Targeting TRPV1-mediated autophagy attenuates nitrogen mustard-induced dermal toxicity

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 CA²⁺ content, and the activities of CA²⁺/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-Ca²⁺-CaMKKβ-AMPK-ULK1 signaling pathway. These results suggest that blocking TRPV1-dependent Autophagy could be a potential treatment strategy for NM-caused cutaneous injury.