Department of Chemical Defense Medicine, School of Military Preventive Medicine, Third Military Medical University (Army Medical University), 30 Gaotanyan Street, Shapingba District, Chongqing, 400038, China.
Institute of Pathology and Southwest Cancer Centre, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, China.
Signal Transduct Target Ther. 2021 Jan 25;6(1):29. doi: 10.1038/s41392-020-00389-z.
Nitrogen mustard (NM) causes severe vesicating skin injury, which lacks effective targeted therapies. The major limitation is that the specific mechanism of NM-induced skin injury is not well understood. Recently, autophagy has been found to play important roles in physical and chemical exposure-caused cutaneous injuries. However, whether autophagy contributes to NM-induced dermal toxicity is unclear. Herein, we initially confirmed that NM dose-dependently caused cell death and induced autophagy in keratinocytes. Suppression of autophagy by 3-methyladenine, chloroquine, and bafilomycin A1 or ATG5 siRNA attenuated NM-induced keratinocyte cell death. Furthermore, NM increased transient receptor potential vanilloid 1 (TRPV1) expression, intracellular Ca content, and the activities of Ca/calmodulin-dependent kinase kinase β (CaMKKβ), AMP-activated protein kinase (AMPK), unc-51-like kinase 1 (ULK1), and mammalian target of rapamycin (mTOR). NM-induced autophagy in keratinocytes was abolished by treatment with inhibitors of TRPV1 (capsazepine), CaMKKβ (STO-609), AMPK (compound C), and ULK1 (SBI-0206965) as well as TRPV1, CaMKKβ, and AMPK siRNA transfection. In addition, an mTOR inhibitor (rapamycin) had no significant effect on NM-stimulated autophagy or cell death of keratinocytes. Finally, the results of the in vivo experiment in NM-treated skin tissues were consistent with the findings of the in vitro experiment. In conclusion, NM-caused dermal toxicity by overactivating autophagy partially through the activation of TRPV1-Ca-CaMKKβ-AMPK-ULK1 signaling pathway. These results suggest that blocking TRPV1-dependent autophagy could be a potential treatment strategy for NM-caused cutaneous injury.
氮芥（NM）可引起严重的水疱性皮肤损伤，目前缺乏有效的靶向治疗方法。主要的限制是 NM 诱导的皮肤损伤的具体机制尚不清楚。最近，自噬在物理和化学暴露引起的皮肤损伤中发挥了重要作用。然而，自噬是否有助于 NM 诱导的皮肤毒性尚不清楚。在此，我们初步证实 NM 剂量依赖性地引起角质形成细胞的细胞死亡和自噬。3-甲基腺嘌呤、氯喹和巴弗洛霉素 A1 或 ATG5 siRNA 抑制自噬可减轻 NM 诱导的角质形成细胞死亡。此外，NM 增加瞬时受体电位香草醛 1（TRPV1）表达、细胞内 Ca 含量和 Ca/钙调蛋白依赖性激酶激酶β（CaMKKβ）、AMP 激活的蛋白激酶（AMPK）、非典型蛋白激酶 1（ULK1）和雷帕霉素靶蛋白（mTOR）的活性。TRPV1 抑制剂（辣椒素）、CaMKKβ 抑制剂（STO-609）、AMPK 抑制剂（compound C）和 ULK1 抑制剂（SBI-0206965）以及 TRPV1、CaMKKβ 和 AMPK siRNA 转染均可消除 NM 诱导的角质形成细胞自噬。此外，mTOR 抑制剂（雷帕霉素）对 NM 刺激的角质形成细胞自噬或细胞死亡没有显著影响。最后，NM 处理皮肤组织的体内实验结果与体外实验结果一致。总之，NM 通过过度激活自噬引起皮肤毒性，部分通过激活 TRPV1-Ca-CaMKKβ-AMPK-ULK1 信号通路。这些结果表明，阻断 TRPV1 依赖性自噬可能是治疗 NM 引起的皮肤损伤的一种潜在治疗策略。
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