New biflavonoids isolated from Xylia kerrii leaves extract with selective cytotoxicity against MH7A human rheumatoid arthritis synovial fibroblast cells.

Three new biflavonoids (1-3) and two known flavonoids (4, 5) were isolated from Xylia kerrii collected in Thailand. Compounds 1-5 showed selective cytotoxicity against the rheumatoid fibroblast-like synovial MH7A cell line, and these compounds showed weak cytotoxicity against the human lung synovial fibroblast WI-38 VA13 sub 2 RA cell line. Notably, compound 1 was highly selective toward MH7A cells with an IC50 value of 6.9 µM, whereas the IC50 value for WI-38 VA13 sub 2 RA cells was > 100 µM. The western blotting analysis of MH7A cells treated with compound 1 showed increased CDKN2A /p16INK4A and caspase-8 levels.

Fangji Huangqi Decoction alleviates rheumatoid arthritis through regulating HIF-1α mediated the angiogenesis and the balance between autophagy and apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Fangji Huangqi Decoction (FHD) is frequently prescribed for the clinical treatment of wind-cold and wind-dampness pathogenic superficial deficiency syndrome. It also has a notable curative effect on rheumatoid arthritis (RA). AIM OF THE STUDY: The study aimed to explore the possible mechanism of FHD against RA and provided a theoretical basis for alternative therapies for RA. MATERIALS AND METHODS: We used UPLC-Q-TOF-MS to analysis the ingredients and absorbed blood components of FHD. At the same time, the collagen-induced arthritis (CIA) rat model was established to estimate the therapeutic effects on FHD by considering body weight, arthritis score, paw swelling, autonomous movement ability, and synovial microvessel counts. Subsequently, immunofluorescence, immunohistochemistry, and Western blot were employed to detect the anti-angiogenic capacity of FHD in vivo, as well as the levels of apoptosis and autophagy in the synovial tissue. In addition, flow cytometry and Western blot were used to assess the effects of FHD on apoptosis and autophagy in MH7A cells. The effects of FHD on the proliferation and migration of MH7A cells were measured by CCK8 assay, cell migration and, invasion experiments. Finally, a tube formation assay was performed to evaluate the angiogenic capacity of FHD in co-cultures of MH7A cells and HUVEC cells. RESULTS: Through testing of FHD's original formula, a total of 26 active ingredients have been identified, with 17 of them being absorbed into the bloodstream. FHD significantly improved the pathological symptoms and synovial hyperplasia of CIA rats. FHD could suppress the expression of HIF-1α, promote apoptosis in CIA rat synovial tissue, and suppress autophagy and angiogenesis. In vitro experiments showed that serum containing FHD inhibited the proliferation, migration, and invasion of MH7A cells, and also suppressed the expression of autophagy-related proteins while promoting apoptosis. FHD markedly repressed the expression of HIF-1α protein in TNF-α-stimulated MH7A cells and inhibited the tube formation capacity induced by MH7A cells in HUVEC cells. CONCLUSIONS: The study had proven that FHD played an excellent anti-RA role, which may be attributed to its potential mechanism of regulating the balance between autophagy and apoptosis in RA FLS by suppressing the HIF-1α, thus contributing to its anti-angiogenic activities.

[Mechanism of Yuxuebi Tablets in treating synovial inflammation in rheumatoid arthritis based on transcriptomics].

This study investigated the mechanism of Yuxuebi Tablets(YXB) in the treatment of synovial inflammation in rheumatoid arthritis(RA) based on transcriptomic analysis. Transcriptome sequencing technology was employed to analyze the gene expression profiles of joint tissues from normal rats, collagen-induced arthritis(CIA) rats(an RA model), and YXB-treated rats. Common diffe-rentially expressed genes(DEGs) were subjected to Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses. RA synovial inflammation-related target genes were retrieved from the OMIM and GeneCards databases. Venny 2.1 software was used to identify the intersection of YXB target genes and RA synovial inflammation-related target genes, and GO and KEGG enrichment analyses were performed on the intersecting target genes. Immunohistochemistry was used to assess the protein expression levels of the inflammatory factors interleukin-1ß(IL-1ß) and tumor necrosis factor-α(TNF-α) in rat joint tissues. Western blot analysis was employed to measure the expression levels of key proteins in the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt) signaling pathway. A total of 2 058 DEGs were identified by intersecting the genes from the normal group vs model group and the model group vs YXB treatment group. A search in OMIM and GeneCards databases yielded 1 102 RA synovial inflammation-related target genes. After intersecting with the DEGs in the YXB treatment group, 204 intersecting target genes were identified, primarily involving biological processes such as immune response, signal transduction, and inflammatory response; cellular components including plasma membrane, extracellular space, and extracellular region; molecular functions like protein binding, identical protein binding, and receptor binding. These target genes were mainly enriched in signaling pathways such as PI3K/Akt, cytokine-cytokine receptor interaction, and Janus kinase/signal transducer and activator of transcription(JAK/STAT). Western blot results showed that YXB at low, medium, and high doses could significantly inhibit the expression levels of key proteins in the PI3K/Akt signaling pathway in rat joint tissues in a dose-dependent manner. Immunohistochemistry further confirmed these findings, showing that YXB not only suppressed the protein expression levels of the inflammatory factors IL-1ß and TNF-α in the joint synovial tissues of CIA rats, but also inhibited p-Akt protein expression. In conclusion, this study used transcriptomic analysis to uncover the key mechanisms of YXB in inhibiting synovial inflammation and alleviating the progression of RA, with a focus on its role in suppressing the PI3K/Akt signaling pathway.

Natural compound library screening to identify berberine as a treatment for rheumatoid arthritis.

OBJECTIVE: Fibroblast-like synoviocytes (FLS) play a critical role on the exacerbation and deterioration of rheumatoid arthritis (RA). Aberrant activation of FLS pyroptosis signaling is responsible for the hyperplasia of synovium and destruction of cartilage of RA. This study investigated the screened traditional Chinese medicine berberine (BBR), an active alkaloid extracted from the Coptis chinensis plant, that regulates the pyroptosis of FLS and secretion of inflammatory factors in rheumatoid arthritis. METHODS: First, BBR was screened using a high-throughput drug screening strategy, and its inhibitory effect on RA-FLS was verified by in vivo and in vitro experiments. Second, BBR was intraperitoneally administrated into the collagen-induced arthritis rat model, and the clinical scores, arthritis index, and joint HE staining were evaluated. Third, synovial tissues of CIA mice were collected, and the expression of NLRP3, cleaved-caspase-1, GSDMD-N, Mst1, and YAP was detected by Western blot. RESULTS: The administration of BBR dramatically alleviated the severity of collagen-induced arthritis rat model with a decreased clinical score and inflammation reduction. In addition, BBR intervention significantly attenuates several pro-inflammatory cytokines (interleukin-1ß, interleukin-6, interleukin-17, and interleukin-18). Moreover, BBR can reduce the pyroptosis response (caspase-1, NLR family pyrin domain containing 3, and gasdermin D) of the RA-FLS in vitro, activating the Hippo signaling pathway (Mammalian sterile 20-like kinase 1, yes-associated protein, and transcriptional enhanced associate domains) so as to inhibit the pro-inflammatory effect of RA-FLS. CONCLUSION: These results support the role of BBR in RA and may have therapeutic implications by directly repressing the activation, migration of RA-FLS, which contributing to the attenuation of the progress of CIA. Therefore, targeting PU.1 might be a potential therapeutic approach for RA. Besides, BBR inhibited RA-FLS pyroptosis by downregulating of NLRP3 inflammasomes (NLRP3, caspase-1) and eased the pro-inflammatory activities via activating the Hippo signaling pathway, thereby improving the symptom of CIA.

Reduction of Edema and Pain in Transcutaneous Electrical Nerve Stimulation Treated-Arthritic Rat.

Transcutaneous electrical nerve stimulation (TENS) has been used to reduce pain or improve motor function in musculoskeletal and neurological disorders in the clinic. Although some studies have suggested electrotherapy as an intervention for edema, the effects and mechanisms of TENS on inflammation-induced edema remain unclear. Thus, we aimed to investigate the effects of TENS on arthritic pain with edema. 1% carrageenan was injected into the right tibiofemoral joint of 69 male Sprague-Dawley rats (200-250 g). After the development of arthritic pain, low-frequency (4-Hz, Low-TENS, n = 25) and high-frequency (100-Hz, High-TENS, n = 25) TENS with sub-motor threshold or placebo-TENS (n = 19) was applied for 20-min to medio-lateral part of the ipsilateral side. Weight bearing and knee-bend tests were used to assess pain-like behaviors. Also, we examined the size of edema and measured tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) levels in the synovium by western blot. Eight rats in each of the two TENS groups were injected with Naloxone. Edema was reduced in the low- and high-frequency TENS groups at 6-h. TENS-treated rats showed reduced pain in the knee-bend test at 6-h. We observed decreased weight load shifts on the ipsilateral side in TENS groups. Naloxone reduced these effects. TNF-α and IL-1ß expression decreased in the synovial membrane at 6-h. These results suggest that low- and high-frequency TENS have acutely positive effects on inflammatory edema, with the management of arthritic pain and reduction in pro-inflammatory mediators. Therefore, Low-TENS and High-TENS may be useful in treating acute inflammatory pain and edema.

Isorhamnetin Downregulates MMP2 and MMP9 to Inhibit Development of Rheumatoid Arthritis through SRC/ERK/CREB Pathway.

OBJECTIVE: To investigate the effect of isorhamnetin on the pathology of rheumatoid arthritis (RA). METHODS: Tumor necrosis factor (TNF)- α -induced fibroblast-like synoviocytes (FLS) was exposed to additional isorhamnetin (10, 20 and 40 µ mol/L). Overexpression vectors for matrix metalloproteinase-2 (MMP2) or MMP9 or SRC were transfected to explore their roles in isorhamnetin-mediated RA-FLS function. RA-FLS viability, migration, and invasion were evaluated. Moreover, a collagen-induced arthritis (CIA) rat model was established. Rats were randomly divided to sham, CIA, low-, medium-, and high-dosage groups using a random number table (n=5 in each group) and administed with normal saline or additional isorhamnetin [2, 10, and 20 mg/(kg·day)] for 4 weeks, respectively. Arthritis index was calculated and synovial tissue inflammation was determined in CIA rats. The levels of MMP2, MMP9, TNF-α, interleukin-6 (IL-6), and IL-1 ß, as well as the phosphorylation levels of SRC, extracellular regulated kinase (ERK), and cyclic adenosine monophosphate response element-binding (CREB), were detected in RA-FLS and synovial tissue. Molecular docking was also used to analyze the binding of isorhamnetin to SRC. RESULTS: In in vitro studies, isorhamnetin inhibited RA-FLS viability, migration and invasion (P<0.05). Isorhamnetin downregulated the levels of MMP2, MMP9, TNF-α, IL-6, and IL-1 ß in RA-FLS (P<0.05). The overexpression of either MMP2 or MMP9 reversed isorhamnetin-inhibited RA-FLS migration and invasion, as well as the levels of TNF-α, IL-6, and IL-1 ß (P<0.05). Furthermore, isorhamnetin bound to SRC and reduced the phosphorylation of SRC, ERK, and CREB (P<0.05). SRC overexpression reversed the inhibitory effect of isorhamnetin on RA-FLS viability, migration and invasion, as well as the negative regulation of MMP2 and MMP9 (P<0.05). In in vivo studies, isorhamnetin decreased arthritis index scores (P<0.05) and alleviated synovial inflammation. Isorhamnetin reduced the levels of MMP2, MMP9, TNF-α, IL-6, and IL-1 ß, as well as the phosphorylation of SRC, ERK, and CREB in synovial tissue (P<0.05). Notably, the inhibitory effect of isorhamnetin was more pronounced at higher concentrations (P<0.05). CONCLUSION: Isorhamnetin exhibited anti-RA effects through modulating SRC/ERK/CREB and MMP2/MMP9 signaling pathways, suggesting that isorhamnetin may be a potential therapeutic agent for RA.

Coptisine inhibits aggressive and proliferative actions of fibroblast like synoviocytes and exerts a therapeutic potential for rheumatoid arthritis.

OBJECTIVE: Coptisine, a natural bioactive small molecular compound extracted from traditional Chinese herb Coptis chinensis, has been shown to exhibit anti-tumor effect. However, its contribution to autoimmune diseases such as rheumatoid arthritis (RA) is unknown. Here, we evaluate the effect of coptisine in controlling fibroblast-like synoviocytes (FLS)-mediated synovial proliferation and aggression in RA and further explore its underlying mechanism(s). METHODS: FLS were separated from synovial tissues obtained from patients with RA. Protein expression was measured by Western blot or immunohistochemistry. Gene expression was detected by quantitative RT-PCR. The EdU incorporation was used to measure cell proliferation. Migration and invasion were determined by Boyden chamber assay. RNA sequencing analysis was used to seek for the target of coptisine. The in vivo effect of coptisine was evaluated in collagen-induced arthritis (CIA) model. RESULTS: Treatment with coptisine reduced the proliferation, migration, and invasion, but not apoptosis of RA FLS. Mechanistically, we identified PSAT1, an enzyme that catalyzes serine/one-carbon/glycine biosynthesis, as a novel targeting gene of coptisine in RA FLS. PSAT1 expression was increased in FLS and synovial tissues from patients with RA compared to healthy control subjects. Coptisine treatment or PSAT1 knockdown reduced the TNF-α-induced phosphorylation of p38, ERK1/2, and JNK MAPK pathway. Interestingly, coptisine administration improved the severity of arthritis and reduced synovial PSAT1 expression in mice with CIA. CONCLUSIONS: Our data demonstrate that coptisine treatment suppresses aggressive and proliferative actions of RA FLS by targeting PSAT1 and sequential inhibition of phosphorylated p38, ERK1/2, and JNK MAPK pathway. Our findings suggest that coptisine might control FLS-mediated rheumatoid synovial proliferation and aggression, and be a novel potential agent for RA treatment.

Fluid-attenuated inversion-recovery sequence with fat suppression as an alternative to contrast-enhanced MRI in pediatric synovitis.

BACKGROUND: Non-contrast magnetic resonance imaging (MRI) fluid-attenuated inversion-recovery sequence (FLAIR) with fat suppression (FS) has not been validated in children. OBJECTIVE: Compare FLAIR to T1-weighted post contrast (T1CE) in the detection of knee synovitis. METHODS AND MATERIALS: Institutional review board (IRB) waived consent. Children who underwent T1CE and FLAIR sequences of the knee on a 3-T magnet from April 2021 to December 2021 were included. Two pediatric radiologists assessed axial FLAIR and T1CE images for synovitis and synovial thickness. Reliability and agreement were assessed. Sensitivities, specificities, and accuracy were calculated for FLAIR using T1CE as reference standard. RESULTS: In total, 42 knees (39 patients) were assessed (median age 12.9 years (2.3-17.8 years); 62% male, 38% female). Readers judged 20/42 (48%) knees to have synovitis. Sensitivity of FLAIR for reader 1 was 79% (19/24; 95% CI 0.58, 0.93) and 84% (16/19; 95% CI 0.60, 0.97) for reader 2. Specificity of FLAIR for reader 1 was 94% (17/18; 95% CI 0.73, 1) and 83% (19/23; 95% CI 0.61, 0.95) for reader 2. Accuracy for readers 1 and 2 was 86% (36/42; 95% CI 0.71, 0.95) and 83% (35/42; 95% CI 0.69, 0.93), respectively. Inter-reader reliability was good (0.75-0.90) for synovial measurements for FLAIR (ICC = 0.80; 95% CI 0.71, 0.86) and moderate for T1 CE (ICC = 0.62 (95% CI 0.48, 0.73)). CONCLUSION: FLAIR FS depicts synovium in the pediatric knee with similar reliability to T1 CE and may be an acceptable alternative to contrast in the initial diagnosis of synovitis.

Non-coding RNAs involved in fibroblast-like synoviocyte functioning in arthritis rheumatoid: From pathogenesis to therapy.

Rheumatoid arthritis (RA) is a polygenic autoimmune disorder with an uncertain etiology, primarily impacting the joints. Moreover, the disease may manifest beyond articular involvement, leading to extra-articular manifestations. Fibroblast-like synoviocytes (FLS) are cells of mesenchymal origin that possess crucial physiological significance within the synovium, contributing to the synthesis of specific constituents found in the synovial fluid and articular cartilage. Consequently, there has been a growing focus on FLS as a potential therapeutic target in the context of RA. Recent investigations have revealed that non-coding RNAs (ncRNAs) serve as pivotal regulators of FLS function, with their dysregulated expression patterns being detected within FLS populations. NcRNAs, such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), assume essential functions as regulators of gene expression at both the post-transcriptional and transcriptional levels, and also serve as guiding molecules for chromatin-modifying complexes. Majority of these ncRNAs contribute to various FLS activities including metastasis, proliferation, and cytokine production. In the current work, we comprehensively review the existing literature on ncRNAs, which play pivotal roles in FLS activity and the pathogenesis of RA. Furthermore, this study provides a comprehensive summary and description of the lncRNA/circRNA-miRNA-mRNA regulatory axes in FLS activity, along with potential implications for the RA development. As well, in the final section, we illustrated that therapeutic agents including herbal medicine, and exosomes by modulating ncRNAs regulate FLS activity.

Efficient delivery of the lncRNA LEF1-AS1 through the antibody LAIR-1 (CD305)-modified Zn-Adenine targets articular inflammation to enhance the treatment of rheumatoid arthritis.

BACKGROUNDS: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by synovial hyperplasia. Maintaining a balance between the proliferation and apoptosis of rheumatoid arthritis synovial fibroblasts (RASFs) is crucial for preventing the erosion of bone and cartilage and, ultimately, mitigating the progression of RA. We found that the lncRNA LEF1-AS1 was expressed at low levels in the RASFs and inhibited their abnormal proliferation by targeting PIK3R2 protein and regulating the PI3K/AKT signal pathway through its interaction with miR-30-5p. In this study, we fabricated a nano-drug delivery system for LEF1-AS1 using Zn-Adenine nanoparticles (NPs) as a novel therapeutic strategy against RA. METHODS: The expression levels of LEF1-AS1, miR-30-5p, PIK3R2, p-PI3K, and p-AKT were detected in the primary RASFs and a human fibroblast-like synovial cell line (HFLS). Zn-Adenine nanoparticles (NPs) were functionalized with anti-CD305 antibody to construct (Zn-Adenine)@Ab. These NPs were then loaded with LEF1-AS1 to form (Zn-Adenine)@Ab@lncRNA LEF1-AS1. Finally, the (Zn-Adenine)@Ab@lncRNA LEF1-AS1 NPs were locally injected into a rat model with collagen-induced arthritis (CIA). The arthritic injuries in each group were evaluated by HE staining and other methods. RESULTS: LEF1-AS1 was expressed at low levels in the primary RASFs. High expression levels of LEF1-AS1 were detected in the HFLS cells, which corresponded to a significant downregulation of miR-30-5p. In addition, the expression level of PIK3R2 was significantly increased, and that of p-PI3K and p-AKT were significantly downregulated in these cells. The (Zn-Adenine)@Ab@lncRNA LEF1-AS1 NPs significantly inhibited the proliferation of RASFs and decreased the production of inflammatory cytokines (IL-1ß, IL-6, TNF-α). Intra-articular injection (IAI) of (Zn-Adenine)@Ab@lncRNA LEF1-AS1 NPs significantly alleviated cartilage destruction and joint injury in the CIA-modeled rats. CONCLUSIONS: LEF1-AS1 interacts with miR-30-5p to inhibit the abnormal proliferation of RASFs by regulating the PI3K/AKT signal pathway. The (Zn-Adenine)@Ab NPs achieved targeted delivery of the loaded LEF1-AS1 into the RASFs, which improved the cellular internalization rate and therapeutic effects. Thus, LEF1-AS1 is a potential target for the treatment of RA.

Moxibustion intervention improves synovitis by down-regulating NLRP3/Caspase-1/IL-1ß signaling of synovial tissue in rats with adjuvant arthritis. / 基于NLRP3/Caspase-1/IL-1ßä¿¡å·é€šè·¯æŽ¢è®¨è‰¾ç¸æ”¹å–„ä½å‰‚æ€§å ³èŠ‚ç‚Žå¤§é¼ æ»‘è†œç‚Žçš„æœºåˆ¶.

OBJECTIVES: To observe the effect of moxibustion on activities of NOD-like receptor family protein 3 (NLRP3)/cysteine aspartic acid specific protease-1 (Caspase-1)/interleukin-1ß (IL-1ß) signaling pathway in rats with adjuvant arthritis (AA), so as to explore its mechanisms underlying improvement of rheumatoid arthritis (RA). Me-thods Thirty male Wistar rats were randomly divided into normal control, AA model and moxibustion groups, with 10 rats in each group. The AA model was replicated by raising in wind, cold and damp environment combined with complete Freund's adjuvant injection. In the moxibustion group, moxibustion was applied to bilateral "Shenshu" (BL23) and "Zusanli"(ST36) for 20 min each time, once daily for 21 days. Changes of joint swelling degree (JSD) and arthritis index (AI) in each group were observed. The ultrastructural changes of synovial cells in each group were observed by transmission electron microscopy. The protein expression levels of NLRP3, apoptosis-associated speck-like protein (ASC), Caspase-1, tumor necrosis factor-α (TNF-α) and IL-1ß in the synovial tissues of the knee joint were measured by Western blot. RESULTS: Compared with the normal control group, JSD, AI and the protein expressions of NLRP3, ASC, Caspase-1, TNF-α and IL-1ß in the synovial tissues were significantly increased (P<0.01) in the model group. In comparison with the model group, JSD, AI and the protein expression levels of NLRP3, ASC, Caspase-1, TNF-α and IL-1ß were significantly decreased (P<0.01) in the moxibustion group. Results of transmission electron microscope showed an irregular and vague nuclear membrane of synovial cells, and unclear mitochondrial membrane boundary with sparse, swelling crests in the model group, which was relatively milder in the damage degree in the moxibustion group. CONCLUSIONS: Moxibustion can relieve the inflammatory response in the synovial membrane of AA rats, which may be related to its function in down-regulating synovial NLRP3/Caspase-1/IL-1ß inflammatory signaling.

Myricetin ameliorates the IL-21-induced tumorigenic phenotype of adjuvant-induced arthritis FLS by modulating the choline kinase signaling cascade.

The synovial intimal lining is mainly governed by fibroblast-like synoviocytes (FLS), which portray a transformed tumor-like phenotype in rheumatoid arthritis (RA). Among the diverse cytokines that engender FLS, interleukin-21 (IL-21) was reported to stimulate hyperproliferation and perpetuate inflammation. Recently, choline kinase (ChoKα) has been reported to be an essential enzyme aiding RA-FLS hyperproliferation by altering phosphatidylcholine biosynthesis. The current study aimed to elucidate the therapeutic efficacy of myricetin, a flavonoid, in abating the IL-21-induced tumor-like phenotype of adjuvant-induced arthritis (AIA)-FLS via the ChoKα signaling cascade. Our results showed that myricetin suppressed IL-21 receptor expression and activation of the ChoKα signaling cascade (N-Ras, Ral-GDS, and PI3K) in IL-21-induced AIA-FLS. Consequently, myricetin treatment decreased ChoKα and PLD2 enzymatic activity and inhibited the proliferative, migratory, and invasive properties of AIA-FLSs. Our results demonstrated that myricetin could be a promising anti-arthritic compound by abating IL-21-induced hyperproliferation, migration, and invasive behavior of AIA-FLS by downregulating the ChoKα signaling cascade.

Sesamol serves as a p53 stabilizer to relieve rheumatoid arthritis progression and inhibits the growth of synovial organoids.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease known as a leading cause of disability with considerable mortality. Developing alternative drugs and targets for RA treatment is an urgent issue. Sesamol is a phenolic compound isolated from natural food sesame (Sesamum indicum L.) with various biological activities. PURPOSE: The current research intended to illuminate the bioactivity and mechanisms of sesamol in RA fibroblast-like synoviocytes (FLS), and aimed to estimate the potential clinical application value of sesamol in RA treatment. METHODS: CCK-8, EdU, and flow cytometry assays, as well as transwell tests were applied to observe the effects of sesamol on the abnormal functions of RA-FLS. Moreover, synovial organoids and a collagen-induced arthritis (CIA) mouse model were constructed to further explore the therapeutic capacity of sesamol on RA. Furthermore, RNA sequencing combined with quantitative real-time PCR assay, Western blot as well as co-immunoprecipitation were employed to clarify the mechanism of sesamol in regulating RA progression. RESULTS: Sesamol suppressed the proliferation through inhibiting DNA replication, triggering cell cycle arrest and apoptosis of RA-FLS. Besides, sesamol impaired RA-FLS migration and invasion. Interestingly, sesamol inhibited the growth of constructed synovial organoids and alleviated RA symptoms in CIA mice. Moreover, RNA sequencing further implicated p53 signaling as a downstream pathway of sesamol. Furthermore, sesamol was shown to decrease p53 ubiquitination and degradation, thereby activating p53 signaling. Finally, bioinformatics analyses also highlighted the importance of sesamol-regulated networks in the progression of RA. CONCLUSIONS: Our investigation demonstrated that sesamol served as a novel p53 stabilizer to attenuate the abnormal functions of RA-FLS via facilitating the activation of p53 signaling. Moreover, our study highlighted that sesamol might be an effective lead compound or candidate drug and p53 could be a promising target for the therapy of RA.

Tyro3 receptor tyrosine kinase contributes to pathogenic phenotypes of fibroblast-like synoviocytes in rheumatoid arthritis and disturbs immune cell balance in experimental arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by synovitis and joint damage, the underlying causes of which remain unclear. Our prior investigations revealed a notable correlation between the expression of Tyro3 Protein Tyrosine Kinase (Tyro3TK) and the progression of RA. To further elucidate the pathogenic role of Tyro3TK in RA, we analyzed the influence of Tyro3TK on pathogenic phenotypes of RA fibroblast like synoviocyte (FLS) in vitro and compared disease severity, joint damages and immunological parameters of K/BxN serum transfer arthritis (STA) in Tyro3TK-/- deficient mice and wild type controls. Our findings underscored the remarkable effectiveness of Tyro3TK blockade, as evidenced by diminished secretion of inflammatory cytokines and matrix metalloproteinases (MMPs), curtailed migration and invasiveness of RAFLS, and attenuated differentiation of pathogenic helper T cell subsets mediated by RAFLS. Correspondingly, our in vivo investigations illuminated the more favorable outcomes in Tyro3TK-deficient mice, characterized by reduced joint pathology, tempered synovial inflammation, and restored immune cell equilibrium. These data suggested that Tyro3TK might contribute to aggravated autoimmune arthritis and immunological pathology and act as a potential therapeutic target for RA.

Efficacy of electroacupuncture stimulating Zusanli (ST36) and Xuanzhong (GB39) on synovial angiogenesis in rats with adjuvant arthritis.

OBJECTIVE: To investigate the efficacy of electroacupuncture (EA) stimulating Zusanli (ST36) and Xuanzhong (GB39) on synovial angiogenesis in rats with adjuvant arthritis (AA). METHODS: AA models were established by bilateral injection of Freund's complete adjuvant (FCA) in male Sprague-Dawley rats. Three days after injection, rats were given EA at Zusanli (ST36) and Xuanzhong (GB39) acupoints, once every other day, for 16 d. The arthritis index score, paw volume, and hematoxylin-eosin (HE) staining was performed for each animal. Angiogenesis marker cluster of differentiation 34 (CD34) expression and synovial cell apoptosis in synovial tissue were observed. The levels of Notch1, hairy and enhancer of split homolog-1 (Hes1), transforming growth factor-beta (TGF-ß) and basic fibroblast growth factor (bFGF) were subsequently detected. RESULTS: We found that EA significantly decreased arthritis index scores, paw volume, and HE staining scores. EA could significantly inhibit the expression of CD34, promoting apoptosis of synovial cells in the joint synovial tissue of AA rats. The expression of Notch1 signaling pathway proteins and mRNAs (Notch1, Hes1, TGF-ß, and bFGF) were markedly downregulated by EA treatment. CONCLUSIONS: These results prove that EA attenuates synovial angiogenesis by inhibiting the Notch1 signaling pathway in AA rat models. Based on our findings, we propose that EA is a promising complementary and alternative therapy in rheumatoid arthritis.

Wnt/ß-catenin signaling pathway promotes abnormal activation of fibroblast-like synoviocytes and angiogenesis in rheumatoid arthritis and the intervention of Er Miao San.

BACKGROUND: Er Miao San (EMS) is an important herbal formula and a representative prescription for the treatment of the downwards flow of damp-heat syndrome. Clinical practice has proven that EMS can effectively treat rheumatoid arthritis (RA). Previous studies have demonstrated that EMS regulates the functions of T cells and dendritic cells and affects the polarization of macrophages. However, it is not clear whether the inhibitory effect of EMS on RA is related to the regulation of abnormal synovial activation and angiogenesis. PURPOSE: The aim of this study was to elucidate the effect and potential mechanisms of EMS on the abnormal activation and angiogenesis of fibroblast-like synoviocytes (FLSs) in RA. METHODS: The effect of EMS on rats with adjuvant arthritis (AA) and MH7A cells was examined by X-ray, haematoxylin-eosin (HE) staining, immunohistochemistry (IHC), ELISA and western blotting. Angiogenesis in AA rats was measured by a small animal ultrasound imaging system, immunofluorescence (IF) analysis and ELISA. An exchange between MH7A cells and HUVECs was induced using conditioned media that mimicked the microenvironment in vivo. CCK-8, western blotting, and scratch healing and Transwell migration assays were used to evaluate the effect of EMS on the Wnt/ß-catenin signaling pathway and angiogenesis in the inflammatory microenvironment of RA. RESULTS: Our results showed that EMS had a protective effect on AA rats. On the one hand, there was a decrease in paw swelling, the arthritis index, organ indices and proinflammatory factor levels, as well as relief of joint damage. On the other hand, blood flow, the number of immature blood vessels and proangiogenic factors were decreased. Furthermore, EMS reduced the expression of the Wnt/ß-catenin signaling pathway in the synovial tissue of AA rats and MH7A cells. In the inflammatory microenvonrment of RA, the results were consistent. CONCLUSION: This study demonstrated that EMS could protect against RA by inhibiting the abnormal activation and angiogenesis of FLSs, and the mechanism may be related to inhibiting the activation of the Wnt/ß-catenin signaling pathway.

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats.

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-α was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

Membrane vesicles from Lactobacillus johnsonii delay osteoarthritis progression via modulating macrophage glutamine synthetase/mTORC1 axis.

AIMS: The manipulation of macrophage recruitment and their shift in the M1/M2 ratio is a promising approach to mitigate osteoarthritis (OA). Nevertheless, the current clinical medication available for OA is only palliative and may result in undesirable outcomes. Hence, it is urgent to explore alternative disease-modifying drug supplement that are both safer and more effective in OA treatment, like probiotic and probiotic-derived membrane vesicles. METHODS: The synovial inflammation and cartilage damage in collagenase-induced OA (CIOA) mice were observed using haematoxylin and eosin, saffron O-solid green and immunohistochemical staining. Bipedal balance test and open field test were conducted to determine the effectiveness of L. johnsonii-derived membrane vesicles (LJ-MVs) in reducing joint pain of CIOA mice. Additionally, Transwell, western blot, and immunological testing were used to examine the effect of LJ-MVs on macrophage migration and reprogramming. Furthermore, a 4D label-free proteomic analysis of LJ-MVs and their parent bacterium was performed, and the glutamine synthetase (GS)/mTORC1 axis in macrophage was verified by western blot. RESULTS: L. johnsonii and its membrane vesicles, LJ-MVs, exhibit a novel ability to mitigate inflammation, cartilage damage, and pain associated with OA. This is achieved by their ability to impede macrophage migration, M1-like polarization, and inflammatory mediators secretion, while simultaneously promoting the M2/M1 ratio in synovial macrophages. The mechanism underlying this effect involves the modulation of macrophage GS/mTORC1 pathway, at least partially. SIGNIFICANCE: Owing to their probiotic derivation, LJ-MVs will be a more dependable and potent disease-modifying drugs for the prevention and therapy of OA in the long run.

Lycium ruthenicum Murr. anthocyanins inhibit hyperproliferation of synovial fibroblasts from rheumatoid patients and the mechanism study powered by network pharmacology.

BACKGROUND: Rheumatoid arthritis (RA), is a typical autoimmune disease affecting nearly 1% of the world's population. The dysfunctional hyperproliferation of synovial fibroblast (SF) in articular cartilage of RA patients is considered as the essential etiology. Traditional chemotherapeutic agents for RA treatment are imperfect for their high cost and unpredictable side-effects. L. ruthenicum anthocyanins (LRAC) is a natural product that of potential for therapeutic application against RA. METHODS: LRAC was characterized by UPLC-MS/MS. Bioinformatics analyses based on network pharmacology were applied to predict the potential targets of LRAC, and to select DEGs (differentially expressed genes) caused by RA pathogenesis from GSE77298. Interactions between LRAC and the predicted targets were evaluated by molecular docking. Effects of LRAC on SFs from RA patients were examined by in vitro assays, which were analyzed by flow cytometry and western blotting (WB). RESULTS: LRAC was able to inhibit the abnormal proliferation and aggressive invasion of SFs from RA patients. LRAC was mainly constituted by petunidin (82.7%), with small amount of delphinidin (12.9%) and malvidin (4.4%) in terms of anthocyanidin. Bioinformatics analyses showed that in 3738 RA-related DEGs, 58 of them were collectively targeted by delphinidin, malvidin and delphinidin. AR, CDK2, CHEK1, HIF1A, CXCR4, MMP2 and MMP9, the seven hub genes constructed a central network mediating the signal transduction. Molecular docking confirmed the high affinities between the LRAC ligands and the protein receptors encoded by the hub genes. The in vitro assays validated that LRAC repressed the growth of RASF by cell cycle arresting and cell invasion paralyzing (c-Myc/p21/CDK2), initiating cell apoptosis (HIF-1α/CXCR4/Bax/Bcl-2), and inducing pyroptosis via ROS-dependent pathway (NOX4/ROS/NLRP3/IL-1ß/Caspase-1). CONCLUSION: LRAC can selectively inhibit the proliferation of RASFs, without side-effecting immunosuppression that usually occurred for RA treatment using MTX (methotrexate). These findings demonstrate the potential application of LRAC as a phytomedicine for RA treatment, and provide a valid approach for exploring natural remedies against autoimmune diseases.

Cynanchum komarovii extract for the treatment of rheumatoid arthritis by acting on synovial cells in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Cynanchum komarovii (CK), the northwest Chinese region's common medicinal herb, was traditionally utilized to treat arthritis, toothache, bald sores and cholecystitis. Various forms of arthritis can be treated with CK, based on "Medicinal Plants of Chinese Desert Areas". However, the exact mechanism of action in rheumatoid arthritis (RA) is uncertain. AIM OF THE STUDY: To evaluate the in vitro and in vivo effects of CK extracts on RA and to preliminarily investigate its anti-RA mechanism of action. MATERIALS AND METHODS: The main components of CK extract were analyzed by HPLC method. The effects of CK on the proliferation and apoptosis of human rheumatoid arthritis fibroblast-like synoviocytes (HFLS-RA) cells and the expression of apoptosis-related proteins in HFLS-RA cells were evaluated by CCK8 assay, flow cytometry and WB assay. To verify the anti-RA effect of CK extracts in vivo, a collagen-induced arthritis (CIA) rat model was established. The rats were divided into six groups: normal group, model group, CK high-dose group (1000 mg/kg, CK-H), CK medium-dose group (500 mg/kg, CK-M), CK low-dose group (250 mg/kg, CK-L) and methotrexate-positive drug group (MTX); the drug was administered continuously for 28 days. Body weight changes, joint swelling, arthritis index, bone density, ankle lesions, immune organ index, splenic lesions and inflammatory factor expression were used to evaluate the in vivo anti-RA activity of the extract. RESULTS: The findings of in vitro experiments showed that 10% CK-containing serum decreased the expression level of Bcl-2, increased the expression levels of Bax and Cleaved Caspase-3 in synovial cells, and prevented TNF-α induced aberrant proliferation and apoptotic antagonism in HFLS-RA cells. According to in vivo studies, CK extract at doses above 250 mg/kg was effective in controlling the levels of inflammatory factors, lowering the arthritis index, and improving foot swelling in CIA rats. When administered at doses up to 1000 mg/kg, CK extract significantly improved synovial lesions, increased bone density, and decreased abnormally elevated immune organ index in CIA rats. CONCLUSIONS: CK has significant anti-RA activity, and its anti-RA mechanism of action may be related to the regulation of the expression levels of apoptosis related proteins and the promotion of apoptosis in synovial cells.