BackgroundSystemic lupus erythematosus (SLE) is an autoimmune disease that involves multiple organs. However, the current SLE-related biomarkers still lack sufficient sensitivity, specificity and predictive power for clinical application. Thus, it is significant to explore new immune-related biomarkers for SLE diagnosis and development.MethodsWe obtained seven SLE gene expression profile microarrays (GSE121239/11907/81622/65391/100163/45291/49454) from the GEO database. First, differentially expressed genes (DEGs) were screened using GEO2R, and SLE biomarkers were screened by performing WGCNA, Random Forest, SVM-REF, correlation with SLEDAI and differential gene analysis. Receiver operating characteristic curves (ROCs) and AUC values were used to determine the clinical value. The expression level of the biomarker was verified by RT‒qPCR. Subsequently, functional enrichment analysis was utilized to identify biomarker-associated pathways. ssGSEA, CIBERSORT, xCell and ImmuCellAI algorithms were applied to calculate the sample immune cell infiltration abundance. Single-cell data were analyzed for gene expression specificity in immune cells. Finally, the transcriptional regulatory network of the biomarker was constructed, and the corresponding therapeutic drugs were predicted.ResultsMultiple algorithms were screened together for a unique marker gene, MX2, and expression analysis of multiple datasets revealed that MX2 was highly expressed in SLE compared to the normal group (all P < 0.05), with the same trend validated by RT‒qPCR (P = 0.026). Functional enrichment analysis identified the main pathway of MX2 promotion in SLE as the NOD-like receptor signaling pathway (NES=2.492, P < 0.001, etc.). Immuno-infiltration analysis showed that MX2 was closely associated with neutrophils, and single-cell and transcriptomic data revealed that MX2 was specifically expressed in neutrophils. The NOD-like receptor signaling pathway was also remarkably correlated with neutrophils (r >0.3, P < 0.001, etc.). Most of the MX2-related interacting proteins were associated with SLE, and potential transcription factors of MX2 and its related genes were also significantly associated with the immune response.ConclusionOur study found that MX2 can serve as an immune-related biomarker for predicting the diagnosis and disease activity of SLE. It activates the NOD-like receptor signaling pathway and promotes neutrophil infiltration to aggravate SLE.

Context Er Miao San (EMS) is a formulation that contains Atractylodis Rhizoma and Phellodendri Cortex in 1:1 ratio, and is commonly used to treat rheumatoid arthritis (RA) and other inflammatory diseases. Objective We investigated the mechanism of action and effects of EMS on peritoneal macrophage differentiation in a rat model of adjuvant arthritis (AA). Materials and methods EMS (3, 1.5 and 0.75 g/kg; once daily) and methotrexate (0.5 mg/kg; once every 3 days) were administered orally from days 21 to 35 after immunisation. Paw swelling and arthritis index were measured; pathological changes in the ankle joint were observed using x-ray and haematoxylin eosin staining. The ratio of CD86/CD206 in macrophages was detected by flow cytometry. Examination of the miRNA-33/NLRP3 signalling pathway was examined by RT-qPCR and western blotting. The levels of cytokines in the serum and cell supernatants were tested by ELISA. Results EMS significantly reduced the AA index in rats (from 11.0 to 9.3) and pathological changes in the ankle joint (from 3.8 to 1.4). The ratio of CD86/CD206 was reduced, and polarisation to M1 improved (from 0.9 to 0.6) in macrophages of EMS-treated rats. EMS downregulated the miRNA-33/NLRP3 pathway. Furthermore, EMS treatment increased IL-10 and TGF-β levels in the serum and supernatant of macrophages of AA rats and simultaneously decreased the levels of IL-1β and TNF-α. Discussion and conclusions Our results suggest that EMS may reduce macrophage polarisation to the M1 inflammatory phenotype by downregulating the miRNA-33/NLRP3 pathway in AA rats. These findings may provide new insights into the treatment of RA.

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Background: This study aims to explore the potential molecular mechanism of Er Miao San (EMS) in rheumatoid arthritis (RA) on the basis of network pharmacology and molecular docking. Methods: The active ingredients and targets of EMS, RA and pathways were obtained through multiple databases. Common targets were analyzed by protein-protein interactions (PPIs) with the STRING database. Gene Ontology (GO) and KEGG enrichment analyses were conducted with the DAVID database to annotate targets of RA and common targets. Hub genes were obtained by the degree algorithm with the cytoHubba plug-in. Based on the determined network pharmacology, experimental validation was performed. Results: In this study, 45 active ingredients, 3162 potential drug targets and 343 pathway targets were uncovered via databases. The top 3 hub targets were obtained by the degree algorithm for 34 common target genes, including NFKBIA (encodes the IκBα protein), RELA (also named p65) and TNF. Molecular docking demonstrated that the core components fumarine, berberine and wogonin (Degree: 19, 16, 16, respectively) bind well to hub targets. In vivo experiments showed that EMS has better protective effects on adjuvant arthritis (AA) rats. In vitro studies demonstrated that EMS could inhibit the proliferation of TNF-ɑ-induced FLSs and attenuate the production of inflammatory cytokines in a dose-dependent manner. Moreover, treatment with EMS significantly decreased the ratio of p-NF-κB p65/NF-κB p65 and p-IκBα/IκBα in FLSs. Furthermore, as shown in the investigations, treatment of FLSs with 10% EMS remarkably suppressed TNF-ɑ-induced nuclear translocation of NF-κB p65 to FLSs. Conclusion: The potential mechanisms and therapeutic effects of EMS on RA may be attributed to the regulation of inflammation, which provides a reference for elucidating the mechanism of action of EMS.