Vitamin D3 Ameliorates Nitrogen Mustard-induced Cutaneous Inflammation Via Inactivating NLRP3 Inflammasome Signaling Pathway

Vesicating poisons(blistering agents)as a classic chemical warfare agent still pose a great military threat so far.The alkylating agent nitrogen mustard(NM)is a typical representative of vesicating poisons,which can cause infantry skin damage without effective treatment.Substantial evidence suggests that the underlying mechanisms of cutanous injury caused by NM are diverse and complicated.Recently,it’s believed that the inflammatory response as a key factor runs through the entire process of NM-triggered dermal toxicity.However,the exact mechanism of NM-induced cutaneous inflammatory injury has not yet been elucidated,which hampers the development of specific antidotes.Therefore,clarifying the underlying mechanism of NM-induced skin inflammatory injury and expanding the understanding of inflammatory reactions caused by alkylating agents have important military value for the development of special antidotes.NLR pyrin domain containing 3(NLRP3)inflammasome is an intracellular protein complex that can be activated by a variety of pathogens,macromolecules,and environmental stimuli.Activated NLRP3 inflammasome can promote interleukin-1β(IL-1β)and IL-18 maturation via initiating Caspase-1 activation,and plays a crucial role in the implementation of innate immune system function,especially in inflammatory response.However,excessive activation of NLRP3 inflammasome can cause a severer inflammatory response and result in damage to the body in turn.Recent studies have actually shown that NLRP3 inflammasome-mediated inflammatory damage is proposed to be a key player in the occurrence and development of cutaneous damage caused by external stimuli,such as ultraviolet and dinitrofluorobenzene.Inflammatory injury may be recognized as a new target for preventing and treating such injuries.Vitamin D3(VD3)is a fat-soluble steroid hormone synthesized in skin,and shows significant anti-inflammation,anti-oxidation and metabolic regulation effects.Available data indicate that VD3 can inhibit the release of NM-induced mouse dermal inflammatory factors and promote skin healing.At the same time,it has been found that VD3 has important regulatory effects on NLRP3 inflammasome.However,the precise contribution of the inflammasome to VD3-mediated protection against NM-triggered dermal toxicity remains to be elucidated.In addition,previous studies have confirmed mitochondrial reactive oxygen species(mtROS),the free radicals,are one of the important activators of NLRP3;SIRT3 directly binds to and deacetylates SOD2 to increase SOD2 activity,which is involved in the maintanance of the mtROS homeostasis and regulates NLRP3 inflammasome activity subsequently.Therefore,together with previously published data,we propose the following hypothesis: VD3 may ameliorate nitrogen mustard-induced cutaneous inflammation via inactivating NLRP3 inflammasome by regulating SIRT3-SOD2-mtROS signaling pathway.To verify the hypothesis in current study,by establishing NM-induced dermal injury in vivo and in vitro models,and using multiple inhibitors,siRNA,laser confocal,Western blot and other experimental techniques,the activation status of NLRP3 inflammasome was first detected,and then the possible role of SIRT3-SOD2-mtROS signaling pathway was clarified;furthermore,the role of this signal pathway in the therapeutic application of VD3 to improve NM-induced skin inflammatory damage was exoplored,and the molecular mechanism of VD3 to mitigate NM-induced cutanous injury was dissected.The main results and conclusions of current study are as follows:(1)NM induces inflammatory response in keratinocytes.when the NM concentration is less than or equal to 20 μM,NM does not show significant effect on cell proliferation as well as morphology,but does activate the expression and secretion of IL-1β in a concentration-dependent manner,and up-regulates the expression of COX2.(2)NM induces inflammatory response via activating the NLRP3 inflammasome in keratinocytes.NM can promote the activation of NLRP3 inflammasome in a concentration-dependent manner.Inhibition of NLRP3 inflammasome significantly attenuated NM-induced inflammatory damage.(3)The "SIRT3-SOD2-mtROS" signaling pathway plays an important role in NM-induced NLRP3 inflammasome production in keratinocytes.NM induces the generation of mtROS in cells by inhibiting the activity of SIRT3-SOD2 pathway,and then triggers the production of active NLRP3 inflammasome and inflammatory factors.Depleting mt ROS can significantly inhibit the activation of NM-induced NLRP3 inflammasome activation and ameliorate inflammatory injury.(4)VD3 protects keratinocytes against NM-induced inflammation via inhibiting the NLRP3 inflammasome.VD3 can inhibit the generation of NLRP3 inflammasome and decrease the expression and release of inflammatory factors.(5)VD3 suppresses NM-induced NLRP3 inflammasome activation through the activation of SIRT3-SOD2-mtROS pathway.VD3 can reduce mtROS generation via partially reversing the inhibitory effect of NM on the SIRT3-SOD2 antioxidant pathway,and then inactive NLRP3 inflammasome and decrease inflammatory factors production,thereafter,improve NM-induced keratinocyte inflammatory damage in vitro.In addition,VD3 pre-treatment can restore both the expression and activity of SIRT3 and SOD2,inhibit the generation of skin tissue inflammatory factors,and accelerate the repair of skin damage in vivo.Our present data indicate that SIRT3/NLRP3 signaling pathway plays an important role in the promoting wound healing effect of VD3 in NM-induced dermal injury.In summary,present study demonstrates,for the first time,that vasicant NM induces dermal inflammatory injury via activating NLRP3 inflammasome through the suppression of SIRT3-SOD2-mtROS signaling pathway,and VD3 mitgates the NM-induced dermal injury via inactivating NLRP3 inflammasome by upregulating the same signaling pathway.The results provide new insights into the mechanism of NM-induced skin damage and VD3-mediated therapeutic effect on NM-induced dermal toxicity;meanwhile,it has potential application value for exploring new protective strategies against NM threat.