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 ... -  《-》

Experimental study of the effects of pirfenidone and nintedanib on joint inflammation and pulmonary fibrosis in a rheumatoid arthritis-associated interstitial lung disease mouse model.

Rheumatoid arthritis-associated interstitial lung disease (RA-ILD) is a serious pulmonary complication in rheumatoid arthritis (RA) patients, is one of the leading causes of death in RA patients. This study was designed to determine whether pirfenidone and nintedanib can alleviate joint inflammation and pulmonary fibrosis in a mouse model of RA-ILD. Male DBA/1 mice were injected with bovine type II collagen (bCII) to establish the RA-ILD model. Pirfenidone (20 mg/kg) and nintedanib (60 mg/kg) were administered, and body weight, joint swelling, pathology of the lungs and knees, macrophage polarization in bronchoalveolar lavage fluid (BALF), and the fluorescence intensity of phosphorylated janus kinase 2/phosphorylated signal transducer and activator of transcription 3 (p-Jak2/p-Stat3) in the lungs and knees were determined. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure mRNA, and western blotting was conducted to detect the protein. Macrophage line RAW264.7 were divided into the following groups: the RAW264.7, RAW264.7 + IL-4/IL-13 (IL-4/IL-13, 60 ng/mL), RAW264.7 + IL-4/IL-13 + pirfenidone (0.5 and 1.0 mmol/L), RAW264.7 + IL-4/IL-13 + nintedanib (0.1 and 0.5 µmol/L). Mouse primary fibroblast-like synovial (FLS) cells were divided into the following groups: the FLS, FLS + transforming growth factor-β1 (TGF-β1; 10 µg/L), FLS + TGF-β1 + pirfenidone (0.5 and 1.0 mmol/L), FLS + TGF-β1 + nintedanib (0.1 and 0.5 µmol/L) groups. Proteins in each group were detected. The body weights of the mice in the pirfenidone and nintedanib groups were greater than those in the RA-ILD group (P<0.05), the arthritis scores were also significantly lower (P<0.05). The proportion of M2-type macrophages in the BALF of the nintedanib group significantly decreased (P<0.05). Inflammatory cell infiltration in the lung was reduced in the pirfenidone and nintedanib groups; additionally, decreased levels of synovium, collagen, angiogenesis, and bone destruction of the knee joint and a lower synovitis score were observed (P<0.05). Masson staining revealed that collagen deposition in the lungs in the pirfenidone and nintedanib groups was reduced (P<0.05). P-Jak2/p-Stat3 expression in the lungs and knee joints in the pirfenidone and nintedanib groups was low (P<0.001 in the lung and P<0.005 in the knee joint). The mRNA expression of collagen-IV, Stat3, and Jak2 in the lungs was lower in the pirfenidone and nintedanib (P<0.05); the protein expression levels of p-Jak2/Jak2, p-Stat3/Stat3, p-Smad3/Smad3, and TGF-β receptor 2 (TGF-βR2) in the lungs in the pirfenidone and nintedanib groups decreased (P<0.05). P-Jak2/Jak2, p-Stat3/Stat3, TGF-βR2, cluster of differentiation 206 (CD206), and arginase-1 (ARG-1) were lower in the pirfenidone and nintedanib groups of RAW264.7 cells (at all different concentrations, P<0.05). P-JAK2/JAK2, p-Stat3/Stat3, and TGF-βR2 were lower in the pirfenidone and nintedanib groups of FLS cells (at all different concentrations, P<0.05). Pirfenidone and nintedanib not only reduced the degree of pulmonary fibrosis but also relieved joint symptoms in an RA-ILD mouse model. The mechanisms of action are related to the inhibition of the TGF-β signaling pathway, Jak2/Stat3 signaling pathway, and polarization of macrophages to the M2 phenotype.

Liu J ，Xu L ，Guan X ，Zhang J ... -  《-》

Sappanone A attenuates rheumatoid arthritis via inhibiting PI3K/AKT/NF-κB and JAK2/STAT3 signaling pathways in vivo and in vitro.

Sappanone A (SA), a bioactive compound in Caesalpinia sappan L., has anti-inflammation, antioxidant, and bone protection activities. But its effect on rheumatoid arthritis (RA) and the underlying mechanism are incompletely understood. Candidate targets of SA against RA were screened by network pharmacology and further validated by molecular docking. CIA rats and HFLS-RA were used to explore the effect and mechanism of SA on RA in vivo and in vitro, respectively. Macroscopic inspection (body weight, paw swelling, arthritis index), histological examination and micro-CT were used to evaluate the anti-RA effect of SA in vivo. ELISA and western blotting were used to explore the effects of SA on the levels of inflammatory cytokines in serum and the phosphorylation level of key proteins in tissue, respectively. Moreover, agonists and inhibitors of key proteins were used on HFLS-RA to explore the underlying mechanism of SA. Finally, immunofluorescence was utilized to explore the effects of SA on apoptosis in HFLS-RA. SA significantly reduced arthritis index, alleviated paw swelling, and improved inflammatory cell infiltration and cartilage degradation in CIA rats. The levels of the pro-inflammatory cytokines including TNF-α, IL-1β, IL-6, and IL-17 were decreased while the level of the anti-inflammatory cytokine IL-10 was promoted by SA. The SA also down-regulated the protein phosphorylation levels of JAK2, STAT3, PI3K, AKT and p65 in vivo and in vitro. Furthermore, SA reversed the agonist-induced increase in phosphorylation levels of PI3K/AKT/NF-κB and JAK2/STAT3 pathway-related proteins. In addition, SA acted on the phosphorylation levels of these proteins in the same trend as the pathway inhibitors and dose-dependently reduced the phosphorylation levels of PI3K/AKT/NF-κB pathway proteins. The immunofluorescence results suggested that SA could promote apoptosis in HFLS-RA. SA could inhibit inflammatory symptoms and bone destruction in CIA, and its mechanism may be related to the inhibition of PI3K/AKT/NF-κB and JAK2/STAT3 pathways. Hence, SA could be developed as a potential anti-RA therapeutic drug.

Deng C ，Sun S ，Zhang H ，Liu S ，Xu X ，Hu Y ，Ma H ，Xin P ... -  《-》

Age-associated B cells contribute to the pathogenesis of rheumatoid arthritis by inducing activation of fibroblast-like synoviocytes via TNF-α-mediated ERK1/2 and JAK-STAT1 pathways.

Age-associated B cells (ABCs) are a recently identified B cell subset, whose expansion has been increasingly linked to the pathogenesis of autoimmune disorders. This study aimed to investigate whether ABCs are involved in the pathogenesis and underlying mechanisms of rheumatoid arthritis (RA). ABCs were assessed in collagen-induced arthritis (CIA) mice and patients with RA using flow cytometry. Transcriptomic features of RA ABCs were explored using RNA-seq. Primary fibroblast-like synoviocytes (FLS) derived from the synovial tissue of patients with RA were cocultured with ABCs or ABCs-conditioned medium (ABCsCM). IL-6, MMP-1, MMP-3 and MMP-13 levels in the coculture supernatant were detected by ELISA. Signalling pathways related to ABCs-induced FLS activation were examined using western blotting. Increased ABCs levels in the blood, spleen and inflammatory joints of CIA mice were observed. Notably, ABCs were elevated in the blood, synovial fluid and synovial tissue of patients with RA and positively correlated with disease activity. RNA-seq revealed upregulated chemotaxis-related genes in RA ABCs compared with those in naive and memory B cells. Coculture of FLS with RA ABCs or ABCsCM led to an active phenotype of FLS, with increased production of IL-6, MMP-1, MMP-3 and MMP-13. Mechanistically, ABCsCM-derived TNF-α promoted the upregulation of interferon-stimulated genes in FLS, with elevated phosphorylation of ERK1/2 and STAT1. Furthermore, blockage of ERK1/2 and Janus Kinase (JAK)-STAT1 pathways inhibited the activation of FLS induced by ABCsCM. Our results suggest that ABCs contribute to the pathogenesis of RA by inducing the activation of FLS via TNF-α-mediated ERK1/2 and JAK-STAT1 pathways.

Qin Y ，Cai ML ，Jin HZ ，Huang W ，Zhu C ，Bozec A ，Huang J ，Chen Z ... -  《-》

Mechanism of LMNB1 activating GPR84 through JAK-STAT pathway to mediate M2 macrophage polarization in lung cancer.

It is reported that G protein-coupled receptor 84 (GPR84) can participate in inflammation and immune regulation to repress anti-tumor responses. However, the function of GPR84 in lung cancer (LC) and its potential molecular mechanisms are still largely unknown. Bioinformatics and molecular experiments were employed to assess the expression of GPR84 in LC. The pathways enriched by GPR84 were analyzed by the Kyoto Encyclopedia of Genes and Genomes. Bioinformatics prediction identified the potential upstream regulatory factors of GPR84, which were verified through dual luciferase and chromatin immunoprecipitation experiments. Cell viability was measured by methyl thiazolyl tetrazolium assay. The expression levels of key proteins related to the janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway such as JAK2, p-JAK2, p-STAT3, and STAT3 were detected by western blot. Macrophages were co-cultured with LC cells. Flow cytometry was employed to examine the proportion of mannose receptor-positive cells. The expression levels of M2 polarization marker genes chitinase-like protein 3, arginase-1, and found in inflammatory zone 1 were measured by quantitative reverse transcription polymerase chain reaction. We applied an enzyme-linked immunosorbent assay to determine levels of cytokines (interleukin-10 and transforming growth factor beta) to evaluate the M2 macrophage polarization. GPR84 was highly expressed in LC and substantially enriched in the JAK-STAT pathway. GPR84 facilitated the M2 polarization of macrophages in LC. Adding the JAK-STAT pathway inhibitor weakened the promoting effect of GPR84 overexpression on M2 macrophage polarization. Furthermore, GPR84 also had an upstream regulatory factor lamin B1 (LMNB1). Knocking down LMNB1 blocked the JAK-STAT signaling pathway to repress M2 macrophage polarization in LC, while overexpression of GPR84 reversed the impact of LMNB1 knockdown on macrophage polarization. The project suggested that the LMNB1/GPR84 axis can facilitate M2 polarization of macrophages in LC by triggering the JAK-STAT pathway. Targeting LMNB1/GPR84 or blocking the JAK-STAT pathway may be a novel approach for LC diagnosis and treatment.

Ji Y ，Wang Y ，Zhang N ，Yang J ，Li J ，Zheng H ，Wang L ，Wang W ，Li J ... -  《-》