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.

Gu SL ，Liu XS ，Xu ZS ，Li LL ，Wu XJ ，Li FL ，Huang Y ，Ran X ，Li R ... -  《-》

Shikonin suppresses rheumatoid arthritis by inducing apoptosis and autophagy via modulation of the AMPK/mTOR/ULK-1 signaling pathway.

The overproliferation of fibroblast-like synoviocytes (FLS) contributes to synovial hyperplasia, a pivotal pathological feature of rheumatoid arthritis (RA). Shikonin (SKN), the active compound from Lithospermum erythrorhizon, exerts anti-RA effects by diverse means. However, further research is needed to confirm SKN's in vitro and in vivo anti-proliferative functions and reveal the underlying specific molecular mechanisms. This study revealed SKN's anti-proliferative effects by inducing both apoptosis and autophagic cell death in RA FLS and adjuvant-induced arthritis (AIA) rat synovium, with involvement of regulating the AMPK/mTOR/ULK-1 pathway. SKN's influences on RA FLS were assessed for proliferation, apoptosis, and autophagy with immunofluorescence staining (Ki67, LC3B, P62), EdU incorporation assay, staining assays of Hoechst, Annexin V-FITC/PI, and JC-1, transmission electron microscopy, mCherry-GFP-LC3B puncta assay, and western blot. In AIA rats, SKN's anti-arthritic effects were assessed, and its impacts on synovial proliferation, apoptosis, and autophagy were studied using Ki67 immunohistochemistry, TUNEL, and western blot. The involvement of AMPK/mTOR/ULK-1 pathway was examined via western blot. SKN suppressed RA FLS proliferation with reduced cell viability and decreased Ki67-positive and EdU-positive cells. SKN promoted RA FLS apoptosis, as evidenced by apoptotic nuclear fragmentation, increased Annexin V-FITC/PI-stained cells, reduced mitochondrial potential, elevated Bax/Bcl-2 ratio, and increased cleaved-caspase 3 and cleaved-PARP protein levels. SKN also enhanced RA FLS autophagy, featuring increased LC3B, reduced P62, autophagosome formation, and activated autophagic flux. Autophagy inhibition by 3-MA attenuated SKN's anti-proliferative roles, implying that SKN-induced autophagy contributes to cell death. In vivo, SKN mitigated the severity of rat AIA while also reducing Ki67 expression, inducing apoptosis, and enhancing autophagy within AIA rat synovium. Mechanistically, SKN modulated the AMPK/mTOR/ULK-1 pathway in RA FLS and AIA rat synovium, as shown by elevated P-AMPK and P-ULK-1 expression and decreased P-mTOR expression. This regulation was supported by the reversal of SKN's in vitro and in vivo effects upon co-administration with the AMPK inhibitor compound C. SKN exerted in vitro and in vivo anti-proliferative properties by inducing apoptosis and autophagic cell death via modulating the AMPK/mTOR/ULK-1 pathway. Our study revealed novel molecular mechanisms underlying SKN's anti-RA effects.

Wang XH ，Shen CP ，Wang TT ，Huang Y ，Jin Y ，Zhou MY ，Zhang MY ，Gu SL ，Wang MQ ，Liu ZC ，Li R ，Cai L ... -  《-》

Silencing aquaporin 1 inhibits autophagy to exert anti-rheumatoid arthritis effects in TNF-α-induced fibroblast-like synoviocytes and adjuvant-induced arthritis rats.

Fibroblast-like synoviocytes (FLS) are key players in rheumatoid arthritis (RA) by resisting apoptosis via increased autophagy. Elevated synovial aquaporin 1 (AQP1) affects RA FLS behaviors, but its relationship with FLS autophagy is unclear. We aim to clarify that silencing AQP1 inhibits autophagy to exert its anti-RA effects. We studied the effects and mechanisms of AQP1 silencing on autophagy in TNF-α-induced RA FLS and examined the crucial role of autophagy inhibition in its impacts on RA FLS pathogenic behaviors. We explored whether silencing synovial AQP1 relieved rat adjuvant-induced arthritis (AIA) by reducing synovial autophagy. TNF-α stimulation increased AQP1 expression and autophagy levels in RA FLS, with a positive correlation between them. AQP1 silencing inhibited autophagy in TNF-α-stimulated RA FLS, along with suppressing proliferation, promoting apoptosis, and mitigating inflammation. Notably, the inhibitory effects of AQP1 silencing on RA FLS pathogenic behaviors were cancelled by autophagy activation with rapamycin (Rapa) but enhanced by autophagy inhibition using 3-Methyladenine. Mechanistically, silencing AQP1 enhanced the binding of Bcl-2 to Beclin1 by decreasing Beclin1-K63 ubiquitination, thus inhibiting RA FLS autophagy. In vivo, silencing synovial AQP1 relieved the severity and development of rat AIA, alongside reducing Ki67 expression, promoting apoptosis, and decreasing autophagy within AIA rat synovium. Expectedly, the Rapa co-administration nullified the anti-AIA effects of silencing synovial AQP1. These findings reveal that silencing AQP1 inhibits RA FLS pathogenic behaviors and attenuates rat AIA through autophagy inhibition. This study may help clarify the pathogenic role of AQP1 in enhancing autophagy during RA development.

Zhang MY ，Wang MQ ，Huang Y ，Gu SL ，Zhou MY ，Xu ZS ，Li LL ，Lv M ，Cai L ，Li R ... -  《-》

Dual drug nanoparticle synergistically induced apoptosis, suppressed inflammation, and protected autophagic response in rheumatoid arthritis fibroblast-like synoviocytes.

Rheumatoid arthritis (RA) is a chronic immune-mediated joint inflammatory disorder associated with aberrant activation of fibroblast-like synoviocytes (FLS). Recently, FLS gained importance due to its crucial role in RA pathogenesis, and thus, targeting FLS is suggested as an attractive treatment strategy for RA. FLS-targeted approaches may be combined with disease-modifying antirheumatic drugs (DMARDs) and natural phytochemicals to improve efficacy in RA control and negate immunosuppression. In this study, we assessed the therapeutic effectiveness of DD NP HG in primary RA-FLS cells isolated from the synovial tissue of FCA-induced RA rats. We observed that DD NP HG had good biosafety for healthy FLS cells and, at higher concentrations, a mild inhibitory effect on RA-FLS. The combination therapy (DD NP HG) of MTX NP and PEITC NE in RA-FLS showed a higher rate of apoptosis with significantly reduced LPS-induced expression of pro-inflammatory cytokines (TNF-α, IL-17A, and IL-6) in arthritic FLS. Further, the gene expression studies showed that DD NP HG significantly down-regulated the mRNA expression of IL-1β, RANKL, NFATc1, DKK1, Bcl-xl, Mcl-1, Atg12, and ULK1, and up-regulated the mRNA expression of OPG, PUMA, NOXA and SQSTM1 in LPS-stimulated RA-FLS cells. Collectively, our results demonstrated that DD NP HG significantly inhibited the RA-FLS proliferation via inducing apoptosis, down-regulating pro-inflammatory cytokines, and further enhancing the expression of genes associated with bone destruction in RA pathogenesis. A nanotechnology approach is a promising strategy for the co-delivery of dual drugs to regulate the RA-FLS function and achieve synergistic treatment of RA.

Haloi P ，Choudhary R ，Lokesh BS ，Konkimalla VB ... -  《-》

Bacopaside I, acting as an aquaporin 1 inhibitor, ameliorates rheumatoid arthritis via suppressing aquaporin 1-mediated autophagy.

Aquaporin 1 (AQP1) is a promising target for regulating fibroblast-like synoviocyte (FLS) behaviors in rheumatoid arthritis (RA). Bacopaside I (BSI), the main active compound of the herbal medicine Bacopa monnieri with anti-RA effects, inhibits tumor cell growth by blocking AQP1, but its potential use in RA is unclear. To address BSI's anti-RA effects and elucidate its underlying mechanisms. We investigated BSI's therapeutic effects on TNF-α-induced RA FLS and identified AQP1 as its direct target through molecular docking, cellular thermal shift assay (CETSA), and AQP1 knockdown experiments. We studied BSI's impacts on rat adjuvant-induced arthritis (AIA) and synovial proliferation, apoptosis, and autophagy in AIA rat synovium. We explored the role of autophagy inhibition in BSI's effects in vitro and in vivo by co-treating with the autophagy activator rapamycin (Rapa) and/or the inhibitor 3-methyladenine (3-MA). BSI suppressed proliferation, promoted apoptosis, and reduced autophagy in TNF-α-stimulated RA FLS. Notably, BSI's in vitro effects were reduced by Rapa and enhanced by 3-MA. The molecular docking and CETSA confirmed BSI's binding to AQP1, while AQP1 knockdown invalidated BSI's in vitro effects, further indicating AQP1 as the target of BSI. In vivo, BSI attenuated the severity of rat AIA, alongside reduced synovial proliferation, increased apoptosis, and decreased autophagy within AIA rat synovium. Moreover, Rapa co-treatment negated BSI's effects on synovial proliferation and apoptosis and abolished its anti-AIA activity. BSI, as an AQP1 inhibitor, hindered AQP1-mediated autophagy, causing increased apoptosis, reduced proliferation in RA FLS, and relieved rat AIA symptoms.

Huang Y ，Zhou MY ，Li LL ，Lv M ，Xu ZS ，Wu XJ ，Gu SL ，Zhang MY ，Cai L ，Li R ... -  《-》