Probing The Mechanism Of Neuropeptide Y-induced Vitiligo Recurrence Based On NPYR/NF-κB Pathway

Purpose:The relationship between mental stress and vitiligo recurrence is significant.Nevertheless,it is not clear how this causative factor contributes to the recurrence of vitiligo.The present study aims to explore whether neuropeptide Y（NPY）can promote the release of cytokines related to vitiligo recurrence（CRVR）through hydrogen peroxide-induced oxidative stress in keratinocytes and their signalling mechanisms,as well as to indicate a probable role of cellular autophagy.This is a possible role for autophagy.Methods:In this study,a human keratinocyte cell line（Hacat）was used to induce oxidative damage using hydrogen peroxide,and the appropriate concentration of H₂O₂for Hacat treatment was established using crystalline violet staining,flow cytometry for apoptosis and CCK-8assay for cell viability and inhibition.Then,the effect of NPY on CRVR expression at different concentrations and times was investigated by Western Blot and cellular immunofluorescence staining to determine the effect of NPY on CRVR expression and the activation status of the NPYR-NF-κB signalling pathway in different groups（Control,NPY,H₂O₂and NPY+H₂O₂groups）.inhibited the NF-κB pathway using the NF-κB inhibitor BAY 11-7082 to understand the expression level of CRVR under reverse inhibition of the signalling pathway.Finally,flow cytometry was used to further investigate the effects of NPY on oxidative damage to intracellular ROS levels and levels of cellular autophagy and apoptosis in keratinocytes,and techniques such as Western Blot and reactive oxygen ROS fluorescent probes were used to examine the possible role of cellular autophagy in this.Results:（1）.We selected H₂O₂treatment for Hacat cells in the range from low to high concentrations,and 100μmol/L was specified as the treatment concentration of H₂O₂.（2）Treatment of hacat cells with NPY from low to high concentrations for 24,48 and 72 h was undertaken.It was determined the appropriate concentration（12.5μg/ml）and duration of action（24h）for NPY to work in the Hacat cells.（3）Both the expression of NPY4R/NF-κB signaling pathway and CRVR were significantly upregulated in the H₂O₂+NPY group relative to the control and H₂O₂groups,（p<0.05）,but CXCL9 and NPY2R expression levels were not significantly different in the various subgroups（p>0.05）.（4）Inhibition of Hacat cell survival was observed at concentrations of 2.5 and 5 n M/ml of BAY 11-7082（p<0.05）.Down-regulation of IL-15,IL15Rα,CXCL9 and CXCL10 expression was measured with increasing concentrations of BAY 11-7082 inhibitor relative to the control group.（5）There was a significant increase in the apoptosis ratio in the H₂O₂group relative to the control group（p<0.05）,in contrast to a decrease in the apoptosis ratio in the H₂O₂group+NPY group compared to the H₂O₂group（p<0.05）.Relative to the control group,there was an upregulation of the LC3-II/LC3-Ⅰratio in both the H₂O₂group and the H₂O₂group+NPY group（p<0.05）.Moreover,the LC3-II/LC3-Ⅰratio was highest in the H₂O₂group+NPY group compared to the H₂O₂group,and the difference was significant（p<0.05）.The upregulation of reactive oxygen species ROS was most significant in the H₂O₂group relative to the control group（p<0.05）.On the contrary,the expression level of ROS was down-regulated in the H₂O₂group+NPY group compared to the H₂O₂group（p<0.05）.Conclusions:In the present study,it is evidenced that the expression of CRVR in oxidatively damaged keratinocytes is regulated by NPY through NPY4R/NF-κB signalling and can be inhibited by NF-κB inhibitors.Furthermore,they also revealed that ROS levels in oxidatively damaged keratinocytes could be reduced by NPY through promoting cellular autophagy,thereby inhibiting apoptosis.Taken together,the literature and our findings suggested that by promoting the expression of CRVR in vitiligo keratinocytes,NPY could activate CD8+Trm cells and induce melanocyte destruction.Our findings clarify the particular molecular mechanisms and signalling pathways that initiate memory immune responses,which contribute to revealing the intrinsic link between neuroendocrine,immune responses and apoptosis,which further enriches and refines the pathogenesis of vitiligo.