[Mechanism of bulleyaconitine A in inhibiting bone destruction of rheumatoid arthritis via Src/PI3K/Akt signaling pathway].

Based on the sarcoma receptor coactivator(Src)/phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt) signaling pathway, the mechanism of action of bulleyaconitine A in the treatment of bone destruction of experimental rheumatoid arthritis(RA) was explored. Firstly, key targets of RA bone destruction were collected through GeneCards, PharmGKB, and OMIM databa-ses. Potential targets of bulleyaconitine A were collected using SwissTargetPrediction and PharmMapper databases. Next, intersection targets were obtained by the Venny 2.1.0 platform. Protein-protein interaction(PPI) network and topology analysis were managed by utilizing the STRING database and Cytoscape 3.8.0. Then, Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses were conducted in the DAVID database. AutoDock Vina was applied to predict the molecular docking and binding ability of bulleyaconitine A with key targets. Finally, a receptor activator of nuclear factor-κB(RANKL)-induced osteoclast differentiation model was established in vitro. Quantitative real-time polymerase chain reaction(qRT-PCR) was used to detect the mRNA expression levels of related targets, and immunofluorescence and Western blot were adopted to detect the protein expression level of key targets. It displayed that there was a total of 29 drug-disease targets, and Src was the core target of bulleyaconitine A in anti-RA bone destruction. Furthermore, KEGG enrichment analysis revealed that bulleyaconitine A may exert an anti-RA bone destruction effect by regulating the Src/PI3K/Akt signaling pathway. The molecular docking results showed that bulleyaconitine A had better bin-ding ability with Src, phosphatidylinositol-4,5-diphosphate 3-kinase(PIK3CA), and Akt1. The result of the experiment indicated that bulleyaconitine A not only dose-dependently inhibited the mRNA expression levels of osteoclast differentiation-related genes cathepsin K(CTSK) and matrix metalloproteinase-9(MMP-9)(P<0.01), but also significantly reduced the expression of p-c-Src, PI3K, as well as p-Akt in vitro osteoclasts(P<0.01). In summary, bulleyaconitine A may inhibit RA bone destruction by regulating the Src/PI3K/Akt signaling pathway. This study provides experimental support for the treatment of RA bone destruction with bulleyaconitine A and lays a foundation for the clinical application of bulleyaconitine A.

[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.

[Mechanism of aqueous extract of Strychni Semen in improving pain in rats with rheumatoid arthritis based on TLR4/TNF-α/MMP-9 signaling pathway].

This study aims to explore the mechanism of aqueous extract of Strychni Semen(SA) in relieving pain in the rat model of rheumatoid arthritis(RA) via Toll-like receptor 4(TLR4)/tumor necrosis factor-α(TNF-α)/matrix metalloproteinase-9(MMP-9) signaling pathway. Firstly, the main chemical components of Strychni Semen were searched against TCMSP, TCMID, ETCM, and related literature, and the main targets of the chemical components were retrieved from TargetNet and SwissTargetPrediction. The main targets of RA and pain were searched against GeneCards, Online Mendelian Inheritance in Man(OMIM), and Therapeutic Target Database(TTD). Venny 2.1.0 was used to obtain the common targets shared by Strychni Semen, RA, and pain, and STRING and Cytoscape 3.6.1 were used to build the protein-protein interaction network. Then, molecular docking was carried out in AutoDock Vina. Finally, the rat model of type Ⅱ collagen-induced arthritis(CIA) was established. The up-down method and acetone method were employed to examine the mechanical pain threshold and cold pain threshold of rats, and the pain-relieving effect of SA on CIA rats was evaluated comprehensively. Hematoxylin-eosin(HE) staining was employed to evaluate the histopathological changes of joints in CIA rats. The expression levels of key target proteins was determined by immunohistochemistry and Western blot, and the mRNA levels of key targets were determined by real-time fluorescence quantitative polymerase chain reaction(real-time PCR). The results of network prediction showed that Strychni Semen may act on the TLR4/TNF-α/MMP-9 signaling pathway to exert the pain-relieving effect. The results of molecular docking showed that brucine, the main active component of SA, had strong binding ability to TLR4, TNF-α, and MMP-9. The results of animal experiments showed that SA improved the mechanical and cold pain sensitivity(P<0.05, P<0.01) and reduced the joint histopathological score of CIA rats(P<0.01). In addition, medium and high doses of SA down-regulated the protein and mRNA levels of TNF-α, TLR4, and MMP-9(P<0.05,P<0.01). In conclusion, SA alleviated the mechanical pain sensitivity, cold pain sensitivity, and joint histopathological changes in CIA rats by inhibiting the over activation of TLR4/TNF-α/MMP-9 signaling pathway.

Brucine alleviates fibroblast-like synoviocytes dysfunction and inflammation by regulating YY1 during rheumatoid arthritis.

Brucine is a weak alkaline indole alkaloid with wide pharmacological activities and has been identified to protect against rheumatoid arthritis (RA) process. Circular RNAs (circRNAs) are also reported to be involved in the pathogenesis of RA. Here, we aimed to probe the role and mechanism of Brucine and circ_0139658 in RA progression. The fibroblast-like synoviocytes of RA (RA-FLSs) were isolated for functional analysis. Cell proliferation, apoptosis, invasion, migration, as well as inflammatory response were evaluated by CCK-8 assay, EdU assay, flow cytometry, transwell assay, and ELISA analysis, respectively. qRT-PCR and western blotting analyses were utilized to measure the levels of genes and proteins. The binding between miR-653-5p and circ_0139658 or Yin Yang 1 (YY1), was verified using dual-luciferase reporter and RNA pull-down assays. Brucine suppressed the proliferation, migration, and invasion of RA-FLSs, and alleviated inflammation by reducing the release of pro-inflammatory factors and macrophage M1 polarization. RA-FLSs showed increased circ_0139658 and YY1 levels and decreased miR-653-5p levels. Circ_0139658 is directly bound to miR-653-5p to regulate YY1 expression. Brucine treatment suppressed circ_0139658 and YY1 expression but increased YY1 expression in RA-FLSs. Functionally, circ_0139658 overexpression reversed the suppressing effects of Brucine on RA-FLS dysfunction and inflammation. Moreover, circ_0139658 silencing alleviated the dysfunction and inflammation in RA-FLSs, which were reverted by YY1 overexpression. Brucine suppressed the proliferation, migration, invasion, and inflammation in RA-FLSs by decreasing YY1 via circ_0139658/miR-653-5p axis.

Identification of Hedyotis diffusa Willd-specific mRNA-miRNA-lncRNA network in rheumatoid arthritis based on network pharmacology, bioinformatics analysis, and experimental verification.

Hedyotis diffusa Willd (HDW) possesses heat-clearing, detoxification, anti-cancer, and anti-inflammatory properties. However, its effects on rheumatoid arthritis (RA) remain under-researched. In this study, we identified potential targets of HDW and collected differentially expressed genes of RA from the GEO dataset GSE77298, leading to the construction of a drug-component-target-disease regulatory network. The intersecting genes underwent GO and KEGG analysis. A PPI protein interaction network was established in the STRING database. Through LASSO, RF, and SVM-RFE algorithms, we identified the core gene MMP9. Subsequent analyses, including ROC, GSEA enrichment, and immune cell infiltration, correlated core genes with RA. mRNA-miRNA-lncRNA regulatory networks were predicted using databases like TargetScan, miRTarBase, miRWalk, starBase, lncBase, and the GEO dataset GSE122616. Experimental verification in RA-FLS cells confirmed HDW's regulatory impact on core genes and their ceRNA expression. We obtained 11 main active ingredients of HDW and 180 corresponding targets, 2150 RA-related genes, and 36 drug-disease intersection targets. The PPI network diagram and three machine learning methods screened to obtain MMP9, and further analysis showed that MMP9 had high diagnostic significance and was significantly correlated with the main infiltrated immune cells, and the molecular docking verification also showed that MMP9 and the main active components of HDW were well combined. Next, we predicted 6 miRNAs and 314 lncRNAs acting on MMP9, and two ceRNA regulatory axes were obtained according to the screening. Cellular assays indicated HDW inhibits RA-FLS cell proliferation and MMP9 protein expression dose-dependently, suggesting HDW might influence RA's progression by regulating the MMP9/miR-204-5p/MIAT axis. This innovative analytical thinking provides guidance and reference for the future research on the ceRNA mechanism of traditional Chinese medicine in the treatment of RA.

MHC-DRB alleles with amino acids Val11, Phe13, and the shared epitopes promote collagen-induced arthritis and a rapid IgG1 response in Filipino cynomolgus macaques.

Macaques are useful animal models for studying the pathogenesis of rheumatoid arthritis (RA) and the development of anti-rheumatic drugs. The purpose of this study was to identify the major histocompatibility complex (MHC) polymorphisms associated with the pathology of collagen-induced arthritis (CIA) and anti-collagen IgG induction in a cynomolgus macaque model, as MHC polymorphisms affect the onset of CIA in other animal models. Nine female Filipino cynomolgus macaques were immunized with bovine type II collagen (b-CII) to induce CIA, which was diagnosed clinically by scoring the symptoms of joint swelling over 9 weeks. MHC polymorphisms and anti-b-CII antibody titers were compared between symptomatic and asymptomatic macaques. Four of 9 (44%) macaques were defined as the CIA-affected group. Anti-b-CII IgG in the affected group increased in titer approximately 3 weeks earlier compared with the asymptomatic group. The mean plasma IgG1 titer in the CIA-affected group was significantly higher (p < 0.05) than that of the asymptomatic group. Furthermore, the cynomolgus macaque MHC (Mafa)-DRB1*10:05 or Mafa-DRB1*10:07 alleles, which contain the well-documented RA-susceptibility five amino acid sequence known as the shared epitope (SE) in positions 70 to 74, with valine at position 11 (Val11, V11) and phenylalanine at position 13 (Phe13, F13), were detected in the affected group. In contrast, no MHC polymorphisms specific to the asymptomatic group were identified. In conclusion, the presence of V11 and F13 along with SE in the MHC-DRB1 alleles seems essential for the production of IgG1 and the rapid induction of severe CIA in female Filipino cynomolgus macaques.

Analysis of Enrichment Pathway, Hub Gene, and Protein-Protein Interaction Network in Rheumatoid Arthritis and Construction of Molecular Subtypes in Peripheral Blood.

Objective: To analyze the enrichment pathway, hub gene, and Protein-protein interaction (PPI) network of rheumatoid arthritis (RA) and construct peripheral blood subtypes based on integrated bioinformatics analysis. Methods: Suitable datasets were screened from the GEO database based on titles and abstracts, batch positive analysis was performed using R language, and KEGG enrichment analysis and GO enrichment analysis were performed. After screening the differential genes, the PPI network was constructed, and the hubba plug-in of Cytoscape software was used to obtain the top 10 hub genes(key regulatory genes). hub genes were used as the typing condition to identify the molecular subtypes of synovial tissue and peripheral blood of arthritis. Results: GSE12021 and GSE93272 have been chosen for analysis. GSE12021 presents the transcriptome analysis of human joint synovial tissue, comprising 12 samples from patients with rheumatoid arthritis and 9 samples from normal healthy individuals. On the other hand, GSE93272 includes human peripheral blood samples, comprising 232 samples from patients with rheumatoid arthritis and 43 samples from normal healthy individuals. The main results of GSE12021 KEGG enrichment were Parathyroid hormone synthesis, Relaxin signaling pathway, TNF signaling pathway, Rheumatoid arthritis, T cell receptor signaling pathway, Th1 and Th2 cell differentiation, Th17 cell differentiation, Toll-like receptor signaling pathway and so on. The main results of GSE12021 GO enrichment were regulation of feeding behavior, regulation of neuron death, positive regulation of cell-cell adhesion, and positive regulation of leukocyte activation. The top 10 hub genes were CD8A, JUN, CTLA4, CD19, LCK, FOS, CCL5, IL7R, CCR7 and CD247. Synovial tissue and peripheral blood subtypes of rheumatoid arthritis showed that the two classification methods maintained consistency. Conclusion: Identifying the Hub gene in peripheral blood helps screen molecular subtypes of rheumatoid arthritis.

The Causal Association between Alcohol, Smoking, Coffee Consumption, and the Risk of Arthritis: A Meta-Analysis of Mendelian Randomization Studies.

Objective: To evaluate the genetic causality between alcohol intake, smoking, coffee consumption, and arthritis. Methods: Mendelian randomization (MR) studies with alcohol, smoking, and coffee consumption behaviors as exposures, and osteoarthritis (OA) and rheumatoid arthritis (RA) as outcomes were retrieved from up to July 2023. Two researchers with relevant professional backgrounds independently assessed the quality and extracted data from the included studies. Meanwhile, we applied MR analyses of four lifestyle exposures and five arthritis outcomes (two for OA and three for RA) with gene-wide association study (GWAS) data that were different from the included studies, and the results were also included in the meta-analysis. Statistical analyses were performed using Stata 16.0 and R software version 4.3.1. Results: A total of 84 studies were assessed. Of these, 11 were selected for meta-analysis. As a whole, the included studies were considered to be at a low risk of bias and were of high quality. Results of the meta-analysis showed no significant genetic causality between alcohol intake and arthritis (odds ratio (OR): 1.02 (0.94-1.11)). Smoking and arthritis had a positive genetic causal association (OR: 1.44 (1.27-1.64)) with both OA (1.44 (1.22-1.71)) and RA (1.37 (1.26-1.50)). Coffee consumption and arthritis also had a positive genetic causal association (OR: 1.02 (1.01-1.03)). Results from the subgroup analysis showed a positive genetic causality between coffee consumption and both OA (OR: 1.02 (1.00-1.03)) and RA (OR: 1.56 (1.19-2.05)). Conclusion: There is positive genetic causality between smoking and coffee consumption and arthritis (OA and RA), while there is insufficient evidence for genetic causality between alcohol intake and arthritis.

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.

Effect of acupuncture on arthritic pathological injury in rats with type â ¡ collagen-induced rheumatoid arthritis. / é’ˆåˆºæ”¹å–„â ¡åž‹èƒ¶åŽŸè›‹ç™½è¯±å¯¼æ€§å ³èŠ‚ç‚Žå¤§é¼ ç‚Žæ€§ååº”çš„æœºåˆ¶ç ”ç©¶.

OBJECTIVES: To observe the effect of acupuncture stimulation of "Yanglingquan"(GB34), "Zusanli"(ST36) and "Xuanzhong" (GB39) on arthritis index (AI), joint synovial membrane pathology, serum-related immunoinflammatory factors, and expressions of tumor suppressor gene mt-p53, nuclear factor kappa B (NF-κB) and peroxisome proliferator activated receptor gamma (PPARγ) in knee joint synovial tissue of rats with type Ⅱ collagen-induced arthritis (CIA), so as to explore its possible mechanisms underlying improvement of rheumatoid arthritis (RA). METHODS: Male SD rats were used in the present study. The CIA model was established by subcutaneous injection of collagen emulsion (200 µL/rat) in the tail root region on the first day and repeat (100 µL/rat) once on the 9th day. Eighteen successful CIA rats were randomized into model, medication and acupuncture groups, with 6 rats in each group. Other 6 normal rats were used as the normal control group. For rats of the medication group, leflunomide (1.9 mg/kg) was administrated by gavage, once a day, and for rats of the acupuncture group, manual acupuncture stimulation was applied to bilateral GB34, ST36, GB39 for 30 min, once a day, for 12 weeks. The arthritis index (AI) score (0-4 points) was evaluated once every week. The contents of IL-6, IL-17 and TNF-α in the serum were determined by ELISA. Histopathological changes of the ankle joint were observed by H.E. staining. The protein and mRNA expression levels of mt-p53, NF-κB p65, and PPARγ in the knee joint synovial tissue were determined by Western blot and quantitative real time PCR, separately. RESULTS: Compared with the normal control group, the AI scores at different time-points after modeling, contents of serum TNF-α, IL-6 and IL-17, expression levels of mt-p53, NF-κB p65, PPARγ proteins and mRNAs were significantly increased in the model group (P<0.01, P<0.05). In comparison with the model group, the AI scores at the 10th week in the medication group and at the 3rd, 9th and 10th week in the acupuncture group, contents of serum TNF-α, IL-6 and IL-17, and the expression levels of mt-p53 and NF-κB p65 proteins in both medication and acupuncture groups, as well as mt-p53 and NF-κB p65 mRNAs in the medication group were apparently decreased (P<0.01, P<0.05), while the expression levels of PPARγ protein in both medication and acupuncture group and PPARγ mRNA in the medication group were significantly up-regulated (P<0.05, P<0.01). No significant differences were found between the acupuncture and medication groups in down-regulating the AI score and serum TNF-α, IL-6 and IL-17 contents. The effect of acupuncture was weaker than that of medication in down-regulating the expression of mt-p53 and NF-κB p65 proteins and mRNAs and in up-regulating PPARγ mRNA (P<0.01). H.E. results showed ankle cartilage hyperplasia, reduced joint cavity, mild fibroproliferation and inflammatory cell infiltration in the surrounding soft tissue of the ankle joint in rats of the model group, which was milder in both medication and acupuncture groups. CONCLUSIONS: Acupuncture stimulation can improve the degree of joint inflammation and swelling in CIA rats, which may be related to its effects in inhibiting the overexpression of immunoinflammatory factors in serum and regulating expression of mt-p53, NF-κB p65, PPARγ mRNAs and proteins in the synovial tissue.

[Total triterpenes of Euphorbium alleviates rheumatoid arthritis via Nrf2/HO-1/GPX4 pathway].

This study aims to investigate the effect and mechanism of total triterpenes of Euphorbium in treating rheumatoid arthritis(RA). The rat model of RA was established with Freund's complete adjuvant(FCA). Male rats were randomly assigned into control, model, Tripterygium glycosides(7.5 mg·kg~(-1)), and low-, medium-, and high-dose total triterpenes of Euphorbium(32, 64, and 128 mg·kg~(-1), respectively) groups, with 10 rats in each group. In other groups except the control group, 0.2 mL FCA was injected into the right hind toe. Rats in the intervention groups were administrated with corresponding drugs by gavage, and the control group and the model group with the same volume of 0.5% CMC-Na solution once a day. During the treatment period, the swelling degree of the hind paw was measured and the arthritis was scored until day 30. At the end of drug administration, the pathological changes of the joint tissue were observed by hematoxylin-eosin staining. The content of malondialdehyde(MDA), glutathione(GSH), and Fe~(2+) and the activity of superoxide dismutase(SOD) in the joint tissue were measured by biochemical colorimetry. RT-PCR was performed to determine the mRNA levels of nuclear factor erythroid 2-related factor 2(Nrf2), glutathione peroxidase 4(GPX4), and acyl-CoA synthetase long chain family member 4(ACSL4) in the joint tissue. Western blot was employed to determine the protein levels of Nrf2, Kelch-like ECH-associated protein 1(Keap1), heme oxygenase-1(HO-1), NAD(P)H quinone oxidoreductase 1(NQO1), SOD2, GPX4, and ACSL4 in the joint tissue. The results showed that the treatment with Tripterygium glycosides(7.5 mg·kg~(-1)) and total triterpenes of Euphorbium(32, 64, and 128 mg·kg~(-1)) alleviated the swelling degree of bilateral hind limbs, decreased the arthritis score, reduced joint tissue lesions and the content of MDA and Fe~(2+) in the joint tissue, and increased GSH content and SOD activity. Furthermore, the interventions up-regulated the protein and mRNA levels of Nrf2 and GPX4, down-regulated the protein and mRNA levels of ACSL4, and up-regulated the protein levels of Keap1, NQO1, HO-1, and SOD2 in the Nrf2/HO-1/GPX4. In summary, the total triterpenes of Euphorbium can treat RA by inhibiting lipid peroxidation and abnormal ferroptosis, which may involve the Nrf2/HO-1/GPX4 signaling pathway.

[Mechanism of Chaenomelis Fructus in treatment of rheumatoid arthritis based on network pharmacology and experimental verification].

The material basis and mechanism of Chaenomelis Fructus in the treatment of rheumatoid arthritis(RA) were explored by network pharmacology, and the potential anti-RA targets of Chaenomelis Fructus were verified by molecular docking and animal experiments. The active components and targets of Chaenomelis Fructus were searched against the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform. GeneCards, DisGeNET, and OMIM were used to obtain RA-related targets. The common targets shared by Chaenomelis Fructus and RA were considered as the potential targets of Chaenomelis Fructus in the treatment of RA. Cytoscape 3.9.0 was employed to establish a "traditional Chinese medicine-active component-common target-disease" network. The protein-protein interaction(PPI) network was established by STRING, and the core genes were visualized by RStudio 4.1.0. DAVID was used for Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment to predict and visualize the involved signaling pathways. Molecular docking was carried out with the active components screened out as ligands and RA core genes as the targets. Finally, the prediction results were verified by animal experiments. Four main active components of Chaenomelis Fructus were obtained, which corresponded to 137 targets. Chaenomelis Fructus and RA shared 37 common targets. GO annotation yielded 239 terms(P<0.05), and KEGG pathway enrichment analysis screened out 94 signaling pathways(P<0.05), mainly involving interleukin-17(IL-17), tumor necrosis factor, Toll-like receptor, and nuclear factor-kappa B(NF-κB) signaling pathways. Molecular docking results showed that the main active components of Chaenomelis Fructus bound well with the core targets of RA. The results of animal experiments proved that Chaenomelis Fructus can alleviate joint swelling in the mice with RA. The results of ELISA showed that Chaenomelis Fructus lowered the levels of interleukin-6(IL-6) and interleukin-1ß(IL-1ß). Western blot showed that Chaenomelis Fructus down-regulated the protein level of vascular endothelial growth factor A(VEGFA). Chaenomelis Fructus exerts anti-inflammatory effect and reduces pannus formation by regulating the core targets such as VEGFA, IL-1ß, and IL6 in the treatment of RA. The findings of this study provide new ideas for the future treatment of RA with Chaenomelis Fructus.

[Effect of Juanbi Qianggu Formula on biological behaviors of fibroblast-like synoviocytes in rheumatoid arthritis by regulating FGFR1 signaling pathway based on network pharmacology and cell function experiments].

This study aimed to explore the molecular mechanism of Juanbi Qianggu Formula(JBQGF), an empirical formula formulated by the prestigious doctor in traditional Chinese medicine, in the treatment of rheumatoid arthritis based on network pharmacology and cell function experiments. The main active components and targets of JBQGF were obtained through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and Encyclopedia of Traditional Chinese Medicine(ETCM), and the core targets underwent functional enrichment analysis and signaling pathway analysis. Cytoscape 3.6.0 was used to construct a visualized "active component-target-signaling pathway" network of JBQGF. After screening, nine potential pathways of JBQGF were obtained, mainly including G protein-coupled receptor signaling pathway and tyrosine kinase receptor signaling pathway. As previously indicated, the fibroblast growth factor receptor 1(FGFR1) signaling pathway was highly activated in active fibroblast-like synoviocytes(FLS) in rheumatoid arthritis, and cell and animal experiments demonstrated that inhibition of the FGFR1 signaling pathway could significantly reduce joint inflammation and joint destruction in collagen-induced arthritis(CIA) rats. In terms of the tyrosine kinase receptor signal transduction pathway, the analysis of its target genes revealed that FGFR1 might be a potential target of JBQGF for rheumatoid arthritis treatment. The biological effect of JBQGF by inhibiting FGFR1 phosphorylation was preliminarily verified by Western blot, Transwell invasion assay, and pannus erosion assay, thereby inhibiting matrix metalloproteinase 2(MMP2) and receptor activator of nuclear factor-κB ligand(RANKL) and suppressing the invasion of fibroblasts in rheumatoid arthritis and erosive effect of pannus bone. This study provides ideas for searching potential targets of rheumatoid arthritis treatment and TCM drugs through network pharmacology.

Comparison of the effect of moxibustion at "Zusanli" (ST36) on the polarization of synovial macrophages of knee joints in rats with knee osteoarthritis and rheumatoid arthritis. / 艾灸"足三里"å¯¹è†å ³èŠ‚éª¨å ³èŠ‚ç‚Žä¸Žç±»é£Žæ¹¿æ€§å ³èŠ‚ç‚Žå¤§é¼ è†å ³èŠ‚æ»‘è†œå·¨å™¬ç»†èƒžæžåŒ–çš„å½±å“çš„æ¯”è¾ƒç ”ç©¶.

OBJECTIVES: To observe the similarities and differences of effects of moxibustion at "Zusanli" (ST36) on target tissues and macrophages polarization in knee osteoarthritis (KOA) and rheumatoid arthritis (RA) rats, and to summarize its efficacy and characteristics. METHODS: Thirty rats were equally and randomly divided into control, KOA, RA, KOA+Moxi and RA+Moxi groups. The KOA model and RA model were induced by injection of sodium monoiodoacetate or Freund's complete adjuvant into the rats' knee joints, respectively. Rats of the KOA+Moxi and RA+Moxi groups received moxibustion stimulation at bilateral ST36 for 30 min, once a day for 21 days, beginning from the 7th day on after modeling. The contents of serum interleukin(IL)-1ß and IL-10 were detected by ELISA. Histopathological changes (Markin score of the knee cartilage and synovial pathology score) of the knee joints were observed after HE staining. The polarization state of M1 and M2 macrophages in the synovial tissue of the knee joints was assessed by detecting the expression of CD86 and CD206 after immunofluorescence staining. RESULTS: Compared with the control group, the content of serum IL-1ß, synovial pathology score, and synovial CD86 expression were significantly increased (P<0.01, P<0.05), while the content of serum IL-10 and synovial CD206 expression markedly decreased (P<0.01) in both KOA and RA groups；the Markin score was increased (P<0.01) in the KOA group. In comparison with the KOA group, the Markin score was obviously decreased (P<0.01), while the content of serum IL-10 and CD206 expression were apparently increased (P<0.01) in the KOA+Moxi group. No significant changes were found in the content of serum IL-1ß, synovial pathology score and CD86 expression in the KOA+Moxi group relevant to the KOA group. In comparison with the RA group, the content of serum IL-1ß, synovial pathology score, and CD86 expression were considerably decreased (P<0.01) in the RA+Moxi group. No marked differences were found in the serum IL-10 level, Markin score, and CD206 expression between RA+Moxi and RA model groups. The increased Markin score was significantly higher in the KOA group than in the RA group (P<0.01), but the increased synovial pathology score was significantly lower in the KOA group than in the RA group (P<0.01). Correspondingly, the effect of moxibustion at ST36 was significantly better in RA model than in KOA model in reducing serum IL-1ß (P<0.05). CONCLUSIONS: Moxibustion at ST36 can effectively reduce cartilage injury of knee joint in rats with KOA and reduce synovial injury in rats with RA, which may be related with its effects in lowering IL-1ß level in RA model by inhibiting the polarization of M1 macrophages, and up-regulating level of IL-10 in KOA model by promoting the polarization of M2 macrophages. However, the relevant mechanism needs to be further studied.

Dissecting the Imbalance of Synovial Macrophages in Rheumatoid Arthritis.

Objective: This study sought to identify candidate genes of rheumatoid arthritis (RA) synovial macrophages using bioinformatics and to explore their pathways in the pathogenesis of RA. Methods: The microarray datasets GSE10500 and GSE97779 were obtained from the Gene Express Omnibus and analyzed with synovial macrophages of 14 RA patients and 8 healthy donors. The researchers used R software to identify differentially expressed genes and determine functional enrichment pathways. A protein-protein interaction network was then constructed using STRING and Cytoscape. Gene expression was validated with the GSE71370 dataset and RT-qPCR analysis. Results: 102 DEGs were identified in RA synovial macrophages relative to normal samples. Of these, 72 were upregulated; 30 were downregulated. GO and KEGG pathway analyses suggested that DEGs mainly regulated the immune response and signaling pathways associated with inflammatory activation, apoptosis, and cancer. The top five hub genes and top 1 gene module from the PPI network of DEGs were VEGFA, MMP9, FN1, IGF1, CXCL9, ISG20, RSAD2, IFI27, GBP2, and GBP1. The GSE71370 dataset and RT-qPCR analysis showed that CXCL9 and GBP1 were significantly upregulated (P ≤ .05). Conclusions: CXCL9 and GBP1 may contribute to RA pathogenesis and serve as potential biomarkers and therapeutic targets for RA.

[Heat-tonifying acupuncture relieves pain and synovial inflammatory injury by regulating Keap1-Nrf2/ARE/ HO-1 signaling pathway in rabbits with cold syndrome type rheumatoid arthritis].

OBJECTIVE: To observe the effect of heat-tonifying needling on Keap1-Nrf2/ARE/HO-1 signal transduction pathway in knee synovium in rabbits with cold syndrome type rheumatoid arthritis (RA), so as to explore its mechanisms underl-ying improvement of RA. METHODS: New Zealand rabbits were randomly divided into normal control, RA model, uniform reinforcing-reducing acupuncture, twisting reinforcing acupuncture and heat-tonifying acupuncture groups, with 6 rabbits in each group. The cold syndrome type RA model was established by subcutaneous injection of mixture fluid of ovalbumin and Freund's complete adjuvant at the shoulder-back as well as injection of mixture of ovalbumin and normal saline into knee-joint cavity combined with ice-compress freezing. Acupuncture stimulation (uniform reinforcing-reducing, or twisting reinforcing or heat-tonifying) was applied to bilateral "Zusanli"(ST36) for 1 min with the needle retained for 30 min, once a day for 7 consecutive days. The general conditions of rabbits in each group were recorded, the thermal pain threshold (TPT) and perimeter of knee joints was measured. Conditions of the synovium in the knee cavity, hydrops, blood flow signal, articular surface, and related muscles were observed by using a color Doppler ultrasonic diagnostic apparatus, and the blood flow signals inside the synovium (image scores) were divided into 0 (no signals), I (1 or 2 dot-like signal), II (less than half) ad III (more than half). After H.E. staining, the pathological changes (0-3 points) were assessed according to the state of inflammatory cell infiltration, and hyperplasia of synovial matrix and coating cells. The expression levels of Keap1, Nrf2, HO-1 and GSH-PX1 mRNAs in the knee synovium were detected by quantitative real-time PCR, and the expression of knee synovial HO-1 protein was measured by Western blot. RESULTS: In comparison with the normal control group, the model group had a significant increase in the perimeter, pathological score, expression of Nrf2, HO-1 mRNAs and HO-1 protein (P<0.05), and an obvious decrease in the TPT, expression levels of Keap1 and GSH-PX1 mRNAs (P<0.05). Relevant to the model group, all the three acupuncture maneuvers reversed modeling-induced increase of perimeter and pathological score (P<0.05), decrease of TPT and expression of GSH-PX1 mRNA(P<0.05), further down-regulated expression of Keap1 mRNA (P<0.05), further up-regulated the expression of Nrf2, HO-1 mRNAs and HO-1 protein (P<0.05). The heat-reinforcing manipulation was significantly superior to uniform reinforcing-reducing and twirling reinforcing manipulations in up-regulating TPT, and expression of Nrf2 mRNA, GSH-PX1 mRNA, HO-1 mRNA and protein (P<0.05), and in down-regulating pathological score and Keap1 mRNA expression (P<0.05). CONCLUSION: Heat-tonifying, uniform reinforcing-reducing and twirling reinforcing needling manipulations may relieve pain and improve pathological state in RA rabbits, which may be associated with their functions in raising the ability of anti-oxidative stress by regulating Keap1-Nrf2/ARE/ HO-1 signaling pathway, the therapeutic effect of heat-tonifying needling is superior to that of uniform reinforcing-reducing and twirling reinforcing needling.

Astragalosides inhibit proliferation of fibroblast-like synoviocytes in experimental arthritis by modulating LncRNA S56464.1/miR-152-3p/Wnt1 signaling axis.

AIM: Astragalus membranaceus (Fisch.) Bunge., the dried root of the plant A. membranaceus, is widely used in the treatment of rheumatoid arthritis (RA) in many Chinese herbal remedies. Astragalosides (AST) is the primary medicinal ingredient of A. membranaceus and has a therapeutic effect on RA, but the specific mechanism of this effect has yet to be elucidated. METHODS: In this study, MTT and flow cytometry were used to determine the effects of AST on fibroblast-like synoviocyte (FLS) proliferation and cell cycle progression. Additionally, real-time quantitative polymerase chain reaction and Western blotting were used to determine the effects of AST on the LncRNA S56464.1/miR-152-3p/Wnt1 signaling axis and on critical genes that are essential to the Wnt pathway. RESULTS: The data showed that after the administration of AST, FLS proliferation and LncRNA S56464.1, ß-catenin, C-myc, Cyclin D1, and p-GSK-3ß(Ser9)/GSK-3ß expression were significantly reduced, and miR-152 and SFRP4 expression was notably increased. CONCLUSION: These results suggest that AST can inhibit FLS proliferation by modulating the LncRNA S56464.1/miR-152-3p/Wnt1 signaling axis and that AST may be a potential therapeutic drug for RA.

Effect of curcumin on the expression of NOD2 receptor and pro-inflammatory cytokines in fibroblast-like synoviocytes (FLSs) of rheumatoid arthritis (RA) patients.

BACKGROUND: Previous studies has shown that nucleotide-binding and oligomerization domain-containing protein 2 (NOD2) is expressed in Fibroblast-like synoviocytes (FLSs) of rheumatoid arthritis (RA) patients which is stimulated by muramyl dipeptide (MDP) present in the joint environment and induces inflammation via the NF-κB pathway. Also, other studies have shown that curcumin inhibits proliferation, migration, invasion, and Inflammation and on the other hand increases the apoptosis of RA FLSs. In this study, we aim to evaluate the effect of curcumin, a natural anti-inflammatory micronutrient, on the expression of NOD2 and inflammatory cytokines. METHODS: Synovial membranes were collected from ten patients diagnosed with RA and ten individuals with traumatic injuries scheduled for knee surgery. The FLSs were isolated and treated with 40 µM curcumin alone or in combination with 20.3 µM MDP for 24 h. mRNA was extracted, and real-time PCR was performed to quantitatively measure gene expression levels of NOD2, p65, IL-6, TNF-α, and IL-1ß. RESULTS: The study findings indicate that administering MDP alone can significantly increase the mRNA expression levels of IL-6 and IL-1ß in the trauma group and TNF-α in the RA group. Conversely, administering curcumin alone or in combination whit MDP can significantly reduce mRNA expression levels of P65 and IL-6 in FLSs of both groups. Moreover, in FLSs of RA patients, a single curcumin treatment leads to a significant reduction in NOD2 gene expression. CONCLUSION: This study provides preliminary in vitro evidence of the potential benefits of curcumin as a nutritional supplement for RA patients. Despite the limitations of the study being an investigation of the FLSs of RA patients, the results demonstrate that curcumin has an anti-inflammatory effect on NOD2 and NF-κB genes. These findings suggest that curcumin could be a promising approach to relieve symptoms of RA.

Identification and validation of metabolism-related genes signature and immune infiltration landscape of rheumatoid arthritis based on machine learning.

Rheumatoid arthritis (RA) causes irreversible joint damage, but the pathogenesis is unknown. Therefore, it is crucial to identify diagnostic biomarkers of RA metabolism-related genes (MRGs). This study obtained transcriptome data from healthy individuals (HC) and RA patients from the GEO database. Weighted gene correlation network analysis (WGCNA), the least absolute shrinkage and selection operator (LASSO), and random forest (RF) algorithms were adopted to identify the diagnostic feature biomarker for RA. In addition, biomarkers were verified by qRT-PCR and Western blot analysis. We established a mouse model of collagen-induced arthritis (CIA), which was confirmed by HE staining and bone structure micro-CT analysis, and then further verified the biomarkers by immunofluorescence. In vitro NMR analysis was used to analyze and identify possible metabolites. The correlation of diagnostic feature biomarkers and immune cells was performed using the Spearman-rank correlation algorithm. In this study, a total of 434 DE-MRGs were identified. GO and KEGG enrichment analysis indicated that the DE-MRGs were significantly enriched in small molecules, catabolic process, purine metabolism, carbon metabolism, and inositol phosphate metabolism. AKR1C3, MCEE, POLE4, and PFKM were identified through WGCNA, LASSO, and RF algorithms. The nomogram result should have a significant diagnostic capacity of four biomarkers in RA. Immune infiltration landscape analysis revealed a significant difference in immune cells between HC and RA groups. Our findings suggest that AKR1C3, MCEE, POLE4, and PFKM were identified as potential diagnostic feature biomarkers associated with RA's immune cell infiltrations, providing a new perspective for future research and clinical management of RA.

[Effect of heat-reinforcing needling on the inflammation and necrotizing apoptosis of synovial cells in synovial tissues of knee joint in rabbits with cold syndrome rheumatoid arthritis].

OBJECTIVE: To observe the effect of heat-reinforcing needling (HRN) on inflammatory factors and necrotizing apoptosis of synovial cells in synovial tissues of knee joint in rabbits with cold syndrome rheumatoid arthritis (RA), so as to explore its underlying mechanisms in treating RA. METHODS: By using the random number table method, 40 New Zealand rabbits were randomly divided into normal, model, antagonist(AG), twist-reinforceing needling (TRN) and HRN groups, with 8 rabbits in each group. The model of cold syndrome RA was established by ovalbumin induction combined with Freund's complete adjuvant injection and cryogenic freezing method. In the AG group, the antagonist TAK-632 (25 mg/kg) was administered intragastrically, once every 2 days, for a total of 7 times. Rabbits of TRN and HRN groups were treated with corresponding acupuncture techniques on bilateral "Zusanli" (ST36) for 30 min, once a day for 14 days. After intervention, the changes of knee skin temperature and circumference were measured. Color Doppler ultrasound was used to observe the joint cavity effusion, synovial thickness and internal blood flow signal. The histomorphological changes of synovial tissues were observed after HE staining. ELISA was used to detect the contents of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6 in serum. Transmission electron microscope was used to observe the ultrastructure, necrosis and apoptosis of synovial cells. Western blot was used to detect the protein expressions of receptor-interacting protein kinase1 (RIPK1), RIPK3, mixed lineage kinase domain-like protein (MLKL), and phosphorylation （p）-MLKL in synovial tissues. RESULTS: Compared with the normal group, the synovial was diffusely hyperplasia, joint cavity effusion and abnormal blood flow signal were obvious, inflammatory cells were clustered, arranged closely and disordered in the model group. The findings of transmission electron microscopy showed disruption of cell membrane integrity, swollen or ruptured mitochondria, obviously ruptured nucleus, condensed and pyknotic chromatin and nucleolus in the model group. Also, the skin temperature of the knee joint was significantly decreased (P<0.01), while the circumference of the knee joint, the contents of TNF-α, IL-1ß and IL-6 in serum, the protein expressions of RIPK1, RIPK3, p-MLKL and MLKL in synovial tissues were significantly increased (P<0.01) in the model group. Compared with the model group, synovial tissue hyperplasia, joint cavity effusion, abnormal blood flow signals, synovial cell proliferation, inflammatory cell infiltration, disruption of cell membrane integrity, cell swelling, cell rupture, and nuclear pyknosis were reduced to different degrees in the AG, TRN and HRN groups. Additionally, the skin temperature of the knee joint was increased (P<0.01, P<0.05), while the circumference of the knee joint, the contents of TNF-α, IL-1ß and IL-6 in serum, the expressions of RIPK1, RIPK3, p-MLKL and MLKL in synovial tissues were decreased (P<0.01, P<0.05) in the AG, TRN and HRN groups. The effects of HRN and AG were notably superior to that of TRN in up-regulating skin temperature of the knee joint, and down-regulating the circumference of the knee joint, the contents of TNF-α, IL-1ß and IL-6 in serum, the expressions of RIPK1, RIPK3, p-MLKL and MLKL in synovial tissues (P<0.01, P<0.05). CONCLUSION: HRN can reduce synovial inflammation of knee joint in rabbits with cold syndrome RA, which may be related to its function in inhibiting the necrotizing apoptosis of synovial cells.