Nitrogen mustard (NM) causes severe skin injury with an obvious inflammatory response, which is lack of effective and targeted therapies. Vitamin D3 (VD3) has excellent anti‐inflammatory properties and is considered as a potential candidate for the treatment of NM‐induced dermal toxicity; however, the underlying mechanisms are currently unclear. Cyclooxygenase‐2 (COX2; a widely used marker of skin inflammation) plays a key role in NM‐induced cutaneous inflammation. Herein, we initially confirmed that NM markedly promoted COX2 expression in vitro and in vivo. NM also increased NOD‐like receptor family pyrin domain containing 3 (NLRP3) expression, caspase‐1 activity, and interleukin‐1β (IL‐1β) release. Notably, treatment with a caspase‐1 inhibitor (zYVAD‐fmk), NLRP3 inhibitor (MCC950), and NLRP3 or caspase‐1 siRNA attenuated NM‐induced NLRP3 inflammasome activation, with subsequent suppression of COX2 expression and IL‐1β release in keratinocytes. Meanwhile, NM increased mitochondrial reactive oxygen species (mtROS) and decreased manganese superoxide dismutase 2 (SOD2) and sirtuin 3 (SIRT3) activities. Mito‐TEMPO (a mtROS scavenger) ameliorated NM‐caused NLRP3 inflammasome activation in keratinocytes. Moreover, VD3 improved SIRT3 and SOD2 activities, decreased mtROS contents, inactivated the NLRP3 inflammasome, and attenuated cutaneous inflammation induced by NM in vitro and in vivo. The beneficial activity of VD3 against NM‐triggered cutaneous inflammation was enhanced by the inhibitors of IL‐1, mtROS, NLRP3, caspase‐1, and NLRP3 or caspase‐1 siRNAs, which was abolished in SIRT3 inhibitor or SIRT3 siRNA‐treated keratinocytes and skins from SIRT3−/− mice. In conclusion, VD3 ameliorated NM‐induced cutaneous inflammation by inactivating the NLRP3 inflammasome, which was partially mediated through the SIRT3–SOD2–mtROS signaling pathway.

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Sulfur mustard (a type of vesicant) can directly damage lung bronchial epithelium via aerosol inhalation, and prevalent cell death is an early event that obstructs the respiratory tract. JNK/c-Jun is a stress response pathway, but its role in cell death of the injured cells is not clear. Here, we report that JNK/c-Jun was activated in immortalized human bronchial epithelial (HBE) cells exposed to a lethal dose (20 μM) of nitrogen mustard (NM, a sulfur mustard analog). c-Jun silencing using small-interfering RNA (siRNA) rendered the cells resistant to NM-mediated cell death by blocking poly(ADP-ribose) polymerase 1 (PARP1) cleavage and DNA fragmentation. In addition, the transduction of upstream extrinsic (Fasl-Fas-caspase-8) and intrinsic (loss of Bcl-2 and mitochondrial membrane potential, ΔΨm) apoptosis pathways, as well as phosphorylated (p)-H2AX (Ser139), an epigenetic marker contributing to DNA fragmentation and PARP1 activity, was partially suppressed. To mimic the detachment of cells by NM, HBE cells were trypsinized and seeded on culture plates that were pre-coated with poly-HEMA to prevent cell adhesion. The JNK/c-Jun pathway was found to be activated in the detached cells. In conclusion, our results indicate that JNK/c-Jun pathway activation is necessary for NM-caused HBE cell death and further suggest that c-Jun silencing may be a potential approach to protect HBE cells from vesicant damage.

Nitrogen mustard (NM) can alkylate nucleophilic proteins and DNA, causing severe cell damage. However, there are no reports on NM-induced proteomics dynamic changes. In this study, nuclear and cytoplasmic proteins of 16HBE cell were separated and the components and amounts were detected and analyzed. The amount of DNA protein cross-linking (DPC) and the function of p97 were also explored. One-hour-NM-exposure caused a tremendous number of proteins entered into the nucleus and DPC formation. As repair progressed, proteins exited. Although the protein influx at 1 h was delayed by si-p97 intervention, it continued to 24 h after NM withdrawal. In the early damage, the affected pathways mainly included spliceosome, ribosome biogenesis in eukaryotes, and mRNA surveillance, which switched to protein processing in endoplasmic reticulum and energy production in presumed repair stage. Si-p97 aggravated ferroptosis, cysteine and methionine metabolism at beginning of the damage, followed by downward ranking the transcription related pathways at 24 h. NM caused DPC and H2AX increases at 1 h. Si-p97 suppressed them at 1 h and extended the increase time to 24 h. MG132 effected similar to si-p97. Si-p97 and si-DVC1 increased the cytoplasmic level of proteasome (PSMD2). Si-DVC1 also increased the DPC content. These results suggest that NM caused a severe and rapid protein influx and crosslink in the nucleus in the early stage of injury, followed by the formation of secondary double-strand breaks. P97 was involved in the clearance of proteins in nucleus and DPC for repair, which required the participation of DVC1 and proteasome.