雷公藤红素对恶性胸膜间皮瘤的预防效应及其分子机制的研究

Malignant pleural mesothelioma (MPM) is a kind of threatening occupational cancer with extremely poor prognosis. It is clear that asbestos is a kind of carcinogen of MPM. However, until now effective methods for prevention and treatment of MPM have not been successfully established. So it is a key question for MPM occupational protection to identify preventive drugs specific to and effective in MPM prevention. Oxidative damage of mesothelial cells as well as chronic inflammation in pleural cavity are regarded as key factors contributing to the development and progression of MPM. We previously found that celastrol, a natural product derived from the plant Tripterygium wilfordii Hook. f., could significantly inhibit asbestos-induced oxidative damage and inflammation at low doses. In consideration of previous reports, we hypothesize that celastrol may prevent the carcinogenesis and progression of MPM via activating the antioxidate Nrf2 (nuclear respiratory factor 2)-ARE (antioxidant response element) pathway while inhibiting the inflammatory NLRP3 (NOD-like receptor protein3) inflammasome and NF-κB pathways. We aim to evaluate the efficacy of Celastrol in prevention of MPM and explore the mechanisms involved in this process by using multiple in vitro and in vivo models mimicking the whole process of the development, growth disorder and progression of asbestos-induced MPM. By comparing the effects of celastrol on the Nrf2 –ARE, NLRP3 and NF-kB pathways and on the DNA methylation status of related genes in the presence or absence of celastrol, we will provide insights into the molecular mechanism for asbestos-induced MPM and preventive actions of celastrol. This study may identify celastrol as a new preventive drugs for asbestos-induced MPM and will provide theoretical basis for the occupational protection and management of MPM.

恶性胸膜间皮瘤(MPM)是危害大、预后差的职业性肿瘤，石棉是明确的致病因素，目前尚无有效的防治手段。故寻找针对性的预防药物是MPM职业防护的重要科学问题。间皮细胞DNA氧化损伤和胸膜腔慢性炎症被认为是MPM发生发展过程中的关键因素。申请者发现雷公藤红素(Cel)在较低剂量下对石棉诱导的氧化损伤和炎性反应均有明显抑制作用。因此提出科学假说：Cel激活Nrf2-AER通路、抑制NLRP3炎症小体和NF-κB途径，从而阻断MPM发生发展。本项目拟采用涵盖MPM的启动、促长和进展全过程的体内外研究模型，考察Cel对MPM预防效应及其分子机制。首先验证Cel对体内外间皮细胞恶性转化的抑制程度，明确预防效果；进而通过比较Nrf2-AER、NLRP3炎症小体和NF-κB通路活性程度及相关基因甲基化水平，从分子水平揭示其作用机制。通过以上研究，以期发现MPM新的预防药物，并为未来建立防治策略提供科学依据。

多壁碳纳米管(multi-walled carbon nanotubes,MWCNTs)呼吸暴露进入肺组织造成生物毒性，常规物理防护难以起到有效的保护作用。利用化学药物对其进行防护逐渐成为一个可供选择的研究方向。现有研究表明，MWCNTs进入肺组织造成氧化应激及炎症损伤；MWCNTs诱导细胞产生过量ROS，形成氧化损伤，激活NF-κB信号通路造成炎症损伤。利用上述分子机制对其进行化学防护成为现实可能。既往文献报道及研究基础提示雷公藤红素可以通过激活Nrf2信号通路抑制ROS过量产生，并能抑制NF-κB信号通路的激活。于是本研究选择雷公藤红素从上述两方面机制入手，对MWCNTs生物毒性进行化学防护。本研究：①采用体外Cel抑制MWCNTs诱导BEAS-2B细胞氧化损伤细胞模型；②通过气管灌注给予C57BL/6小鼠不同剂量的MWCNTs，从动物水平上考察Cel对MWCNTs诱导肺组织氧化损伤及炎症损伤的抑制作用；③利用NF-κB基因敲除小鼠(P50-/- C57BL/6),反面证实Cel通过NF-κB信号通路抑制MWCNTs诱导的炎症损伤的分子机制。体外实验结果:①在MWCNTs的诱导作用下，细胞产生高浓度的ROS产生氧化应激损伤；②体外MWCNTs诱导下，敲低Keap1后ROS产生量下降，敲低Nrf2后ROS产生量升高；③Cel治疗MWCNTs诱导的细胞后，细胞ROS产生量下降，细胞毒性降低。Cel通过溶酶体途径促进Keap1降解，促进Nrf2入核，刺激HO-1表达。动物实验结果:①Cel减轻MWCNTs诱导的氧化损伤，恢复抗氧化酶水平；②Cel抑制NF-κB信号通路减轻MWCNTs诱导炎症。NF-κB基因敲除后，MWCNTs诱导的炎症损伤减轻。以上结果表明:①雷公藤红素可以通过Keap1/Nrf2/HO-1信号通路降低ROS产生，抑制MWCNTs诱导的氧化应激损伤；②雷公藤红素可以通过抑制NF-κB信号通路的激活减轻MWCNTs诱导的肺部炎症损伤。从而说明，雷公藤红素有望成为一种可以减轻MWCNTs诱导的生物毒性的可供选择的化学药物。