TY  - EJOU
AU  - Zhang, Zheng 
AU  - Huang, Qingchun 

TI  - Luteolin Reverses Methotrexate Resistance in Rheumatoid Arthritis via Targeting Sp1-DHFR Axis and p38 MAPK/STAT3 Signaling
T2  - BIOCELL

PY  - 
VL  - 
IS  - 
SN  - 1667-5746

AB  - <b>Background</b>: 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. <b>Methods</b>: An MTX-resistant human fibroblast-like synoviocyte (FLS) model was established <i>in vitro</i> 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 <i>in vivo</i> using a collagen-induced arthritis (CIA) mouse model treated with MTX alone or in combination with luteolin. <b>Results</b>: Compared to parental cells, MTX-resistant FLS showed ~15-fold higher DHFR mRNA (<i>p</i> &lt; 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 (<i>p</i> &lt; 0.001) and increasing total apoptosis to 16.0% vs. 13.7% with MTX alone (<i>p</i> &lt; 0.01). Mechanistically, luteolin suppressed Sp1 binding to the DHFR promoter by ~35% (<i>p</i> &lt; 0.01), reducing DHFR promoter activity and downregulating DHFR mRNA/protein. Concurrently, luteolin inhibited p38 MAPK/STAT3 phosphorylation by ~40–50% (<i>p</i> &lt; 0.01). In CIA mice, luteolin + MTX outperformed MTX monotherapy: arthritis score decreased from 1.5 to 0.67 (<i>p</i> &lt; 0.001), serum IL-6 from 150 pg/mL to 50 pg/mL (<i>p</i> &lt; 0.001), and synovial DHFR expression was reduced. These improvements indicate clinically relevant attenuation of disease severity. <b>Conclusion</b>: 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.
KW  - Luteolin; methotrexate resistance; rheumatoid arthritis; dihydrofolate reductase; specificity protein 1

DO  - 10.32604/biocell.2026.083358