Effect And Mechanism Of Minocycline On Oxidative Stress-induced Keratinocyte CXCL16 Production

Objective:Through the construction of an in vitro cell model,the effects and mechanisms of minocycline in oxidative stress-induced CXCL16 of keratinocyte were explored;and the understanding of the pharmacological mechanism of minocycline was expanded and deepened,providing a solid theoretical basis for further development and application of minocycline as a new treatment for vitiligo diseases in the future.Methods:Cell model was established.Human keratinocyte line（HaCat）was cultured in DMEM medium containing 10%FBS.Different concentrations of H₂O₂（125、250、500、1000、10000 uM）;different concentrations of minocycline（125、250、500、1000 uM）;1000 um H₂O₂+minocycline（500、1000 uM）were on HaCat cells;PBS treatment group was as blank control group.After treatment,CCK8method was used to test cell viability.Chemokine chip was used to detect the cytokines in the test.In the same experimental group,the total RNA was extracted,and then reverse transcripted to cDNA,and the expression of CXCL16 mRNA was detected by real-time qRT-PCR.The culture supernatants of the above groups were collected and the level of CXCL16 was detected by ELISA.The chemotaxis of PBMC was detected by transwell and flow cytometry.Western blot was used to detect the phosphorylation expression of eIF2a and NF-κB in HaCat cell.Results:CCK8 method was used to test the cell viability.The results of chemokine chip detection showed that compared with the blank control group,the release of CXCL16 increased after H₂O₂ treatment on HaCat cells,while the expression of CXCL16 decreased significantly after minocycline treatment.As a result,the CXCL16,the most obvious cytokine,was selected.There was no significant effect on the proliferation and apoptosis of HaCat cells in each treatment group.Compared with the H₂O₂ positive control group,after the treatment of HaCat cells by H₂O₂（1000 uM） and minocycline（500、1000 u M）,the results of q RT-PCR and ELISA showed that the expression of CXCL16 was significantly decreased,and the chemotaxis of CD8⁺T cells in peripheral blood was significantly decreased by transwell chemotaxis and flow cytometry;WB detection showed that minocycline significantly inhibited the phosphorylation expression of eIF2a、and NF-κB.Conclusion:1.Under the induction of oxidative stress,eIF2a signaling pathway activates NF-κB to participate in the production of CXCL16 in human keratinocytes,which drives toxic CD8⁺T cells to infiltrate around melanocytes and cause damage.Through our experiments,we found that minocycline can reduce oxidative stress of keratinocytes and impair melanocyte reduction by inhibiting the activation of this signaling pathway.2.This study provides insights into the molecular mechanisms underlying the beneficial effects of minocycline in the treatment of vitiligo.Our study suggests that minocycline may be a promising drug to prevent melanocyte loss and promote re-pigmentation in vitiligo patients.