Syringin inhibits the crosstalk between macrophages and fibroblast-like synoviocytes to treat rheumatoid arthritis via PDE4.

Syringin (SRG) is well-known for its anti-inflammatory effects. However, its pharmacological mechanisms against rheumatoid arthritis (RA) are not fully understood. We assessed the anti-RA effects of SRG using a collagen-induced arthritis (CIA) rat model. And, we employed single-cell RNA sequencing (scRNA-seq) to analyze the changes in cell types and gene expression in the synovial tissues. Building on these observations, we investigated the effects of SRG on M1 macrophage polarization and RA-fibroblast-like synoviocytes (FLS) proliferation. Our findings highlighted the anti-RA effects of SRG on CIA rat. scRNA-seq of rat synovial tissues revealed significant changes in M1 and RA-FLS. Specifically, SRG decreased the levels of inflammatory factors in the supernatants of LPS and IFN-γ induced THP-1 cells and downregulated M1-polarized markers in these cells. Further analysis indicated that SRG's regulation of phosphodiesterase 4 (PDE4) and its associated factors was crucial for its anti-M1 polarization effects. Besides, we found that SRG inhibited the activation of FLS in vivo but showed no direct effects on RA-FLS in vitro. However, in RA-FLS, co-cultured with supernatant from SRG-treated M1-polarized THP-1 cells exhibited lower ability of cell proliferation and activation as compared to co-cultured with supernatant from M1-polarized THP-1 cells. By integrating scRNA-seq analysis with in vivo and in vitro validations, our study revealed that SRG achieved its anti-RA effects by blocking the interaction between macrophages and RA-FLS, with PDE4 playing a central role. This study may provide a novel research paradigm in studying the multi-cell regulatory mechanisms of natural compounds.

Cong S ，Wang N ，Pei H ，Li Z ，Meng Y ，Maimaitituersun S ，Zhao X ，Wan R ，Wan Q ，Luo L ，Bian Y ，Wen W ，Cui H ... -  《-》

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 ... -  《-》

Suberosin attenuates rheumatoid arthritis by repolarizing macrophages and inhibiting synovitis via the JAK/STAT signaling pathway.

Rheumatoid arthritis (RA) is a systemic disease that primarily manifests as chronic synovitis of the symmetric small joints. Despite the availability of various targeted drugs for RA, these treatments are limited by adverse reactions, warranting new treatment approaches. Suberosin (SBR), isolated from Plumbago zeylanica-a medicinal plant traditionally used to treat RA in Asia-possesses notable biological activities. This study aimed to investigate the effects and potential underlying pathways of SBR on RA. Tumor necrosis factor-alpha (TNF-α) induced inflammation in RA-derived fibroblast-like synoviocytes (RA-FLS), and the expression of proinflammatory mediators was assessed using q-RT PCR and ELISA after treatment with various SBR concentrations. Bone marrow-derived macrophages (BMDMs) were induced to differentiate into M1 and M2 macrophages, followed by treatment with various SBR concentrations and macrophage polarization assessment. Low-dose (0.5 mg/kg/d) and high-dose (2 mg/kg/d) SBR regimens were administered to a collagen-induced arthritis (CIA) mouse model for 21 days, and the anti-arthritic effects of SBR were evaluated. Network pharmacology and molecular docking analyses were used to predict the anti-arthritic targets of SBR. The effect of SBR on the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway was evaluated. SBR suppressed macrophage polarization toward the M1 phenotype while enhancing their polarization toward the M2 phenotype. SBR reduced the levels of proinflammatory mediators in TNF-α-induced RA-FLS. Mechanistically, SBR inhibited the phosphorylation of the JAK1/STAT3 signaling pathway in RA-FLS and M1 macrophages and promoted the phosphorylation of the JAK1/STAT6 pathway in M2 macrophages, enhancing M2 polarization. In vivo, prophylactic treatment of low-dose SBR reduced M1 macrophage infiltration into synovial tissue, increased the proportion of M2 macrophages, and decreased the expression of inflammatory mediators in the serum and synovial tissue, alleviating synovial inflammation. SBR significantly alleviated arthritis in CIA mice through macrophage repolarization and inhibition of inflammation. SBR significantly reduced clinical symptoms, joint pathological damage, and expression inflammatory cytokine expression in CIA mice. SBR exhibited anti-arthritic effects via the JAK1/STAT3 and JAK1/STAT6 signaling pathways, inhibiting synovial tissue inflammation and M1 macrophage polarization while promoting M2 macrophage polarization. Therefore, SBR may be an effective candidate for RA treatment.

Liu H ，Li Q ，Chen Y ，Dong M ，Liu H ，Zhang J ，Yang L ，Yin G ，Xie Q ... -  《-》

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 ... -  《-》

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 ... -  《-》