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 ... -  《-》

Therapeutic effects of shikonin on adjuvant-induced arthritis in rats and cellular inflammation, migration and invasion of rheumatoid fibroblast-like synoviocytes via blocking the activation of Wnt/β-catenin pathway.

Shikonin (SKN), the main bioactive component isolated from Lithospermum erythrorhizon Sieb et Zucc, has multiple activities including anti-rheumatic effect, but its specific roles and the precise mechanisms in regulating biological properties of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) are unclear and need further clarification. This study explored the therapeutic roles of SKN on rat adjuvant-induced arthritis (AIA) and cellular inflammation, migration and invasion of TNF-α-induced RA FLS (MH7A cells), and further demonstrated the involved mechanisms. SKN was intraperitoneally given to AIA rats and its therapeutic role was valued. The effects of SKN in vivo and in vitro on the production of pro-inflammatory factors were examined by ELISA and western blot. Wound-healing, transwell and phalloidin staining assay were carried out to evaluate the effects of SKN on TNF-α-induced migration and invasion in RA FLS. The involvement of Wnt/β-catenin pathway was checked by immunohistochemistry or immunofluorescence assay for β-catenin and western blot for pathway-related proteins. SKN treatment in AIA rats reduced paw swelling, arthritis index and pathological damage of ankle joints, indicating its anti-arthritic effect in vivo. SKN had anti-inflammatory roles in vivo and in vitro, evidenced by inhibiting the production of pro-inflammatory factors (like IL-1β, IL-6, IL-8, TNF-α, MMP-2 and MMP-9) in sera and synovium of AIA rats, and in TNF-α-induced MH7A cells. Gelatin zymography result revealed the suppression of SKN on TNF-α-induced MMP-2 activity in vitro. Moreover, SKN inhibited TNF-α-induced migration, invasion and cytoskeletal reorganization in MH7A cells. Mechanistically, SKN suppressed the activation of Wnt/β-catenin signaling in AIA rat synovium and in TNF-α-induced MH7A cells, indicated by the reduced protein levels of Wnt1, p-GSK-3β (Ser9) and β-catenin, the raised protein level of GSK-3β and the decreased nuclear translocation of β-catenin. Interestingly, the combination of LiCl (Wnt/β-catenin agonist) canceled the therapeutic functions of SKN on cellular inflammation, migration and invasion in TNF-α-induced MH7A cells, whereas XAV939 (Wnt/β-catenin inhibitor) enhanced the therapeutic roles of SKN. SKN showed therapeutic effects on rat AIA and cellular inflammation, migration and invasion of TNF-α-stimulated RA FLS via interrupting Wnt/β-catenin pathway.

Liu FY ，Wang MQ ，Liu MM ，Li T ，Wang XH ，Jiang F ，Wu XJ ，Cheng J ，Cai L ，Li R ... -  《-》

Chelerythrine ameliorates rheumatoid arthritis by modulating the AMPK/mTOR/ULK-1 signaling pathway.

Rheumatoid arthritis (RA) is a long-term, progressive, and disabling autoimmune disease. It causes inflammation, swelling and pain in and around the joints and other body organs. Currently, no cure is available for RA. Clinical interventions can only relieve the condition, and at least 30% of RA patients do not respond to first‑line therapy. This means that the development of more effective therapies against RA is urgently needed. This study aimed to assess the anti-rheumatoid arthritis effect of chelerythrine (CLT) and explore its mechanism of action. The cytotoxic effect of CLT on human rheumatoid arthritis fibroblast-like synoviocyte (HFLS-RA) cells and HFLS-normal cells were measured by MTT assay. The growth and migration of HFLS-RA cells were determined by colony-formation and wound-healing assay. The level of intracellular reactive oxygen species (ROS) was detected using the DCFH-DA reagent. Cell apoptosis was measured by flow cytometry, TUNEL staining, caspase 3 activity, as well as the activation of apoptosis related proteins. In addition, the levels of autophagy related markers such as LC3B and P62 were determined by immunocytochemistry and western blotting. Lastly, the anti-RA effect of CLT was evaluated in an Adjuvant-Induced Arthritis(AIA) rat model and the severity of arthritis was detected and quantified using macroscopic inspection and X‑ray imaging. We discovered that treatment with CLT effectively inhibited the migration and colony-formation of the HFLS-RA cells and resulted in cell death. Moreover, CLT increased the intracellular level of ROS and the apoptotic rate of HFLS-RA by activating the AMPK/mTOR/ULK-1 signaling pathways. In vivo study showed CLT effectively ameliorated AIA in rats, protecting them from inflammation and bone damage. Our study shows CLT is an effective agent for ameliorating RA in vitro and in vivo by modulation of the AMPK/mTOR/ULK-1 signaling pathway. These findings indicate that CLT is a great potential candidate for development as a therapeutic agent for the prevention and treatment of RA.

Cai J ，Zhang LC ，Zhao RJ ，Pu LM ，Chen KY ，Nasim AA ，Leung EL ，Fan XX ... -  《-》

Penta-acetyl geniposide induces apoptosis of fibroblast-like synoviocytes from adjuvant-induced arthritis rats in vitro, associated with inhibition of NF-κB activation.

Approaches promoting fibroblast-like synoviocytes (FLS) apoptosis are considered as a meaningful strategy for rheumatoid arthritis (RA) treatment. We have previously reported the anti-arthritic effect of penta-acetyl geniposide ((Ac)5GP, an active derivative of geniposide) on adjuvant-induced arthritis (AIA) rats in vivo. The present study aimed to investigate the pro-apoptotic effect of (Ac)5GP on AIA FLS in vitro and the underlying molecular mechanisms. Rat AIA was induced by complete Freund's adjuvant, and FLS were primary-cultured from synovial tissues. AIA FLS were treated with (Ac)5GP (50, 100 and 200 μM) for 48 h and cell proliferation and apoptosis were respectively examined. The involvement of apoptosis-related proteins (Bax, Bcl-2 and caspase 3) and nuclear factor kappa B (NF-κB) signaling pathway was checked. (Ac)5GP inhibited the viability of AIA FLS and reduced the percentage of Ki67-positive cells in AIA FLS. Particularly, (Ac)5GP promoted AIA FLS apoptosis in vitro by inducing apoptotic nuclear morphology, facilitating DNA ladder formation and increasing percentages of both early and late apoptotic cells. (Ac)5GP treatment on AIA FLS decreased Bcl-2 protein level whereas increased the levels of Bax and caspase 3 proteins. Moreover, (Ac)5GP reduced the degradation and phosphorylation of IκBα, down-regulated NF-κB p65 protein level in nucleus and inhibited NF-κB p65 nuclear translocation. (Ac)5GP had a potent pro-apoptotic effect on AIA FLS in vitro, which is associated with regulating apoptosis-related proteins and inhibiting NF-κB activation.

Cai L ，Li CM ，Chen WN ，Qiu YY ，Guo YL ，Li R ... -  《-》

[Berberine inhibits autophagy and promotes apoptosis of fibroblast-like synovial cells from rheumatoid arthritis patients through the ROS/mTOR signaling pathway].

Zong S ，Zhou J ，Cai W ，Yu Y ，Wang Y ，Song Y ，Cheng J ，Li Y ，Gao Y ，Wu B ，Xian H ，Wei F ... -  《-》