Cold Plasma Irradiation Attenuates Atopic Dermatitis Via Enhancing HIF-1α-induced MANF Transcription Expression

Atopic dermatitis(AD)is a chronic disease of skin with characteristics of relapse and skin inflammation,which has a rising incidence worldwide.Cold atmospheric plasma(CAP)has been widely applied in medical treatment clinically,especially skin diseases.However,the mechanism of CAP on the treatment of skin diseases is still undefined.In this study,dinitrofluorobenzene(DNFB)-induced AD mice model was constructed,followed by CAP irradiation.CAP irradiation inhibited cells apoptosis,relieve skin inflammation,endoplasmic reticulum(ER)stress and oxidative stress caused by dinitrofluorobenzene stimulation,which was mediated by CAP-induced the expression of mesencephalic astrocyte-derived neurotrophic factor(MANF).In terms of mechanism,hypoxia-inducible factor-1α(HIF-1α)expression was increased intracellularly after CAP irradiation,which further bound to the promoter region of manf gene to activate the transcriptional regulation of MANF and further promote MANF expression.This study revealed the effect and mechanism of CAP irradiation for AD mice,which indicates the potential medical application of CAP irradiation for AD treatment.Objective:Atopic dermatitis is a chronic inflammatory skin disease.The current clinical treatment has limitations and adverse reactions.This study investigated the effect and mechanism of CAP irradiation in DNFB-induced AD mice.Methods:Firstly,DNFB-induced AD mice model were constructed,and the degree of skin injury was detected by HE staining.Moreover,skin tissue of AD mice was irradiated by CAP irradiation and we detected related factors of apoptosis,inflammatory,endoplasmic reticulum stress and oxidative stress in the skin tissue of mice by HE staining,IHC,TUNEL staining,WB and qPCR.The levels of inflammatory factors in peripheral blood were detected by ELISA,and the numbers of monocytes and granulocytes in serum were detected by flow cytometry.To explore the mechanism of CAP irradiation on DNFB-induced AD mice,we injected hrMANF protein by tail vein and MANF neutralizing antibody subcutaneously.The related factors of inflammatory,ER stress and oxidative stress were detected by HE staining,IHC,WB and qPCR.ELISA was used to detect the levels of inflammatory factors in peripheral blood.Finally,HacatT cells were used for in vitro experiments to clarify the mechanism of CAP irradiation for AD mice by WB,CHIP and dual-luciferase reporter assay.Results:1.CAP irradiation inhibited DNFB-induced apoptosis.In this study,the degree of skin injury in AD mice after CAP irradiation was detected,and HE staining showed that the degree of skin injury in AD mice was relieved after CAP irradiation.Similarly,it was determined by IHC,WB and TUNEL staining that CAP irradiation inhibited DNFB-induced cell apoptosis.2.CAP irradiation relieved DNFB-induced skin inflammation,ER stress and oxidative stress in mice.This study examined the related factors of inflammation,ER stress,and oxidative stress in mouse skin tissue by IHC,WB and qPCR.The levels of inflammatory cytokines,ROS and NO in serum were determined by ELISA,and the number of monocytes and granulocytes in peripheral blood were determined by flow cytometry.The results of IHC and WB showed that,compared with DNFB group,the expressions of HMGB1,TNF-α,IL-1β and CCL2 were decreased,the MANF expressions were increased,the expressions of Bip and CHOP were reduced,the HO-1 expressions were up-regulated,and the mRNA and protein levels were consistent after CAP irradiation.Moreover,compared with DNFB group,the levels of TNF-α,IL-1β,ROS and NO in serum decreased,while IL-10 levels were increased.There were lower proportions of CD11b+Ly6Chi monocytes and CD11b+Ly6G+neutrophils in peripheral blood of mice after CAP irradiation.3.CAP irradiation enhanced MANF expression to reduce DNFB-induced skin inflammatory injury,ER stress and oxidative stress in mice.Mice were injected hrMANF protein by tail vein and MANF neutralizing antibody subcutaneously.HE staining showed that hrMANF protein treatment significantly relieved DNFB-induced skin injury,which was consistent with the effect of CAP irradiation.However,the skin injury was aggravated after MANF antibody treatment.IHC and WB results showed that,compared with MANF neutralizing antibody treatment,the expressions of HMGB1,TNF-α,IL-1β,CCL2,Bip and CHOP were decreased,and HO-1 expressions were increased after hrMANF protein treatment,mRNA and protein levels were consistent.ELISA results indicated lower TNF-α,IL-1β,ROS and NO levels,and higher levels of IL-10 in the serum of mice after hrMANF protein treatment,compared with the MANF neutralizing antibody treatment.4.CAP irradiation induced MANF transcriptional expression via increasing HIF-1αlevel.The expression levels of HIF-1α and MANF in the skin tissue of mice after CAP irradiation were examined by IHC and WB.The results showed that CAP irradiation significantly promoted HIF-1α and MANF expressions in skin tissues of mice in a time-dependent way.In addition,in vitro experiments in vitro by using HaCaT cells,and WB results showed a positive correlation between HIF-1α and MANF expressions in HaCaT cells.HIF-1α binds to the MANF promoter region by CHIP.Finally,we further confirmed that HIF-1α regulates transcriptional activation of MANF by dual luciferase reports.Conclusion:1)CAP irradiation inhibited DNFB-induced cell apoptosis of AD mice;2)CAP irradiation enhanced MANF expression to reduce DNFB-induced skin inflam matory injury,ER stress and oxidative stress in mice;3)CAP promotes the increase of HIF-1α that binds to MANF promoter region for MANF transcriptional activation and expression.