Narciclasine Ameliorates Psoriasis-like Dermatitis By Regulating Phospholipid Metabolism Via STAT3/PLA2 Axis

Background:Psoriasis is a common chronic inflammatory skin disease recognized by the World Health Organization as "an incurable chronic,non-infectious,painful,disfiguring and disabling disease." Metabolic syndrome(MetS)is one of the most common and important comorbidities of psoriasis,suggesting an important role of lipid metabolism in the pathogenesis of psoriasis.Although there are a variety of treatments for psoriasis,they have different degrees of side effects,poor response rate and drug resistance.Therefore,it is still of clinical and practical significance to find new safe and effective treatments.Narciclasine(Ncs)is an alkaloid isolated from Amaryllidaceae whose biological activities include anti-tumor,anti-bacterial,anti-inflammatory,anti-angiogenesis as well as promoting energy expenditure and improving diet-related obesity.Exploring the therapeutic role of Ncs in psoriasis may improve both inflammatory responses and metabolic disorders.Objective:To investigate the therapeutic effect and possible mechanism of Ncs on IMQ-induced psoriasis-like skin inflammatidon in mice.Methods:(1)To examine the therapeutic effect of Ncs on IMQ-induced psoriatic dermatitis in mice:Female BALB/C mice aged 6-8 weeks were randomly divided into 4 groups:Con group,IMQ group and Ncs group(1mg/kg and 2mg/kg).In short,after hair removal,mice were treated with partial application of IMQ to the back skin for psoriasis modeling,and the control group was treated with Vaseline cream.During modeling,the Ncs group was injected with Ncs solution intradermally,and the IMQ and Con groups were injected with vehicle control solution.On the 7th day of modeling,the psoriasis severity of mice in each group was evaluated by PASI score.The skin thickening and the proportion of Ki67 and CD4+cells were observed by HE staining and immunohistochemistry.The expression levels of inflammatory molecules in mouse skin and serum were determined by RT-PCR and CBA inflammatory molecule kit.The proportion of Th cells in mouse spleen was detected by flow cytometry.(2)To detect the inhibitory effect of Ncs on keratinocyte inflammation:The effect of Ncs on HaCaT cell proliferation was detected by CCK8 assay.The effect of Ncs on HaCaT cell apoptosis and cell cycle was analyzed by flow cytometry.The effects of Ncs on cell cycle-related proteins CDK1,CDK2,CyclinA2 and CyclinB2 were detected by Western Blot.The expression of inflammatory molecules in LPS-stimulated HaCaT cells by Ncs was detected by RT-PCR.(3)Analysis of the target of Ncs by transcriptomics:After the modeling,skin tissues of each group were collected(n=3),and RNA sequencing was performed using MGI-SEQ 2000 platform.Data standardization and differential expression analysis were performed using DESeq2 software;GO/KEGG and GSEA enrichment analysis results were visualized using OmicShare cloud platform.mRNA levels of the enriched genes were verified by RT-PCR.(4)Analysis of the regulatory effect of Ncs on lipid metabolism by lipidomics:After the psoriasis model was established,skin tissues of mice in each group(n=5)were collected,and the lipids in the skin tissues were extracted with organic solvents.The characteristics of lipid metabolites in skin were determined by liquid chromatography-mass spectrometry(LC-MS).LipidSearch and mataX software were used to collect and screen the raw data of mass spectrometry.After statistical analysis,different characteristics of Con group,IMQ group and Ncs group were found.(5)Analysis of target cells of Ncs by single-cell sequencing:The original scRNA-seq dataset of psoriasis patients and healthy controls(n=3)was downloaded from the GEO database.Seurat R package was used to filter and analyze the data.PCA and FindClusters function were used to perform dimensionality reduction and cell clustering analysis on the data.The analysis results were visualized using dot plots,violin plots,etc.(6)Verify the target of Ncs:Transcription factor prediction analysis was performed using the ChEA3 database based on differential gene sets derived from GSEA analysis of phospholipid metabolism.Molecular docking was used to analyze the binding energy between Ncs and transcription factors with significant differences,and the target TF was determined according to its function.The expression level of target TF was verified by western blot in skin tissue.The binding site between the target TF and the PLA2 gene promoter region was analyzed through the JASPER database,and primers were designed for ChIP-qPCR experiments to verify.Results:(1)The skin of mice in the IMQ group showed typical psoriasis-like symptoms such as erythema,scaling and thickening,while the skin inflammation of mice in the Ncs group was significantly reduced,and the 2mg/kg dose group was more effective with lower PASI score.HE staining showed that compared with the IMQ group,the skin thickening of mice in the Ncs group was reduced.Immunohistochemical staining showed that Ncs treatment reduced the proportion of Ki67+cells in epidermis and CD4+T cells in dermis.The expression levels of inflammatory cytokines in skin tissue and serum and the proportion of Th17 cells in spleen were decreased in mice treated with Ncs.(2)Ncs inhibited the proliferation of HaCaT cells in a concentration-and time-dependent manner.Ncs induced S phase arrest of HaCaT cell cycle and decreased the protein expressions of CDK1,CDK2,CyclinA2 and CyclinB2.Ncs inhibited the production of LPS-stimulated inflammatory mediators in HaCaT cells including CCL1/2/20 and CXCL1/10,S100A8,and S100A9.(3)Transcriptomic analysis of skin tissues of mice in Con,IMQ and Ncs groups showed that compared with the control group,IMQ treatment up-regulated pathways related to keratinocyte differentiation and inflammation.Differential expression genes in Ncs vs IMQ group were enriched in lipid metabolism,especially in phospholipid metabolism.Several phospholipase A2 family genes were downregulated by Ncs treatment,including PLA2G2F,PLA2G3,PLA2G4B/D/E/F.(4)Lipidomic analysis of the skin tissues of Con group,IMQ group and Ncs group showed phospholipid metabolism disorder in the skin of mice treated with IMQ,while Ncs treatment restored the expression profiles of some lipid molecules to those similar to those of the Con group.Ncs treatment mainly changed the metabolic levels of phosphatidylcholine,phosphatidylethanolamine and sphingolipids,and increased the anti-inflammatory lipid molecules in the skin of mice.(5)Single-cell data analysis of skin from psoriasis patients and healthy volunteers showed that PLA2 family genes were mainly expressed in keratinocytes and up-regulated in psoriasis;RT-PCR analysis showed that Ncs could reduce the expression of PLA2 family genes induced by TNFα and IL-17A to varying degrees.(6)The potential TFs were identified by TF prediction analysis using the ChEA3 database,and the target TF STAT3 was determined in combination with their functions.WB showed that compared with IMQ group,Ncs treatment reduced STAT3 expression and phosphorylation level in psoriasis mice skin.Molecular docking also showed strong interaction between Ncs and STAT3,and the binding site was located in the SH2 domain of STAT3.The results of ChIP-qPCR showed that STAT3 could bind the promoter region of PLA2 genes.Conclusion:Our findings suggest that Narciclasine ameliorates psoriasis-like dermatitis by regulating phospholipid metabolism via STAT3/PLA2 axis.Ncs has the potential to be further developed as a novel anti-psoriasis drug.