Eupalinolide B alleviates rheumatoid arthritis through the promotion of apoptosis and autophagy via regulating the AMPK/mTOR/ULK-1 signaling axis.

The excessive proliferation of fibroblast-like synoviocytes (FLS) leads to synovial hyperplasia, a key pathological hallmark of rheumatoid arthritis (RA). Eupalinolide B (EB), a sesquiterpene lactone of Eupatorium lindleyanum DC., has anti-inflammatory effects and anti-proliferative activity in tumor cells. However, its potential use in RA treatment is unclear. This study explored EB's anti-rheumatoid activities by promoting apoptosis and autophagy in RA-FLS and the synovium of adjuvant-induced arthritis (AIA) rats, focusing on its regulation of the AMPK/mTOR/ULK-1 axis. Our findings revealed that EB inhibited proliferation, induced apoptosis, and promoted autophagy in RA-FLS. Autophagy inhibition using 3-methyladenine (3-MA) diminished EB's anti-proliferative effects, suggesting that EB promotes RA-FLS autophagy as a death mechanism. Z-VAD-FMK, a pan-caspase inhibitor, decreased EB-induced autophagy, while 3-MA co-treatment reduced caspase-3 activity, demonstrating that EB-induced apoptosis and autophagy promoted each other to support its anti-proliferative effects. In vivo, EB exhibited clear anti-arthritic effects in AIA rats, as shown by reduced paw swelling, arthritis index, serum levels of TNF-α, IL-1β, and MCP-1, and joint damage, along with decreased Ki67 expression, increased apoptosis, and enhanced autophagy in AIA rat synovium. Mechanistically, EB regulated the AMPK/mTOR/ULK-1 axis in RA-FLS and AIA rat synovium, as evidenced by higher expression of p-AMPK and p-ULK-1 and lower levels of p-mTOR. Notably, co-treatment of the AMPK inhibitor compound C negated EB's beneficial effects in RA-FLS and AIA rats. Collectively, EB demonstrated exact anti-RA effects by inducing apoptosis and autophagy via the regulation of the AMPK/mTOR/ULK-1 axis, highlighting its potential for RA therapy.