Metabolomics Study Of Dupilumab In The Treatment Of Atopic Dermatitis And The Effect And Mechanism Of Butyrate On Atopic Dermatiti

Objectives1.To Investigate comprehensive metabolomic and lipidomic alteration in patients with moderate to severe atopic dermatitis(AD)before and after treatment with dupilumab.To explore potential metabolic alterations associated with dupilumab therapeutic efficacy or side effects.2.To evaluate the therapeutic effects of butyrate on AD from TNF-α/IFN-γ-induced inflammation cell model and DNFB-induced AD mouse model,and explore the potential molecular mechanism of SB’s therapeutic effect.MethodsPart 1.Metabolomics study of patients with atopic dermatitis in response to Dupilumab①A total of 33 patients with AD were included in the current study,with serum samples collected before and after treatment with dupilumab.Both Liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry were applied to investigate comprehensive metabolomic and lipidomic of serum.②Patients were further classified into well responders and poor responders group based on EASI-75.Based on the variable importance in projection value of the first principal component in the orthogonal partial least squares discriminant analysis model and the independent sample t test(p<0.05)as the conditions to screen the differential metabolites between before and after treatment in different efficacy groups.The pathway enrichment analysis was performed on the differential metabolites in well response group based on KEGG database.③The baseline serum metabolites related to the efficacy of Dupilumab were screened out based on the independent sample t test(p<0.05).Receiver operating characteristic curve(ROC)curve analysis was performed to examine the ability of candidate metabolites to predict efficacy.Part 2.The effect and mechanism of short-chain fatty acids sodium butyrate on atopic dermatitis①The AD cell model was established by stimulating HaCaT cells and primary keratinocytes with TNF-α/IFN-γ.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the mRNA expression levels of cytokines and chemokines,detecting the expression level of tight junction proteins(Claudin-1,ZO-1,Occludin)to evaluate the barrier function,indirect immunofluorescence(IFA)and WB methods were used to detect the nuclear translocation of NF-κB p65 and the phosphorylation levels of NF-κB p65,AKT,and ERK 1/2 proteins.②Establish dinitrofluorobenzene(DNFB)-induced wild-type and GPR109a-/-AD mouse models.Using Legend Plex multifactor quantitative analysis method to detect the level of Th1/Th2 cell-related inflammatory factors in mouse serum;hematoxylin-eosin staining(H&E)and toluidine blue to evaluate the pathological changes of skin tissue;immunofluorescence histochemistry to detect Occludin protein expression and distribution in skin tissue.ResultsPart 1 Metabolomics study of Dupilumab in the treatment of AD①A total of 849 metabolites and 1753 lipids were detected.OPLS-DA analysis showed that there was a significant separation between before and after treatment group,indicating that the metabolic profile of patients changed significantly after Dupilumab treatment.②Compared with the poor response group,the metabolic characteristics of the patients in the well response group changed more significantly.Pathway enrichment analysis revealed that differential metabolites in the well response group were mainly involved in glycerophospholipid metabolism,valine,leucine,and isoleucine biosynthesis,citric acid cycle,arachidonic acid metabolism,pyrimidine metabolism,and sphingolipid metabolism.③Thirty-six baseline serum metabolites related to efficacy were screened out,and ROC analysis showed that all candidate metabolites were insufficient as biomarkers to predict efficacy.Part 2 The effect and mechanism of short-chain fatty acids sodium butyrate on atopic dermatitis①The results of qRT-PCR showed that SB down-regulated the mRNA expression levels of pro-inflammatory cytokines(IL-1β,IL-6 and IL-18)and chemokines(IL-8)induced by TNF-α/IFN-γ;SB could significantly inhibit the increase in permeability(decrease in TEER value)and the decrease in the expression level of tight junction proteins(Claudin-1,ZO-1)caused by TNF-α/IFN-γ stimulation;the results of IFA and WB showed that SB could inhibit TNF-α/IFN-γ-induced nuclear translocation of NF-κB p65 and phosphorylation of NF-κB p65 and AKT proteins.②The results of animal models showed that topical application of SB could improve typical AD-like skin lesions in DNFB-sensitized mice,reducing skin score and ear thickness,decreasing the frequency of scratching in mice,inhibiting epidermal thickening and mast cell infiltration in AD mice,and reducing serum Inflammatory cytokine levels,improving epidermal barrier function,however,had no effects on GPR109a-/-mice.WB results indicated that SB may regulate NF-κB and AKT signaling pathways by activating GPR109a receptor.Conclusions1.This study explored the changes of metabolome and lipidomics in patients with moderate to severe AD treated with Dupilumab for 16 weeks before and after treatment,highlighting the differential metabolites and related metabolic pathways of patients in the good response group,providing clues to understand the effect of Dupilumab on the metabolism of patients.2.In this study,we confirmed the protective effect of SB on TNF-α/IFN-γ-induced AD cell model and DNFB-induced AD mouse model,and explored its possible mechanism,which provides an experimental basis for the prevention and treatment of AD in the future.