The Mechanism Of Epidermal Nav1.8 Involved In The Pathogenesis Of Inflammatory Skin Diseases

Reactive oxygen species(ROS)-activated proinflammatory signals in keratinocytes play a crucial role in the immunoregulation of inflammatory skin diseases,including rosacea and psoriasis.Nav1.8 is a voltage-gated sodium ion channel,and its abnormal expression in the epidermal layer contributes to pain hypersensitivity in the skin.However,whether and how epidermal Nav1.8 is involved in skin immunoregulation remains unclear.Part I Nav1.8 in keratinocytes contributes to ROS-mediated inflammation in inflammatory skin diseasesObjective:To explore the role and mechanisms of action of the epidermal sodium channel Navl.8 in the pathogenesis of inflammatory skin diseases.Methods:1.LL37-induced rosacea-like mouse model and IMQ-induced psoriasis mouse model were constructed by collecting skin lesions from rosacea and psoriasis patients,and the expression levels of RNA and protein of Nav1.8 in rosacea and psoriasis skin lesions were examined.2.The LL37-induced rosacea-like mouse model and IMQ-induced psoriasis mouse model were constructed after SiNavl.8 silencing of Nav1.8.24h after the end of modeling,the skin thickness and erythema area on the back of mice were recorded.The histopathological changes of mouse skin were observed by HE staining.The expression of chemokines and cytokines were detected by Real-time PCR,and the expression of immune cells in skin tissue was detected by immunofluorescence and flow cytometry.3.The genes that are aberrantly expressed in rosacea-like skin lesions in mice and regulated by Nav1.8 were identified by RNAseq,and the expression of the relevant genes in skin lesions was verified again by immunofluorescence and other technical means.In vitro,a TNFa-induced inflammatory model of keratinocytes was constructed to investigate whether the above aberrantly expressed genes were also regulated by Nav1.8 in vitro.4.To explore the molecular mechanism of Nav1.8 involved in inflammatory skin diseases,we divided Nav1.8 into different segments and identified the proteins that could interact with each segment by IP-MS.5.In vivo,the expression levels of ROS in the LL37 and IMQ-induced mouse models were detected by DHE staining.In vitro,the expression levels of ROS in the inflammatory cell model were examined by DHE staining,DCFH-DA technique.The expression of ROS levels and inflammatory cytokines in keratin-forming cells was probed after reducing ROS levels in keratin-forming cells using the antioxidant MitoTempo.Results:1.Nav1.8 expression was increased in the epidermis of rosacea and psoriasis lesions.2.Nav1.8 was involved in LL37/IMQ-induced skin inflammation in mice,and silencing Nav1.8 improved the degree of skin erythema,lesion thickness and inflammatory infiltration in mice.3.PPI network analysis and epidermal transcriptome data suggest that IL6 and ILlβ are pivotal genes in Nav1.8-mediated skin inflammation in rosacea and psoriasis;IL1β and IL-6 expression is increased in mouse models of inflammatory skin disease and inflammatory cell models,and knockdown of Nav1.8 reduces IL1β and IL-6 expression.4.The antioxidant activity-related enzyme SOD2 is a potential protein that interacts with the Nav1.8-C-terminus.5.ROS expression levels were significantly elevated in mouse models of inflammatory skin disease.overexpression of Navl.8-C or TNFa treatment increased ROS expression in keratin-forming cells,and knockdown of Nav1.8 significantly reduced ROS expression levels in animal and cellular models.mtROS scavenger reduced Navl.8-C-induced ROS accumulation and increased IL-1β and IL-6 expression in keratin-forming cells.and IL-6 expression in keratinocytes.Conclusions:Nav1.8 upregulates ILlβ and IL-6 expression in keratinocytes by promoting ROS accumulation,which in turn promotes the development of the inflammatory skin disease rosacea/psoriasis.Part II Nav1.8 Direct targeting of SOD2 regulates cytokine production and ROS accumulationObjective:To investigate the mechanism of interaction between the epidermal sodium channel Nav1.8 and SOD2.Methods:1.To verify the interaction between Nav1.8 and SOD2 by immunoprecipitation(Co-IP),immunofluorescence and other techniques.2.The effects of TNFα treatment,knockdown of Nav1.8 or overexpression of Navl.8-C and A803467(Nav1.8 inhibitor)treatment on the expression and activity of SOD2 in cells were examined by protein blotting and superoxide dismutase assay.3.Overexpression of Nav1.8-C or Nav1.8 knockdown,detection of SOD2 transport and acetylation expression by WB and co-localization of SOD2 with mitochondria by immunofluorescence.Results:1.Nav1.8 interacts with SOD2 to induce ROS accumulation.2.Knockdown of Nav1.8 increased SOD2 activity in keratin-forming cells,overexpression of Nav1.8-C or TNFa treatment decreased SOD2 activity in keratin-forming cells,and A-80367 treatment did not affect SOD2 activity.3.Overexpression of Navl.8-C reduced SOD2 translocation from cytoplasm to mitochondria and increased SOD2 acetylation in keratinocytes;knockdown of Nav1.8 increased SOD2 accumulation in mitochondria and reduced SOD2 acetylation in keratinocytes.Conclusions:Nav1.8 reduces SOD2 activity by decreasing mitochondrial transport and increasing the acetylation level of SOD2 in keratinocytes,thereby regulating cytokine production and ROS accumulation.