Collagen-induced Arthritis Rats Research Articles

The Miao medicine Sidaxue alleviates rheumatoid arthritis in rats possibly by downregulating matrix metalloproteinases

To explore the inhibitory effect of Sidaxue, a traditional Miao herbal medicine formula, on articular bone and cartilage destruction and synovial neovascularization in rats with collagen-induced arthritis (CIA). In a SD rat model of CIA, we tested the effects of daily gavage of Sidaxue at low, moderate and high doses (10, 20, and 40 g/kg, respectively) for 21 days, with Tripterygium glycosides (GTW) as the positive control, on swelling in the hind limb plantar regions by arthritis index scoring. Pathologies in joint synovial membrane of the rats were observed with HE staining, and serum TNF-α and IL-1β levels were detected with ELISA. The expressions of NF-κB p65, matrix metalloproteinase 1 (MMP1), MMP2 and MMP9 at the mRNA and protein levels in the synovial tissues were detected using real-time PCR and Western blotting. Network pharmacology analysis was conducted to identify the important target proteins in the pathways correlated with the therapeutic effects of topical Sidaxue treatment for RA, and the core target proteins were screened by topological analysis. Treatment with GTW and Sidaxue at the 3 doses all significantly alleviated plantar swelling, lowered arthritis index scores, improved cartilage and bone damage and reduced neovascularization in CIA rats (P<0.05), and the effects of Sidaxue showed a dose dependence. Both GTW and Sidaxue treatments significantly lowered TNF-α, IL-1β, NF-κB p65, MMP1, MMP2, and MMP9 mRNA and protein expressions in the synovial tissues of CIA rats (P<0.05). Network pharmacological analysis identified MMPs as the core proteins associated with topical Sidaxue treatment of RA. Sidaxue alleviates articular bone and cartilage damages and reduces synovial neovascularization in CIA rats possibly by downregulating MMPs via the TNF-α/IL-1β/NF-κB-MMP1, 2, 9 signaling pathway, and MMPs probably plays a key role in mediating the effect of Sidaxue though the therapeutic pathways other than oral administration.

β-Indole-3-acetic acid attenuated collagen-induced arthritis through reducing the ubiquitination of Foxp3 via the AhR-TAZ-Tip60 pathway.

Massive evidence shows that intestinal tryptophan metabolites affected by intestinal flora can modulate the progression of rheumatoid arthritis (RA). However, the effects and mechanisms of intestinal tryptophan metabolites on RA are not yet detailed. Herein, we investigated the protective effects of intestinal tryptophan metabolites on RA and its detailed mechanisms. In this study, the collagen-induced arthritis (CIA) rat model was established. Based on metabolomics analysis, the contents of β-indole-3-acetic acid (IAA), indolylpropionic acid, and indole-3-β-acrylic acid in the sera of CIA rats were significantly less compared with those of the normal rats. Under the condition of Treg or Th17 cell differentiation, IAA significantly promoted the differentiation and activation of Treg cells instead of Th17 cells. Intestinal tryptophan metabolites are well-known endogenic ligands of aryl hydrocarbon receptor (AhR). Not surprisingly, IAA increased the level of Foxp3 through activating the AhR pathway. Interestingly, IAA had little impact on the level of Foxp3 mRNA, but reducing the ubiquitination and degradation of Foxp3. Mechanically, IAA reduced the expression of the transcriptional coactivator TAZ, which was almost completely reversed by either AhR antagonist CH223191 or siRNA. In vitro, IAA decreased the combination of TAZ and the histone acetyltransferase Tip60, while it increased the combination of Tip60 and Foxp3. In CIA rats, oral administration of IAA increased the number of Treg cells and relieved the inflammation. A combined use with CH223191 almost abolished the effect of IAA. Taken together, IAA attenuated CIA by promoting the differentiation of Treg cells through reducing the ubiquitination of Foxp3 via the AhR-TAZ-Tip60 pathway.

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

Ethnopharmacological relevanceFangji 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 studyThe study aimed to explore the possible mechanism of FHD against RA and provided a theoretical basis for alternative therapies for RA. Materials and methodsWe 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. ResultsThrough 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. ConclusionsThe 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.

Host-microbiota interactions in collagen-induced arthritis rats treated with human umbilical cord mesenchymal stem cell exosome and ginsenoside Rh2

Mesenchymal stem cell exosome (MSCs-exo) is a class of products secreted by mesenchymal stem cells (MSCs) that contain various biologically active substances. MSCs-exo is a promising alternative to MSCs due to their lower immunogenicity and lack of ethical constraints. Ginsenoside Rh2 (Rh2) is a hydrolyzed component of the primary active substance of ginsenosides. Rh2 has a variety of pharmacological functions, including anti-inflammatory, anti-tumor, and antioxidant. Studies have demonstrated that gut microbiota and metabolites are critical in developing rheumatoid arthritis (RA). In this study, we constructed a collagen-induced arthritis (CIA) model in rats. We used MSCs-exo combined with Rh2 to treat CIA rats. To observe the effect of MSCs-exo combined with Rh2 on joint inflammation, rat feces were collected for 16 rRNA amplicon sequencing and untargeted metabolomics analysis. The results showed that the arthritis index score and joint swelling of CIA rats treated with MSCs-exo in combination with Rh2 were significantly lower than those of the model and MSCs-exo alone groups. MSCs-exo and Rh2 significantly ameliorated the disturbed gut microbiota in CIA rats. The regulation of Candidatus_Saccharibacteria and Clostridium_XlVb regulation may be the most critical. Rh2 enhanced the therapeutic effect of MSCs-exo compared with the MSCs-exo -alone group. Furthermore, significant changes in gut metabolites were observed in the CIA rat group, and these differentially altered metabolites may act as messengers for host-microbiota interactions. These differential metabolites were enriched into relevant critical metabolic pathways, revealing possible pathways for host-microbiota interactions.

Nuciferine alleviates collagen-induced arthritic in rats by inhibiting the proliferation and invasion of human arthritis-derived fibroblast-like synoviocytes and rectifying Th17/Treg imbalance

Rheumatoid arthritis (RA) is a chronic autoimmune disorder marked by persistent synovial inflammation and joint degradation, posing challenges in the development of effective treatments. Nuciferine, an alkaloid found in lotus leaf, has shown promising anti-inflammatory and anti-tumor effects, yet its efficacy in RA treatment remains unexplored. This study investigated the antiproliferative effects of nuciferine on the MH7A cell line, a human RA-derived fibroblast-like synoviocyte, revealing its ability to inhibit cell proliferation, promote apoptosis, induce apoptosis, and cause G1/S phase arrest. Additionally, nuciferine significantly reduced the migration and invasion capabilities of MH7A cells. The therapeutic potential of nuciferine was further evaluated in a collagen-induced arthritis (CIA) rat model, where it markedly alleviated joint swelling, synovial hyperplasia, cartilage injury, and inflammatory infiltration. Nuciferine also improved collagen-induced bone erosion, decreased pro-inflammatory cytokines and serum immunoglobulins (IgG, IgG1, IgG2a), and restored the balance between T helper (Th) 17 and regulatory T cells in the spleen of CIA rats. These results indicate that nuciferine may offer therapeutic advantages for RA by decreasing the proliferation and invasiveness of FLS cells and correcting the Th17/Treg cell imbalance in CIA rats.

Study on mechanism of Aconitum pendulum in treating rheumatoid arthritis based on toxicity efficacy evaluation and metabonomics

The study aims to investigate the effects and potential mechanism of raw and processed Aconitum pendulum Busch on rheumatoid arthritis(RA) and analyze their toxicity attenuating and efficacy retaining effects. The bovine type Ⅱ collagen-induced arthritis(CIA) rat model was established. The weight, cardiac index, immune organ index, and arthritis index of the rats were recorded and calculated after administration. ELISA was used to measure the expressions of creatine kinase(CK), cardiac troponin T(cTnT), and multiple factors. The pathological morphological changes in heart tissue and ankle joint tissue were observed by hematoxylin-eosin staining. Connexin 43(Cx43) expression in the hearts of CIA rats was detected via immunohistochemical method. The levels of endogenous metabolites in the serum of CIA rats were detected by UPLC-Q-TOF-MS. Potential biomarkers were screened, and related metabolic pathways were analyzed. The results showed that raw A. pendulum could induce local myocardial fiber degeneration and necrosis, increase the cardiac index, decrease the average positive area of Cx43 expression significantly, and increase the expressions of CK and cTnT in cardiac tissue of rats. Meanwhile, raw A. pendulum could decrease the immune organ index, interleukin-6(IL-6), and other inflammatory cytokine contents in the serum and improve the damaged synovium and joint surface of CIA rats, with toxicity and efficacy coexisting. The Zanba stir-fired A. pendulum could reduce the index of arthritis, immune organ index, and content of IL-6 and inflammatory cytokines in serum and improve damaged synovium and joint surface of CIA rats with no obvious cardiac toxicity, showing significant toxicity attenuating and efficacy retaining effects. A total of 19 potential biomarkers of raw A. pendulum and Zanba stir-fired A. pendulum against RA were screened by serum metabolomics, including glycerophospholipid metabolism, glycine, serine, and threonine metabolism, arginine and proline metabolism, and steroid hormone synthesis. In conclusion, Xizang medicine A. pendulum is preventive and curative for RA. Raw A. pendulum has certain cardiotoxicity, and Zanba stir-fired A. pendulum has significant toxicity attenuating and efficacy retaining effects. The anti-RA mechanism may be related to the regulation of glycerophospholipid and amino acid metabolism.

Exploring the Mechanism of Acupuncture's Influence on the Protease Activity and Free Radical Damage in Synovial Fluid of Rheumatoid Arthritis Rats Induced by Type II Collagen from the Release of Active Oxygen.

Acupuncture was studied to investigate the mechanism of its effect on protease vitality and free radical damage in Type I CIA rats induced by type II collagen. The study divided rats into a control group (injected with physiological saline, n = 10), a model group (injected with type II collagen, n = 10), and an intervention group (injected with type II collagen + acupuncture ST36 and GB39, 3 times a week, for a total of 4weeks, n = 10) based on the different injected drugs. Then, various indicators of the mice were experimentally tested using joint index scoring, H&E histological staining, protein blotting, and immunohistochemistry staining methods. Acupuncture ST36 and GB39 can reduce arthritis scores, histological staining scores, and increase MVD in CIA rats. And reduce protease levels, alleviate inflammation, synovial hyperplasia, and angiogenesis. In addition, the intervention group TNF-α, IL-1β and IL-6 mRNA were reduced, and the clearance rates of hydrogen peroxide free radicals and nitric oxide free radicals were increased. The expression levels of ROS and MDA decrease, while the expression levels of SOD increase It has been proved that acupuncture at ST36 and GB39 can inhibit the release of ROS, reduce protease activity, inflammation, synovial hyperplasia, angiogenesis and free radical damage, thus reducing the severity of CIA (Collagen-Induced Arthritis) in rats.

Exosomes Derived from Bone Marrow Mesenchymal Stem Cells Alleviate Rheumatoid Arthritis Symptoms via Shuttling Proteins.

Our prior investigations have evidenced that bone marrow mesenchymal stem cell (BMSC) therapy can significantly improve the outcomes of rheumatoid arthritis (RA). This study aims to conduct a comprehensive analysis of the proteomics between BMSCs and BMSCs-Exos, and to further elucidate the potential therapeutic effect of BMSCs-Exos on RA, so as to establish a theoretical framework for the prevention and therapy of BMSCs-Exos on RA. The 4D label-free LC-MS/MS technique was used for comparative proteomic analysis of BMSCs and BMSCs-Exos. Collagen-induced arthritis (CIA) rat model was used to investigate the therapeutic effect of BMSCs-Exos on RA. Our results showed that some homology and differences were observed between BMSCs and BMSCs-Exos proteins, among which proteins highly enriched in BMSCs-Exos were related to extracellular matrix and extracellular adhesion. BMSCs-Exos can be taken up by chondrocytes, promoting cell proliferation and migration. In vivo results revealed that BMSCs-Exos significantly improved the clinical symptoms of RA, showing a certain repair effect on the injury of articular cartilage. In short, our study revealed, for the first time, that BMSCs-Exos possess remarkable efficacy in alleviating RA symptoms, probably through shuttling proteins related to cell adhesion and tissue repair ability in CIA rats, suggesting that BMSCs-Exos carrying expressed proteins may become a useful biomaterial for RA treatment.

Biomimetic nanoparticles for effective Celastrol delivery to targeted treatment of rheumatoid arthritis through the ROS-NF-κB inflammasome axis

Previous study has indicated that Celastrol (Cel) has various physiological and pharmacological effects, including antibacterial, antioxidant, pro-apoptotic, anticancer and anti-rheumatoid arthritis (RA) effects. However, low water solubility, low oral bioavailability, narrow treatment window, and high incidence of systemic adverse reactions still limit the further clinical application of Cel. Here, aiming at effectively overcome those shortcomings of Cel to boost its beneficial effects for treating RA, we developed the leukosome (LEUKO) coated biomimetic nanoparticles (NPs) for the targeted delivery of Cel to arthritis injury area in RA. LEUKO were synthesized using membrane proteins purified from activated J774 macrophage. LEUKO and Cel-loaded LEUKO (Cel@LEUKO) were characterized using dynamic light scattering and transmission electron microscopy. Our results demonstrated that Cel@LEUKO can inhibit the inflammatory response of lipopolysaccharide (LPS) induced mouse monocyte macrophage leukemia cells (RAW264.7 cells) and human rheumatoid arthritis synovial fibroblasts (MH7A) cells through the inhibition of reactive oxygen species (ROS)-NF-κB pathway. In addition, research has shown that LEUKO effectively targets and transports Cel to the inflammatory site of RA, increased drug concentration in affected areas, reduced systemic toxicity of Cel, and reduced clinical symptoms, inflammatory infiltration, bone erosion, and serum inflammatory factors in collagen-induced arthritis (CIA) rats.

Cayratia albifolia C.L.Li exerts anti-rheumatoid arthritis effect by inhibiting macrophage activation and neutrophil extracellular traps (NETs)

BackgroundCayratia albifolia C.L.Li (CAC), commonly known as “Jiao-Mei-Gu” in China, has been extensively utilized by the Dong minority for several millennia to effectively alleviate symptoms associated with autoimmune diseases. CAC extract is believed to possess significant anti-inflammatory properties within the context of Dong medicine. However, an in-depth understanding of the specific pharmaceutical effects and underlying mechanisms through which CAC extract acts against rheumatoid arthritis (RA) has yet to be established.MethodsTwenty-four Sprague–Dawley rats were divided into four groups, with six rats in each group. To induce the collagen-induced arthritis (CIA) model, the rats underwent a process of double immunization with collagen and adjuvant. CAC extract (100 mg/kg) was orally administered to rats. The anti-RA effects were evaluated in CIA rats by arthritis score, hind paw volume and histopathology analysis. Pull-down assay was conducted to identify the potential targets of CAC extract from RAW264.7 macrophage lysates. Moreover, mechanism studies of CAC extract were performed by immunofluorescence assays, real-time PCR and Western blot.ResultsCAC extract was found to obviously down-regulate hind paw volume of CIA rats, with diminished inflammation response and damage. 177 targets were identified from CAC extract by MS-based pull-down assay. Bioinformatics analysis found that these targets were mainly enriched in macrophage activation and neutrophils extracellular traps (NETs). Additionally, we reported that CAC extract owned significant anti-inflammatory activity by regulating PI3K-Akt-mTOR signal pathway, and inhibited NETosis in response to PMA.ConclusionsWe clarified that CAC extract significantly attenuated RA by inactivating macrophage and reducing NETosis via a multi-targets regulation.Graphical

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&lt;0.05, P&lt;0.01) and reduced the joint histopathological score of CIA rats(P&lt;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&lt;0.05,P&lt;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.

Effect and mechanism of aqueous extract of Strychni Semen on bone destruction in rats with type Ⅱ collagen-induced rheumatoid arthritis

To investigate the mechanism of action of aqueous extract of Strychni Semen(SA) on bone destruction in rats with type Ⅱ collagen-induced arthritis(CIA), the SD rats were randomly divided into normal group, model group, low, medium, and high dose(2.85, 5.70, and 11.40 mg·kg~(-1)) groups of SA, and methotrexate group. Except for the normal group, the CIA model was prepared for the other groups. After the second immunization, different doses of SA were given to the low, medium, and high dose groups of SA once a day, and the methotrexate group was given once every three days. 0.3% sodium hydroxymethylcellulose(CMC-Na) was given once a day to the normal and model groups for 28 d. The clinical score of arthritis was evaluated every three days. Micro computed tomography(Micro-CT) method was used to evaluate the degree of bone destruction. Histopathological changes in the joint tissue and the number of osteoclasts in CIA rats were evaluated by hematoxylin-eosin(HE) staining and tartrate-resistant acid phosphatase(TRAP) staining. The expression of interleukin-1β(IL-1β) in the joint tissue of rats was detected by immunohistochemistry. Western blot was used to detect key protein expression in mitogen-activated protein kinase(MAPK) and phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt) signaling pathways in the joint tissue of rats. The results showed that different doses of SA were able to improve the red and swollen inflammatory joint and joint deformity in CIA rats to varying degrees, reduce the clinical score, inhibit synovial inflammation, vascular opacification, cartilage erosion, and bone destruction, and reduce the number of TRAP-positive cells in bone tissue. Micro-CT results showed that the SA was able to increase bone mineral density, bone volume fraction, trabecular reduce, and trabecular number and reduce bone surface/bone volume and trabecular separation/spacing. Different doses of SA could down-regulate the protein expression of IL-1β, p-JNK, p-ERK, p-p38, PI3K, and p-Akt to varying degrees. In conclusion, SA can improve disease severity, attenuate histopathological and imaging changes in joints, and have osteoprotective effects in CIA rats, and its mechanism of action may be related to the inhibition of the overactivation of MAPK and PI3K/Akt signaling pathways.

Role of the CXCR4-Gnαq-Plcβ signaling pathway in the pathogenesis of collagen-induced arthritis in rats

Rheumatoid arthritis (RA) is a chronic autoimmune disease in which immune cells and inflammatory cytokines are abnormally activated, leading to immunoregulatory dysfunction in the body and triggering systemic inflammatory responses. The interaction between CXC chemokine receptor 4 (CXCR4) and heterotrimeric G-protein α-subunit Gαq (Gnαq) activates phospholipase Cβ (PLCβ), which influences the expression of downstream effectors and participates widely in the onset and development of various diseases, thus suggesting the potential involvement of these molecules in RA pathogenesis. Therefore, the present study aimed to determine whether the CXCR4-Gnαq-PLCβ signaling pathway participates in the onset and development of RA. Using a collagen-induced arthritis (CIA) rat model, we found that compared with the control (healthy) rat group, CIA rats exhibited highly time-dependent arthritis, with the maximum arthritis score occurring in week 3. In contrast to the splenic and joint tissue of control rats, CIA rats showed obvious hyperplasia in the lymphoid white pulp and main germination centers of the spleen, narrowing of joint cavities, and inflammatory cellular infiltration on articular surfaces. The serum levels of expression of IL-1β, IL-4, IL-6, and TNF-α were significantly elevated (P < 0.05, P < 0.01). Core genes of the CXCR4-Gnαq-PLCβ pathway, namely CXCR4, Gnαq, PLCβ1, MMP1, and MMP3, also showed a significant increase in mRNA and protein expression levels (P < 0.05, P < 0.01). Proteins related to the CXCR4-Gnαq-PLCβ pathway were mainly localized to the red and white pulp regions in the spleen as well as in stromal, endothelial, and subdifferentiated synovial cells in the joints. These results indicated that CXCR4 is dependent on Gnαq for inducing the expression of PLCβ1 and stimulation of secretion of inflammatory cytokines by inflammatory cells. This consequently affects the expression of matrix metalloproteinases (MMPs), which serve as downstream effectors, thereby promoting RA pathogenesis. Our findings play an important role in elucidating the mechanisms of the onset and development of RA.

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-1β(IL-1β) 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-1β 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.

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. 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. 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-1β, 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. 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.

All-trans retinoic acid alleviates collagen-induced arthritis and promotes intestinal homeostasis

All-trans retinoic acid (ATRA) has emerged as a promising adjunctive treatment for rheumatoid arthritis. However, the mechanism by which ATRA mitigates arthritis remains unclear. In this study, we aimed to explore ATRA alleviation of arthritis and the role of ATRA in regulating intestinal homeostasis. Thus, we established a collagen-induced arthritis (CIA) model in Wistar rats. After 6 weeks of ATRA treatment, the arthritis index of CIA rats decreased, synovial inflammation was alleviated, and the disruption of Th17/Treg differentiation in peripheral blood was reversed. Additionally, the Th17/Treg ratio in the mesenteric lymph nodes decreased and the expression of Foxp3 mRNA increased and that of IL-17 mRNA decreased in the colon and ileum. Microscopically, we observed reduced intestinal inflammation. Transmission electron microscopy revealed that ATRA could repair tight junctions, which was accompanied by an increase in the expression of Claudin-1, Occludin and ZO-1. Moreover, ATRA regulated the composition of the gut microbiota, as was characterized based on the reduced abundance of Desulfobacterota and the increased abundance of Lactobacillus. In conclusion, ATRA demonstrates the potential to alleviate arthritis in CIA rats, which might be correlated with modulating the gut microbiota and regulating the intestinal immune response. Our findings provide novel insights into ATRA-mediated alleviation of arthritis.

Fat mass and obesity-associated protein inhibit the pathology of rheumatoid arthritis through the NSUN2/SFRP1/Wnt/β-catenin signal axis.

The purpose of this study is to investigate whether fat mass and obesity-associated protein (FTO) and NOL1/NOP2/Sun domain family member 2 (NSUN2) mediated RNA methylation is associated with RA pathology. We studied the anti-rheumatoid arthritis (RA) mechanism mediated by FTO and NSUN2 in RA samples and collagen-induced arthritis (CIA) rats using real time qPCR (RT-qPCR), western blot, immunofluorescence, and other methods. The expression of NSUN2 was significantly increased in both RA patients and CIA rats compared with normal controls. Knockdown of NSUN2 blocked the Wnt/β-catenin signaling pathway and inhibited RA pathological factors such as MMP3, fibronectin, and interleukins. FTO overexpression inhibited RA by inhibiting the expression of NSUN2, up-regulating the level of SFRP1 protein, and blocking the Wnt/β-catenin signaling pathway. NSUN2 overexpression interfered with the inhibitory effects of FTO on the Wnt/β-catenin signaling pathway and RA pathology, which further verified that FTO inhibited RA through the NSUN2/SFRP1/Wnt/β-catenin signal axis. FTO and NSUN2 are important factors of RA, and this work provides new potential diagnostic biomarkers and therapeutic targets for RA. We also reveal a gene expression regulation pattern of the interaction between m6A and m5C. revealing the pathogenesis of RA from the perspective of RNA methylation.

The therapeutic effect of Loranthus parasiticus lignan derivatives on collagen-induced arthritis in rats through the SHBG/NFκB pathway.

Lignan-rich beans, nuts, and various seeds are the main foods with antioxidative and hormone-modulating activities. Although the role of lignans in mediating hormone-dependent cancers and cardiovascular diseases is well characterized, the function of lignans in anti-arthritic activity and its underlying mechanisms remain unknown. Three new lignan derivatives, (-)-nortrachelogenin, trachelogenin, and matairesinol, were extracted from Loranthus parasiticus. After establishing the collagen-induced arthritis (CIA) model by intradermal injection of collagen, rats were treated with three new lignan derivatives ((-)-nortrachelogenin: 37%; trachelogenin: 27%; matairesinol: 25.7%) at a concentration of 50mg/kg and 100mg/kg, or methotrexate at 0.3mg/kg. Mixed lignan derivatives significantly attenuated the immune responses in the joints of CIA rats, leading to lower levels of proinflammatory cytokines (IL-6 and TNF-α) and higher levels of free androgen in the serum compared to the CIA model. The results of molecular docking using AutoDock Vina showed that the lignan derivative (-)-nortrachelogenin was the most effective compound for binding to sex hormone-binding globulin (SHBG), thus inhibiting the activity of NFκB in LPS-stimulated macrophages. In this study, (-)-nortrachelogenin was identified as a novel natural lignan derivative with previously unrecognized anti-inflammatory activity. Its molecular mechanism appears related to the regulation of the NFκB/SHBG pathway. Our findings suggest that further application of sex hormone-like compounds in the treatment of rheumatoid arthritis and the potential clinical applications of (-)-nortrachelogenin are promising.

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

To investigate the effect of isorhamnetin on the pathology of rheumatoid arthritis (RA). 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 β, 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. 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 β 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 β (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 β, 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). 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.

CircTldc1 increases Tldc1 expression by targeting miR-485–5p to promote fibroblast-like synoviocytes proliferation in collagen-induced arthritis

Abnormalities in the function of fibroblast-like synoviocytes (FLSs) are crucial factors leading to joint damage of rheumatoid arthritis. In recent years, the role of circular RNA (circRNA) in RA has gradually been revealed. However, the functional regulation of FLSs mediated by circRNA and its potential mechanisms remain unclear. In this study, we elucidated the expression profile of circRNA in FLSs, as well as the role and molecular mechanisms of circTldc1. Through sequencing and validation experiments on primary FLSs derived from collagen-induced arthritis (CIA) rats, we found that circTldc1 can promote FLSs proliferation and exacerbate CIA-induced joint damage. The data revealed that circTldc1's parent gene, Tldc1, is homologous to human Tldc1, and circTldc1 is located in the cytoplasm of FLSs, belonging to the exonic circRNA category. The results from bioinformatics analysis, molecular experiments on FLSs (manipulating circTldc1 expression in vitro), and animal experiments (local regulation of circTldc1 expression in vivo) collectively confirmed that circTldc1 promotes Tldc1 expression by targeting miR-485–5p. High expression of Tldc1 further enhances FLSs proliferation and inflammatory responses, thereby worsening joint damage in CIA rats. High expression of circTldc1 and its parent gene Tldc1 may serve as biomarkers for RA. Local regulation of circTldc1 and Tldc1 gene levels in the joint cavity may represent a potential strategy to improve joint damage and inflammation in RA.