骨髓间充质干细胞(BMSCs)治疗芥子气中毒的效应与机制研究

Sulfur mustard (SM) is the typical representative of blister agents and a major chemical threat on the battlefields. SM is also one of the major components in chemical weapons abandoned by Japan in China 70 years ago, which has been imposing great threat to the public health and the environment of the nation as well. Furthermore, SM is regarded as the most possible agent for potential terrorism attack because of its easy synthesis and availability. The mechanism of toxicity for SM is very complicated and has not been elucidated clearly yet, so there are no specific antidotes and the treatment and rescue for SM are very difficult and just symptomatic. We found that bone marrow mesenchymal stem cells (BMSCs) could significantly improve the survival rate on a mice model exposed to SM (P<0.05). In the pathological analysis, the pathological change was aggravated in SM group, however, BMSCs could markedly attenuate the histological alterations in the lungs, decrease wet/dry weight ratio of the lungs and the total protein amount in the bronchoalveolar lavage fluid (BALF) (p<0.01). The levels of MDA, SOP and GSH showed an amelioration of the oxidative stress condition. In the lung tissues of the BMSCs group, remarkable decrease of the pro-inflammatory cytokines TNF-α and IL-1β were observed in the Western blot and real-time PCR analysis (p<0.01), while the level of anti-inflammatory cytokine IL-10 were significantly improved (p<0.01). Therefore, BMSCs could be considered as a promising therapeutic measure for treating SM-induced poisoning. We would continue to investigate the efficacy of BMSCs on SM-induced poisoning and explore the possible mechanism. This project would focus on the mechanism of anti-inflammatory and immunomodulation. The results will provide a strong foundation for the future BMSCs application in SM-induced poisoning. It is of significance for the national public safety and health.

芥子气(sulfur mustard, SM)是糜烂性毒剂的典型代表，是日遗化武中的主要毒剂之一，也是最有可能被用于进行恐怖袭击的毒剂之一，给我国人民的健康和环境构成重大威胁。芥子气中毒机制复杂，防救困难，以对症治疗为主，无特效抗毒药物。我们发现骨髓间充质干细胞（bone marrow mesenchymal stem cells, BMSCs）可显著提高SM中毒小鼠的生存率，降低SM中毒ICR小鼠肺的湿干重比，Western blot及荧光定量PCR结果皆显示其能显著降低肺组织内炎性因子TNF-α、IL-1β表达，提高抑炎因子IL-10的表达。本项目将进一步系统评价BMSCs治疗SM中毒的作用，探讨其可能的机制，特别是抗炎及免疫调节方面的机制，为BMSCs在治疗SM中毒方面的应用打下基础。这将为SM中毒救治提供一种有效的方法，对于国家公共安全和民众健康具有重要意义。

芥子气（sulfur mustard, SM）是一种剧毒化学战剂，可引起急性肺损伤，目前仍无有效治疗方法或解毒剂。骨髓间充质干细胞（BMSC）是一种能够自我更新的多分化潜能干细胞，在抗炎与组织修复等方面发挥重要作用。我们从小鼠骨髓分离BMSC后，尾静脉注射SM暴露小鼠，结果显示BMSC可显著提高SM暴露小鼠生存率，改善SM引起的急性肺损伤病理学评分，降低肺湿干重比及BALF蛋白水平。综合研究发现BMSC可能通过如下途径改善SM所致肺损伤：（1）BMSC可显著降低促炎性M1型巨噬细胞及Th17细胞的表达，升高抗炎性M2型巨噬细胞及Treg细胞的表达；（2）BMSC可降低肺组织中TNF-α、IL-1β、IL-6等炎性因子表达量，升高IL-10、TGF-β等抑炎因子的表达，并降低LIX/CXCL5、IP-10/CXCL10、MIP-1/CCL4、RANTES/CCL5等趋化因子水平，进而达到免疫调节与抗炎效果；（3）BMSC可显著提高SM暴露小鼠肺组织内EGF、FGF、PDGF的表达，提高Ki-67阳性细胞比例，提高肺组织内I型、II型肺泡上皮细胞表面标志蛋白AQP5、SP-C及细胞连接蛋白VE-cadherin、Occludin、Claudin-5、ZO-1等的表达，促进肺泡上皮细胞分化及功能修复，改善肺损伤；（4）BMSCs可通过降低SM中毒小鼠肺组织MDA含量，升高SOD、GSH含量，上调Nrf-2蛋白表达，改善氧化应激状态。（5）采用基因敲除小鼠及细胞进行机制研究发现，TLR4信号通路可能在BMSC改善SM肺损伤的过程中发挥了重要作用。（6）我们进一步证明骨髓间充质干细胞的外泌体（BMSC-Ex）对SM暴露所致急性肺损伤的亦有显著改善作用，并最终确定了G蛋白偶联受体GPRC5A在BMSC-Ex抗凋亡和屏障修复功能中扮演了重要角色，且GPRC5A可能通过激活YAP通路促进了Bcl2和紧密连接功能蛋白表达而发挥作用。总之，BMSC可以通过改善炎症、免疫调节和促进上皮屏障修复改善SM所致急性肺损伤，为进一步理解SM中毒机制提供了新思路，为SM中毒治疗提供了一种有效方法，对于国家公共安全和民众健康具有重要价值。