Inhibiting TRPV1-Mediated Autophagy Attenuates Nitrogen Mustard-Induced Dermal Toxicity

There is currently no satisfactory therapy for nitrogen mustard (NM)-caused dermal toxicity, for poor understanding of the exact mechanisms. Autophagy has been found to play important roles in physical or chemical exposure-caused cutaneous injuries. Herein, we initially confirmed that NM dose-dependently caused cell death in keratinocytes. The LC3B2 (light chain 3 beta 2) formation and SQSTM1/p62 (sequestosome 1) degradation and the formation of autophagosomes were also dose-dependently upregulated by NM. And chloroquine treatment resulted in further accumulation of LC3B2 in NM-treated keratinocytes. Suppression of autophagy by 3-methyladenine and chloroquine or ATG5 siRNA attenuated NM-caused cell death in keratinocytes. Furthermore, NM increased TRPV1 (Transient receptor potential vanilloid 1) expression, intracellular Ca2+ content, and the activity of CaMKKβ (Ca2+/Calmodulin-Dependent Kinase Kinase β), AMP-activated protein kinase (AMPK), ULK1 (unc-51-like kinase 1) and mTOR (mammalian target of rapamycin). NM-induced autophagy in keratinocytes was abolished in the presence of inhibitors of TRPV1 (capsazepine), CaMKKβ (STO-609), or AMPK (compound C) as well as TRPV1, CaMKKβ, and AMPK siRNA transfection. Additionally, mTOR inhibitor (rapamycin) had no significant effect on NM-stimulated autophagy and cell death in keratinocytes. Finally, the results of in vivo study in NM-treated skin tissues were consistent with the findings of in vitro study. In conclusion, NM caused dermal toxicity by overactivating autophagy through the activation of TRPV1-Ca2+-CaMKKβ-AMPK-ULK1 signaling pathway. These suggest that blocking TRPV1-dependent autophagy could be a potential treatment strategy for NM-caused skin injury. Funding: This work was supported by the National Natural Science Foundation of China (grant number: 1703268), National Postdoctoral Program for Innovative Talents (BX20180378) and the Excellent Youth Foundation of the Amy Medical University (2017). Declaration of Interest: The authors declare no conflicts of interest. Ethical Approval: All animal experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health and were approved by the Animal Care and Use Committee of the Amy Medical University (Chongqing, China; approval no. SYXC-2016-00115).