Luteolin Reverses Methotrexate Resistance in Rheumatoid Arthritis via Targeting Sp1-DHFR Axis and p38 MAPK/STAT3 Signaling

Zheng Zhang^1,2, Qingchun Huang^1,*
1 The Second Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou, China
2 Department of Rheumatology, The Affiliated Hospital of Hebei University, Baoding, China
* Corresponding Author: Qingchun Huang. Email: email
(This article belongs to the Special Issue: Novel Drug and Cell-Based Therapies for Chronic Diseases Combination Treatment Strategies)

BIOCELL https://doi.org/10.32604/biocell.2026.083358

Received 02 April 2026; Accepted 16 June 2026; Published online 17 August 2026

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Abstract

Background: Rheumatoid arthritis (RA) is a disabling autoimmune disease where methotrexate resistance, driven by Specificity protein 1 (Sp1)-mediated upregulation of dihydrofolate reductase, poses a key clinical challenge. Preliminary network pharmacology analysis identified Dihydrofolate reductase (DHFR) as a high-confidence target of luteolin in RA. Given that Sp1 is a known transcriptional activator of DHFR, we further investigated whether luteolin modulates the DHFR-Sp1 axis, thereby prompting this study to explore its potential in overcoming Methotrexate (MTX) resistance. Methods: An MTX-resistant human fibroblast-like synoviocyte (FLS) model was established in vitro to investigate whether luteolin can reverse MTX resistance and elucidate the underlying mechanisms. Assessments included cell viability, apoptosis, RT-qPCR, Western blotting, DHFR promoter reporter assays, and chromatin immunoprecipitation (ChIP) for Sp1. Translational relevance was evaluated in vivo using a collagen-induced arthritis (CIA) mouse model treated with MTX alone or in combination with luteolin. Results: Compared to parental cells, MTX-resistant FLS showed ~15-fold higher DHFR mRNA (p < 0.001) and increased p38 MAPK/STAT3 phosphorylation. Co-treatment with luteolin (10 μM) and MTX significantly resensitized resistant cells, reducing viability to 60% of control (p < 0.001) and increasing total apoptosis to 16.0% vs. 13.7% with MTX alone (p < 0.01). Mechanistically, luteolin suppressed Sp1 binding to the DHFR promoter by ~35% (p < 0.01), reducing DHFR promoter activity and downregulating DHFR mRNA/protein. Concurrently, luteolin inhibited p38 MAPK/STAT3 phosphorylation by ~40–50% (p < 0.01). In CIA mice, luteolin + MTX outperformed MTX monotherapy: arthritis score decreased from 1.5 to 0.67 (p < 0.001), serum IL-6 from 150 pg/mL to 50 pg/mL (p < 0.001), and synovial DHFR expression was reduced. These improvements indicate clinically relevant attenuation of disease severity. Conclusion: Luteolin reverses MTX resistance in RA synovial fibroblasts through a dual mechanism: Sp1-dependent suppression of DHFR expression and concurrent inhibition of pro-survival signaling pathways.