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 ... -  《-》

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 ... -  《-》

[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 ... -  《-》

The enhanced mitochondrial dysfunction by cantleyoside confines inflammatory response and promotes apoptosis of human HFLS-RA cell line via AMPK/Sirt 1/NF-κB pathway activation.

Cantleyoside (CA) is a kind of iridoid glycosides in Pterocephalus hookeri (C. B. Clarke) Höeck. The purpose of this study was to investigate the effects of CA on human rheumatoid arthritis fibroblast synovial cells (HFLS-RA). Cell proliferation of HFLS-RA was assessed by CCK-8. ELISA was used to detect cytokines NO, TNF-α, IL-1β/6, MCP-1, MMP-1/3/9 and metabolism-related ATPase activities and ATP levels. JC-1, DCFH-DA, Fluo-3 AM and Calcein AM probes were used to detect mitochondrial membrane potential (MMP), reactive oxygen species (ROS), Ca2+ and mitochondrial permeability conversion pore (MPTP), respectively. Isolated mitochondria assay was used to detect mitochondrial swelling. Oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and real-time ATP production were measured using a Seahorse analyzer. Apoptosis was detected by TUNEL and Hoechst staining. Western blot was used to detect the expressions of AMPK/p-AMPK, Sirt 1, IκBα, NF-κB p65/p-NF-κB p65, Bcl-2 and Bax. Cytoplasmic nuclear isolation was also performed to detect the translocation of NF-κB. CA significantly suppressed cell proliferation and the levels of NO, TNF-α, IL-1β/6, MCP-1 and MMP-1/3/9 in HFLS-RA. In addition, CA promoted the apoptosis of HFLS-RA by increasing TUNEL and Hoechst positive cells and the ratio of Bax/Bcl-2. Inhibition of energy metabolism in HFLS-RA by CA reduced OCR, ECAR and real-time ATP generation rate. Importantly, CA promoted p-AMPK and Sirt 1 expression, inhibited IκBα degradation to reduce p-NF-κB and translocation. The results suggest that CA activates the AMPK/Sirt 1/NF-κB pathway by promoting mitochondrial dysfunction, thereby exerting anti-inflammatory and pro-apoptotic effects.

Bai J ，Xie N ，Hou Y ，Chen X ，Hu Y ，Zhang Y ，Meng X ，Wang X ，Tang C ... -  《-》

Tris (1, 3-dichloro-2-propyl) phosphate induces apoptosis and autophagy in SH-SY5Y cells: Involvement of ROS-mediated AMPK/mTOR/ULK1 pathways.

Tris (1, 3-dichloro-2-propyl) phosphate (TDCIPP), an extensively used organophosphorus flame retardant, is frequently detected in the environment and biota. Recent studies have shown that TDCIPP has neurotoxic effects. We hypothesized that the neurotoxicity might occur via the induction of the apoptosis and autophagy pathways. In the present study, we investigated TDCIPP-induced apoptotic death and autophagy in SH-SY5Y cells. Treatment with TDCIPP induced increased reactive oxygen species (ROS) generation and cell apoptosis, as well as autophagy. The autophagy inhibitor 3-methyladenine (3-MA) markedly decreased the expression of the autophagy marker beclin-1, microtubule-associated protein light chain 3-II (LC3II), p62/sequestosome 1 (SQSTM1) degradation, and promoted apoptosis. Conversely, the autophagy inducer rapamycin (Rapa) alleviated TDCIPP-induced apoptosis and markedly increased the expression of the autophagy markers. Pretreatment with N-acetyl cysteine (NAC) eliminated the increased ROS generation, resulting in increased cell viability. For further examination of the signaling pathways involved in TDCIPP-induced autophagy, compound C, a pharmacological inhibitor of adenosine monophosphate activated protein kinase (AMPK) was used. Western blotting showed that compound C markedly reduced the expression of phospho-AMPK (p-AMPK) and phospho-Unc-51-like kinase 1 (p-ULK1), increased phospho-mammalian target of rapamycin (p-mTOR) expression, and decreased beclin-1 and LC3II expression. These results suggested that the AMPK/mTOR/ULK1 signaling pathway was involved in TDCIPP-induced autophagy. The antioxidant NAC antagonized TDCIPP-induced activation of AMPK and autophagy. Taken together, our findings provide the first evidence that TDCIPP promotes apoptosis and autophagy simultaneously and that this process involves the ROS-mediated AMPK/mTOR/ULK1 pathways. Lastly, the induction of autophagy is a protective mechanism against TDCIPP-induced apoptosis.

Li R ，Zhou P ，Guo Y ，Lee JS ，Zhou B ... -  《-》