b remove treatment can also significantly protect the mice from paraquat. Furthermore, we used mRNA microarray to compare tissues from paraquat-treated wild type mouse lung and paraquat-treated Muc5b-/- lung tissue. GO and Pathway analysis and preliminary study found that NF-κB pathway may involved in the mechanism of Muc5b regulated paraquat-induced lung injury. We plan to perform experiment in vivo and in vitro; clinical samples observing; overexpression and knockdown approaches as well as the pull down assay to clarify the function of Muc5b involved in paraquat-induced lung injury and its mechanism of action with mediated by the NF-κB pathway. Since the role of Muc5b involved in paraquat-induced lung injury was first reported by our team. As currently there is few study among its mechanism of Muc5b, our research will provide important novel insights. This study will reveal potential targets for the intervention and treatment of paraquat-induced lung injury and other cause of lung injury.

急性肺损伤是百草枯中毒最主要的死亡原因，成为我国中毒领域重要医学问题。我们前期研究发现：中毒患者血液样本以及百草枯动物模型可见MUC5B蛋白表达量增加；在百草枯诱导野生型及该基因过表达小鼠出现死亡和严重肺损伤情况下，该基因敲除小鼠的肺损伤明显减轻，死亡率为零；MUC5B清除治疗也对小鼠有明显保护作用。进一步通过基因芯片、生物信息学分析以及初步验证发现，NF-κB信号通路可能参与了MUC5B调控百草枯致肺损伤的机制。本研究拟通过体内外实验及临床样本观察；过表达、抑制结合通路挽救策略；pull down试验深入机制探讨等，阐明NF-κB信号通路介导的MUC5B调控百草枯致肺损伤的科学假说。有关MUC5B在百草枯致肺损伤中的功能由本申请人首次提出并报道，其具体机制尚未见报道，因此本研究具有源头创新性，并极有可能成为百草枯致肺损伤以及其他原因肺损伤的早期防治新线索和潜在干预靶标。

联吡啶类除草剂中毒的高死亡率和高发病率一直是我国中毒领域重要医学问题。我们通过本研究发现分泌型黏蛋白MUC5B参与了百草枯致急性肺损伤及肺纤维化的关键机制：百草枯腹腔注射和气道滴注均可以显著增加WT鼠肺组织、肺泡支气管灌洗液和血浆中Muc5b蛋白表达；全身敲除Muc5b，显著降低了腹腔注射百草枯造成的死亡率且小鼠肺部损伤轻微；气道滴注百草枯造成的死亡率同样获得改善，肺部损伤存在但与WT组相比有显著改善。细胞模型中发现百草枯暴露可引起A549细胞中MUC5B的表达变化，且细胞内的炎症因子（TNF-a和IL-6）mRNA表达与ELISA检测细胞上清的含量也随着百草枯暴露的时间而发生改变；另一方面，百草枯暴露激活了NF-kB通路，引起NF-KB p65发生磷酸化，从而激活炎症，同时发现百草枯暴露激活了NF-kB上游的MAPK通路。抑制剂实验发现提示百草枯暴露导致MUC5B表达增高通过P65、ERK、JNK但并不通过MAPKP38途径。最后，采用NAC干预细胞，观察下调MUC5B和细胞外因子TNF-α和 IL-6的表达，表明NAC通过下调ERK MAPK途径，JNK MAPK途径和NF-κB途径减弱急性炎症。该结果可为MUC5B蛋白参与联吡啶类除草剂导致的肺损伤提供毒理学新依据。