Collagen-induced Arthritis Rats Research Articles

Anti-inflammatory properties and characterization of water extracts obtained from Callicarpa kwangtungensis Chun using in vitro and in vivo rat models

Callicarpa kwangtungensis Chun (CK) is a common remedy exhibits anti-inflammatory properties and has been used in Chinese herbal formulations, such as KangGongYan tablets. It is the main component of KangGongYan tablets, which has been used to treat chronic cervicitis caused by damp heat, red and white bands, cervical erosion, and bleeding. However, the anti-inflammatory effects of CK water extract remains unknown. This study assessed the anti-inflammatory effects of CK in vivo and in vitro, characterized its main components in the serum of rats and verified the anti-inflammatory effects of serum containing CK. Nitric oxide (NO), tumour necrosis factor α (TNF-α) and interleukin-6 (IL-6) release by RAW264.7 cells was examined by ELISA and Griess reagents. Inflammation-related protein expression in LPS-stimulated RAW264.7 cells was measured by western blotting. Furthermore, rat model of foot swelling induced by λ-carrageenan and a collagen-induced arthritis (CIA) rat model were used to explore the anti-inflammatory effects of CK. The components of CK were characterized by LC–MS, and the effects of CK-containing serum on proinflammatory factors levels and the expression of inflammation-related proteins were examined by ELISA, Griess reagents and Western blotting. CK suppressed IL-6, TNF-α, and NO production, and iNOS protein expression in LPS-stimulated RAW264.7 cells. Mechanistic studies showed that CK inhibited the phosphorylation of ERK, P38 and JNK in the MAPK signaling pathway, promoted the expression of IκBα in the NF-κB signaling pathway, and subsequently inhibited the expression of iNOS, thereby exerting anti-inflammatory effects. Moreover, CK reduced the swelling rates with λ-carrageenan induced foot swelling, and reduced the arthritis score and incidence in the collagen-induced arthritis (CIA) rat model. A total of 68 compounds in CK water extract and 31 components in rat serum after intragastric administration of CK were characterized. Serum pharmacological analysis showed that CK-containing serum suppressed iNOS protein expression and NO, TNF-α, and IL-6 release. CK may be an anti-inflammatory agent with therapeutic potential for acute and chronic inflammatory diseases, especially inflammatory diseases associated with MAPK activation.

Mechanism of Jinteng Qingbi granules in the treatment of rheumatoid arthritis using metabolomics analysis.

This study investigated the differential metabolites after rheumatoid arthritis (RA) rats were treated with Jinteng Qingbi granules. Collagen-induced arthritis rats were divided into three groups, namely normal group, model group, and Jinteng Qingbi granules group. Serum compounds were identified, annotated, and classified using metabolomics to explain the physicochemical properties and biological functions. The metabolites were screened using univariate and multivariate statistical analyses. There were differences in serum metabolites between RA and normal rats; Jinteng Qingbi granules improved RA and recovered the metabolite levels to normal. Compared to the normal group, 51 differential ions were screened, and 108 ions were changed in the Jinteng Qingbi granules group compared to the RA model. Eight metabolites were upregulated in the RA model group compared to the normal group, whereas 10 metabolites were downregulated. Treatment with Jinteng Qingbi granules increased the levels of 12 metabolites such as cinnamate and decreased the levels of 16 metabolites such as allamandin in the RA model. Differential ion enrichment was mainly related to the histidine metabolic pathway in amino acid metabolism. Jinteng Qingbi granules resulted in improvements in the RA model, which were mainly associated with lipids and lipid-like molecules, organic acids, and derivatives, providing a new possibility and basis for screening biomarkers for the diagnosis and treatment of RA.

IGF2BP2 regulates the inflammation of fibroblast-like synoviocytes via GSTM5 in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease with an unknown etiology. RA cannot be fully cured and requires lengthy treatment, imposing a significant burden on both individuals and society. Due to the lack of specific drugs available for treating RA, exploring a key new therapeutic target for RA is currently an important task. Activated fibroblast-like synoviocytes (FLSs) play a crucial role in the progression of RA, which release interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α resulting in abnormal inflammatory reaction in the synovium. A previous study has highlighted the correlation of m6A reader insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) with inflammation-related diseases in human. However, the role of IGF2BP2 in the inflammatory reaction of FLSs during RA progression has not been assessed. In this study, IGF2BP2 expression was decreased in the synovial tissues of RA patients and collagen-induced arthritis (CIA) rats. Intra-articular injection of an adeno-associated virus (AAV) vector overexpressing IGF2BP2 relieved paw swelling, synovial hyperplasia and cartilage destruction in CIA rats. IGF2BP2 overexpression also inhibited lipopolysaccharide (LPS)-mediated RA fibroblast-like synoviocytes (RA-FLSs) migration and invasion accompanied by a decreased level of inflammatory factors in vitro. Conversely, IGF2BP2 suppression promoted RA-FLSs migration and invasion with an elevated level of inflammatory factors in vitro. The sequencing result showed that glutathione S-transferase Mu 5 (GSTM5), a key antioxidant gene, was the target mRNA of IGF2BP2. Further experiments demonstrated that IGF2BP2 strengthened the stability of GSTM5 mRNA, leading to weakened inflammatory reaction and reduced expression of matrix metalloproteinase 9 and 13 (MMP9, MMP13). Therefore, IGF2BP2-GSTM5 axis may represent a potential therapeutic target for RA treatment.

Expression of CCL2 signaling pathway genes in patients with periodontitis and atherosclerosis.

Periodontitis and atherosclerosis are chronic inflammatory diseases characterized by leukocyte infiltration. We investigated the expression of CCL4, CCR5, c-Jun, c-Fos, NF-κB, and CCL2 as well as the possible mechanism involved in the regulation of CCL2 in human periodontitis tissues and atherosclerotic aorta based on previous research on the CCL4/CCR5/c-Jun and c-Fos/CCL2 pathway leading to CCL2 expression in collagen-induced arthritis (CIA) rat. Sixty-five volunteers were recruited and the condition of their gingiva and coronary arteries were assessed. The subjects were divided into four groups: healthy control, chronic periodontitis (CP), coronary artery diseases (CAD), and noncoronary artery diseases (non-CAD). Total RNA was isolated from gingiva in periodontitis patients and control populations and from the aorta in patients with and without CAD. PCR was used to examine CCL4, CCR5, c-Jun, c-Fos, NF-κB, and CCL2 levels. The production of CCL2 in the gingiva and aorta was analyzed by immunostaining. PCR revealed that CCL4, CCR5, and CCL2 mRNA levels were increased in CP patients' gingivae and aortas from coronary artery bypass grafting (CABG) patients. Marked c-Jun, c-Fos, and NF-κB gene productions were detected in CP patients' gingivae but did not show statistical differences between the CAD and non-CAD groups. Stronger immunoreactivity against CCL2 was observed in periodontitis gingiva and aorta from CABG patients. Our findings suggest that the CCL4/CCR5/c-Jun and c-Fos/CCL2 pathways may be involved in CCL2 expression in periodontitis. CCL4, CCR5, and CCL2 might act as possible nodes to link the presence of periodontitis and atherosclerosis.

Anti-rheumatoid arthritis effects of traditional Chinese medicine Fufang Xiaohuoluo pill on collagen-induced arthritis rats and MH7A cells.

Fufang Xiaohuoluo pill (FFXHL) is a commonly used prescription in clinical practice for treating rheumatoid arthritis in China, yet its specific mechanism remains unclear. This study aims to elucidate the pharmacological mechanisms of FFXHL using both in vivo and in vitro experiments. The collagen-induced arthritis (CIA) rat model was established to evaluate FFXHL's therapeutic impact. Parameters that include paw swelling, arthritis scores, and inflammatory markers were examined to assess the anti-inflammatory and analgesic effects of FFXHL. Human fibroblast-like synoviocytes (MH7A cells) is activated by tumour necrosis factor-alpha (TNF-α) were used to explore the anti-inflammatory mechanism on FFXHL. Our findings indicate that FFXHL effectively reduced paw swelling, joint pain, arthritis scores, and synovial pannus hyperplasia. It also lowered serum levels of TNF-α, interleukin-1β (IL1β), and interleukin-6 (IL-6). Immunohistochemical analysis revealed decreased expression of nuclear factor-kappa B (NF-κB) p65 in FFXHL-treated CIA rat joints. In vitro experiments demonstrated FFXHL's ability to decrease protein secretion of IL-1β and IL-6, suppress mRNA expression of matrix metalloproteinases (MMP) -3, -9, and -13, reduce reactive oxygen species (ROS) levels, and inhibit NF-κB p65 translocation in TNF-α stimulated MH7Acells. FFXHL also suppressed protein levels of extracellular signal-regulated kinase (ERK), c-Jun Nterminal kinase (JNK), p38 MAP kinase (p38), protein kinase B (Akt), p65, inhibitor of kappa B kinase α/β (IKKα/β), Toll-like receptor 4 (TLR4), and myeloid differentiation primary response 88 (MyD88) induced by TNF-α in MH7A cells. The findings imply that FFXHL exhibits significant anti-inflammatory and antiarthritic effects in both CIA rat models and TNF-α-induced MH7A cells. The potential mechanism involves the inactivation of TLR4/MyD88, mitogen-activated protein kinases (MAPKs), NF-κB, and Akt pathways by FFXHL.

[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.

Sophoridine exerts anti-arthritic effects on fibroblast-like synoviocytes and collagen-induced arthritis in rats.

The discovery of alternative medicines with fewer adverse effects is urgently needed for rheumatoid arthritis (RA). Sophoridine (SR), the naturally occurring quinolizidine alkaloid isolated from the leguminous sophora species, has been demonstrated to possess a wide range of pharmacological activities. However, the effect of SR on RA remains unknown. In this study, the collagen-induced arthritis (CIA) rat model and tumor necrosis factor alpha (TNFα)-induced fibroblast-like synoviocytes (FLSs) were utilized to investigate the inhibitory effect of SR on RA. The anti-arthritic effect of SR was evaluated using the CIA rat model in vivo and TNFα-stimulated FLSs in vitro. Mechanistically, potential therapeutic targets and pathways of SR in RA were analyzed through drug target databases and disease databases, and validation was carried out through immunofluorescence, immunohistochemistry, and Western blot. The in vivo results revealed that SR treatment effectively ameliorated synovial inflammation and bone erosion in rats with CIA. The in vitro studies showed that SR could significantly suppress the proliferation and migration in TNFα-induced arthritic FLSs. Mechanistically, SR treatment efficiently inhibited the activation of MAPKs (JNK and p38) and NF-κB pathways in TNFα-induced arthritic FLSs. These findings were further substantiated by Immunohistochemistry results in the CIA rat. SR exerts an anti-arthritic effect in CIA rats through inhibition of the pathogenic characteristic of arthritic FLSs via suppressing NF-κB and MAPKs (JNK and p38) signaling pathways. SR may have a great potential for development as a novel therapeutic agent for RA treatment.

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.

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.