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 ... -  《-》

Therapeutic Effect of Penta-acetyl Geniposide on Adjuvant-Induced Arthritis in Rats: Involvement of Inducing Synovial Apoptosis and Inhibiting NF-κB Signal Pathway.

Cai L ，Li CM ，Tang WJ ，Liu MM ，Chen WN ，Qiu YY ，Li R ... -  《-》

Penta-acetyl Geniposide Suppresses Migration, Invasion, and Inflammation of TNF-α-Stimulated Rheumatoid Arthritis Fibroblast-Like Synoviocytes Involving Wnt/β-Catenin Signaling Pathway.

We previously reported that penta-acetyl geniposide ((Ac)5GP, an active derivative of geniposide) showed anti-arthritic effect on adjuvant-induced arthritis (AIA) rats by promoting the apoptosis of AIA fibroblast-like synoviocyte (FLS). This study aimed to demonstrate the effects of (Ac)5GP on migration, invasion, and inflammation of TNF-α-stimulated rheumatoid arthritis (RA) FLS (MH7A cell) and to explore the involved mechanisms. MTT assay was used to determine the applied non-cytotoxic doses of (Ac)5GP (12.5, 25, 50 μM) in vitro. Results of wound-healing, transwell, and phalloidin staining assays indicated that (Ac)5GP reduced the migration, invasion, and F-actin cytoskeletal reorganization of TNF-α-stimulated MH7A. Results of ELISA and western blot assays confirmed that (Ac)5GP reduced TNF-α-induced production of pro-inflammatory cytokines (like IL-1β, IL-6, IL-8) and matrix metalloproteinases (MMPs, such as MMP-2 and MMP-9). Moreover, (Ac)5GP inhibited TNF-α-induced activation of Wnt/β-catenin pathway, evidenced by reducing the protein levels of Wnt1, p-GSK-3β (Ser9), and β-catenin and preventing β-catenin nuclear translocation. Importantly, the combination of XAV939 (an inhibitor of Wnt/β-catenin) promoted the actions of (Ac)5GP on TNF-α-induced migration, invasion, and inflammation, further revealing the involvement of Wnt/β-catenin pathway underlying the therapeutic effects of (Ac)5GP on TNF-α-stimulated MH7A. In vivo, (Ac)5GP relieved the progression and severity of rat collagen-induced arthritis, related to reducing the levels of IL-1β, IL-6, IL-8, MMP-2, and MMP-9 as well as inhibiting Wnt/β-catenin pathway in synovial tissues. Collectively, (Ac)5GP could suppress TNF-α-induced migration, invasion, and inflammation in RA FLS involving Wnt/β-catenin pathway and (Ac)5GP might be as a candidate agent for RA treatment.

Cai L ，Mu YR ，Liu MM ，Zhou MY ，Meng B ，Liu FY ，Li R ... -  《-》

Apoptotic Effect of Geniposide on Fibroblast-Like Synoviocytes in Rats with Adjuvant-Induced Arthritis via Inhibiting ERK Signal Pathway In Vitro.

Li R ，Cai L ，Tang WJ ，Lei C ，Hu CM ，Yu F ... -  《-》

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 ... -  《-》