Collagen-induced Arthritis Research Articles

ZIF-8@Pt Nanozyme Used for Scavenging Reactive Oxygen Species in the Treatment of Rheumatoid Arthritis

To formulate a ZIF-8 nano mimetic enzyme conjugated with platinum metal (ZIF-8@Pt) that can scavenge reactive oxygen species (ROS) and to explore its potential applications in the treatment of rheumatoid arthritis (RA). The ZIF-8@Pt nanozyme was created by in situ reduction. Characterization of the nanozyme was then performed and its ability to mimic enzymes was investigated. Cell experiments were conducted using RAW264.7 cells, which were divided into three groups, including the untreated group (UT), the positive control group receiving lipopolysaccharide (LPS), which was designated as the LPS group, and the ZIF-8@Pt group receiving ZIF-8@Pt and LPS treatment. The cell experiments were conducted to evaluate the anti-inflammatory properties of ZIF-8@Pt through scavenging intracellular ROS. On the other hand, a collagen-induced arthritis (CIA) model was induced in rats. Similar to the group designations in the cell experiments, the rats were assigned to three groups, including a healthy control group (the UT group), a positive control group receiving a local injection of PBS solution in the knee joint, which was referred to as the control group, and a treatment group receiving a local injection of ZIF-8@Pt solution in the knee joint, which was referred to as the ZIF-8@Pt group. General evaluation, imaging observation, assessment of inflammatory factors, and pathological evaluation were performed to assess the therapeutic efficacy of ZIF-8@Pt against RA. The in vitro experiment revealed significant difference in the levels of intracellular ROS and LPS-induced M1-type macrophage polarization between the LPS group and the ZIF-8@Pt group (P<0.05). The in vivo experiment showed that significant difference in the levels of inflammatory factors, including interleukin-1β (IL-1β), C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), and arginase-1 (Arg-1) in the knee joints of the CIA rats between the LPS group and the ZIF-8@Pt group (P<0.05). Comparing the findings for the ZIF-8@Pt group and the control group, pathology assessment revealed that ZIF-8@Pt reduced local hypoxia and suppressed osteoclastic activity, neovascularization, and M1-type macrophage polarization (P<0.05). The ZIF-8@Pt enzyme mimetic inhibits macrophage inflammatory polarization by ROS scavenging, thereby improving inflammation in RA. Furthermore, the ZIF-8@Pt nanozyme improves the hypoxic environment and inhibits angiogenesis and bone destruction, demonstrating promising therapeutic efficacy for RA.

Adipose-derived stem cell exosomes loaded with icariin alleviates rheumatoid arthritis by modulating macrophage polarization in rats

Rheumatoid arthritis (RA) is a chronic autoimmune disease marked by synovitis and cartilage destruction. The active compound, icariin (ICA), derived from the herb Epimedium, exhibits potent anti-inflammatory properties. However, its clinical utility is limited by its water insolubility, poor permeability, and low bioavailability. To address these challenges, we developed a multifunctional drug delivery system—adipose-derived stem cells-exosomes (ADSCs-EXO)-ICA to target active macrophages in synovial tissue and modulate macrophage polarization from M1 to M2. High-performance liquid chromatography analysis confirmed a 92.4 ± 0.008% loading efficiency for ADSCs-EXO-ICA. In vitro studies utilizing cellular immunofluorescence (IF) and flow cytometry demonstrated significant inhibition of M1 macrophage proliferation by ADSCs-EXO-ICA. Enzyme-linked immunosorbent assay, cellular transcriptomics, and real-time quantitative PCR indicated that ADSCs-EXO-ICA promotes an M1-to-M2 phenotypic transition by reducing glycolysis through the inhibition of the ERK/HIF-1α/GLUT1 pathway. In vivo, ADSCs-EXO-ICA effectively accumulated in the joints. Pharmacodynamic assessments revealed that ADSCs-EXO-ICA decreased cytokine levels and mitigated arthritis symptoms in collagen-induced arthritis (CIA) rats. Histological analysis and micro computed tomography confirmed that ADSCs-EXO-ICA markedly ameliorated synovitis and preserved cartilage. Further in vivo studies indicated that ADSCs-EXO-ICA suppresses arthritis by promoting an M1-to-M2 switch and suppressing glycolysis. Western blotting supported the therapeutic efficacy of ADSCs-EXO-ICA in RA, confirming its role in modulating macrophage function through energy metabolism regulation. Thus, this study not only introduces a drug delivery system that significantly enhances the anti-RA efficacy of ADSCs-EXO-ICA but also elucidates its mechanism of action in macrophage function inhibition.Graphical abstract

Tridecylcyclohexane in incomplete Freund's adjuvant is a critical component in inducing experimental autoimmune diseases.

Incomplete Freund's adjuvant (IFA) has been used for many years to induce autoimmune diseases in animal models, including experimental autoimmune encephalitis and collagen-induced arthritis. However, it remains unclear why it is necessary to emulsify autoantigen and heat-killed Mycobacterium tuberculosis (HKMtb) with IFA to induce experimental autoimmune diseases. Here, we found that immunization with self-antigen and HKMtb was insufficient to induce autoimmune diseases in mice. Furthermore, IFA or one of its components, mineral oil, but not mannide monooleate, was required for the development of experimental autoimmune disease. Immunization with autoantigen and HKMtb emulsified in mineral oil facilitated innate immune activation and promoted the differentiation of pathogenic CD4+ T cells, followed by their accumulation in neuronal tissues. Several water-soluble hydrocarbon compounds were identified in mineral oil. Of these, immunization with HKMtb and autoantigen emulsified with the same amount of hexadecane or tridecylcyclohexane as mineral oil induced the development of experimental autoimmune encephalitis. In contrast, immunization with HKMtb and autoantigen emulsified with tridecylcyclohexane, but not hexadecane, at doses equivalent to those found in mineral oil, resulted in neuronal dysfunction. These data indicate that tridecylcyclohexane in mineral oil is a critical component in the induction of experimental autoimmune disease.

Regional Changes in Brain Biomolecular Markers in a Collagen-Induced Arthritis Rat Model.

The effects of collagen-induced arthritis (CIA), a model of systemic inflammation, on brain regional molecular markers associated with neurological disorders are uncertain. This study investigated the brain regional molecular changes in markers associated with inflammation and neuronal dysfunction in a CIA model. Fourteen male Sprague Dawley rats were divided into control (n = 5) or CIA (n = 9) groups. 10 weeks after CIA induction, brain tissue was collected. Brain regional mRNA expression of inflammatory markers (IL-1β and IL-6), apoptotic markers (BAX and Bcl2) and neurotrophic factors (BDNF, CREB and TrkB) was determined. Monoamine distribution and abundance in different brain regions were determine by mass spectrometry imaging (MSI). Neuroinflammation was confirmed in the CIA group by increased IL-β mRNA expression, concurrent with an increased BAX/Bcl2 ratio. The mRNA expression of CREB was increased in the midbrain and hippocampus while BDNF was increased and TrkB was decreased across all brain regions in CIA compared to control animals. Serotonin was decreased in the midbrain and hippocampus while dopamine was decreased in the striatum of CIA rats, compared to controls. CIA resulted in neuroinflammation concurrent with an apoptotic state and aberrant expression of neurotrophic factors and monoamines in the brain, suggestive of neurodegeneration.

Dietary pectin and inulin: A promising adjuvant supplement for collagen-induced arthritis through gut microbiome restoration and CD4+ T cell reconstitution

Dietary strategies rich in fiber have been demonstrated to offer benefits to individuals afflicted with rheumatoid arthritis (RA). However, the specific mechanisms through which a high-fiber diet (HFD) mitigates RA's autoimmunity remain elusive. Herein, we investigate the influence of pectin- and inulin-rich HFD on collagen-induced arthritis (CIA). We establish that HFD significantly alleviates arthritis in CIA mice by regulating the Th17/Treg balance. The rectification of aberrant T cell differentiation by the HFD is linked to the modulation of gut microbiota, augmenting the abundance of butyrate in feces. Concurrently, adding butyrate to the drinking water mirrors the HFD's impact on ameliorating CIA, encompassing arthritis mitigation, regulating intestinal barrier integrity, and restoring the Th17/Treg equilibrium. Butyrate reshapes the metabolic profile of CD4+ T cells in an AMPK-dependent manner. Our research underscores the importance of dietary interventions in rectifying gut microbiota for RA management and offers an explanation of how diet-derived microbial metabolites influence RA's immune-inflammatory-reaction.

Selection and Mechanism Study of Q-Markers for Xanthocerais lignum Anti-Rheumatoid Arthritis Based on Serum Spectrum-Effect Correlation Analysis.

To elucidate the chemical profile of Xanthocerais lignum's extracts of different polarities and their impact on rheumatoid arthritis (RA), we identified anti-RA markers and predicted their action mechanisms. A collagen-induced arthritis rat model was established, and UPLC-Q-Exactive Orbitrap MS technology was employed to analyze and identify the chemical constituents within the alcohol extract of Xanthocerais lignum and its various extraction fractions, as well as their translocation into the bloodstream. Serum spectrum-effect correlation analysis was utilized to elucidate the pharmacodynamic material basis of Xanthocerais lignum against RA and to screen for Q-Markers. Finally, the potential anti-RA mechanisms of the Q-Markers were predicted through compound-target interaction data and validated using molecular docking techniques. We identified 71 compounds, with flavan-3-ols and flavanones as key components. Of these, 36 were detected in the bloodstream, including 17 original and 19 metabolized forms. Proanthocyanidin A2, dihydroquercetin, catechin, and epicatechin (plus glucuronides) showed potential anti-RA activity. These compounds, acting as Q-Markers, may modulate ERK, NF-κB, HIF-1α, and VEGF in the HIF-1 pathway. This research clarifies Xanthocerais lignum's pharmacodynamic material basis against RA, identifies 4 Q-Markers, and offers insights into their mechanisms, aiding quality assessment and lead compound development for RA treatment.

Undenatured type II collagen protects against collagen-induced arthritis by restoring gut-joint homeostasis and immunity

Oral administration of harmless antigens can induce suppression of reactive immune responses, a process that capitalises on the ability of the gastrointestinal tract to tolerate exposure to food and commensal microbiome without triggering inflammatory responses. Repeating exposure to type II collagen induces oral tolerance and inhibits induction of arthritis, a chronic inflammatory joint condition. Although some mechanisms underlying oral tolerance are described, how dysregulation of gut immune networks impacts on inflammation of distant tissues like the joints is unclear. We used undenatured type II collagen in a prophylactic regime -7.33 mg/kg three times/week- to describe the mechanisms associated with protective oral immune-therapy (OIT) in gut and joint during experimental Collagen-Induced Arthritis (CIA). OIT reduced disease incidence to 50%, with reduced expression of IL-17 and IL-22 in the joints of asymptomatic mice. Moreover, whilst the gut tissue of arthritic mice shows substantial damage and activation of tissue-specific immune networks, oral administration of undenatured type II collagen protects against gut pathology in all mice, symptomatic and asymptomatic, rewiring IL-17/IL-22 networks. Furthermore, gut fucosylation and microbiome composition were also modulated. These results corroborate the relevance of the gut-joint axis in arthritis, showing novel regulatory mechanisms linked to therapeutic OIT in joint disease.

An Injectable Hydrogel Composing Anti-Inflammatory and Osteogenic Therapy toward Bone Erosions Microenvironment Remodeling in Rheumatoid Arthritis.

Healing bone erosions in rheumatoid arthritis (RA) remains greatly challenging via biomaterial strategies. Given the unsuccessful innate bone erosion healing due to an inflammatory disorder, over-activated osteoclasts, and impaired osteoblasts differentiation, RA pathogenesis-guided engineering of an innovative hydrogel platform is needed for remodeling osteoimmune and osteogenic microenvironment of bone erosion healing. Herein, in situ adaptable and injectable interpenetrating polymer network (IPN) hydrogel is developed through an ingenious combination of a bio-orthogonal reaction between hyaluronic acid (HA) and collagen, along with effective electrostatic interactions leveraging bisphosphonate (BP)-functionalized HA macromers (HABP) and nanorod shaped zinc (Zn)-doped biphasic calcium phosphate (ZnBCP). IPN hydrogel exhibits exceptional adaptability to the local shape complexity at bone erosions, and by integrating ZnBCP and HABP, a multi-stage releasing platform is engineered, facilitating controlled cargo delivery for remodeling more anti-inflammatory M2 cells and reducing over-activated osteoclastic activities, thereby reconstructing the bone regeneration microenvironment. Sustainedly co-delivering multiple ions (calcium and phosphate) can display excellent osteogenic properties and be conducive to the bone formation process, by effects of osteogenesis-associated cell differentiation. Overall, the introduced bioactive IPN hydrogel therapy remodels the osteoimmune environment by synergistic pro-inflammation-resolving, osteogenesis, and anti-osteoclastic activities, displaying excellent bone reconstruction in the collagen-induced arthritis rabbit model.

Immunological mechanism of Ermiao Powder improving inflammation in rats with collagen-induced arthritis through α7nAChR-JAK2/STAT3 pathway

This study investigated the immunological mechanisms of Ermiao powder in the treatment of rheumatoid arthritis rats through the alpha 7 nicotinic acetylcholine receptor(α7nAChR)-Janus kinases 2(JAK2)/signal transducer and activator of transcription 3(STAT3) signaling pathway. A total of 56 female Wistar rats were randomly divided into the normal group(HG, n=8), collagen-induced arthritis(CIA) model group(CM, n=8), vagotomy group(VA, n=8), sham group(SH, n=8), Ermiao Powder treatment model group(EM, n=8), Ermiao Powder treatment for vagotomy group(EV, n=8) and Ermiao Powder treatment for sham group(ES, n=8). Following the establishment of CIA models in all groups except the HG group, the rats underwent unilateral vagotomy and sham operation(only the vagus nerve was separated). Drug treatment was started 7 days after surgery and continued for 35 days. The body weight and joints of rats were recorded, the pathological changes of the spleen of rats were observed, the contents of interleukin-6(IL-6), interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) in serum were detected by enzyme-linked immunosorbent assay(ELISA), and the mRNA and protein expression of α7nAChR-JAK2/STAT3 pathway core genes in spleen were detected by qRT-PCR, Western blot and immunohistochemistry. RESULTS:: showed that CM group(compared with HG group) and VA group(compared with CM group and SH group) had significantly decreased body weight(P&lt;0.05, P&lt;0.01), increased arthritis score(P&lt;0.05, P&lt;0.01), swollen ankle joints with deformity, and increased and enlarged lymph nodes in the spleen. There were also notable increases in the serum levels of IL-6, IL-1β and TNF-α(P&lt;0.05, P&lt;0.01), and in the mRNA expressions of JAK2 and STAT3 in the spleen(P&lt;0.05, P&lt;0.01). The protein levels of phosphorylated JAK2(p-JAK2)/JAK2 and phospho-STAT3(p-STAT3)/STAT3 were significantly increased(P&lt;0.05, P&lt;0.01), and the number of JAK2, p-JAK2, STAT3 and p-STAT3 cells increased(P&lt;0.05, P&lt;0.01). EM group(compared with CM group) and ES group(compared with SH group) exhibited significantly increased body weight(P&lt;0.01), decreased arthritis scores(P&lt;0.05, P&lt;0.01), reduced swelling of ankle joint, and decreased number and volume of lymph nodes in the spleen. Furthermore, serum levels of IL-6, IL-1β, and TNF-α decreased(P&lt;0.05, P&lt;0.01), the mRNA expression of JAK2 and STAT3 in spleen decreased(P&lt;0.05, P&lt;0.01), the protein levels of p-JAK2/JAK2 and p-STAT3/STAT3 decreased(P&lt;0.05, P&lt;0.01), and the number of JAK2, p-JAK2, STAT3 and p-STAT3 cells decreased(P&lt;0.05, P&lt;0.01), whereas the mRNA and protein expressions of α7nAChR were significantly increased(P&lt;0.01). Compared with the VA group, there was no significant differences in weight gain and arthritis scores in the EV group. The number of lymph nodes in the spleen was not significantly reduced and the volume was still large, suggesting the inflammation was not significantly improved. The serum levels of IL-6, IL-1β and TNF-α were not significantly different, and there were no significant differences in α7nAChR, JAK2, and STAT3 mRNA expression in the spleen. The protein expression levels of p-JAK2/JAK2 and α7nAChR in spleen were lower(P&lt;0.05, P&lt;0.01), while p-STAT3/STAT3 protein expression was not significantly different. Besides, the two groups had no significant difference in the number of JAK2, p-JAK2, STAT3, and p-STAT3 cells. The results suggested that unilateral vagotomy promoted the increase of phosphorylated JAK2 and STAT3 expressions and exacerbated inflammation. In contrast, Ermiao Powder alleviated the inflammation in rheumatoid arthritis rats by activating the α7nAChR-mediated JAK2/STAT3 pathway through the vagus nerve, suggesting that the α7nAchR-JAK2/STAT3 pathway may be a potential target for the treatment of rheumatoid arthritis.

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.

Long-acting bulleyaconitine A microspheres via intra-articular delivery for multidimensional therapy of rheumatoid arthritis

Bulleyaconitine A (BLA) is a promising candidate for treating rheumatoid arthritis (RA) with diverse pharmacological activities, including anti-inflammatory, analgesic and bone repair. Herein, the long-acting bulleyaconitine A microspheres (BLA-MS) were developed to treat RA comprehensively by forming drug reservoirs in joint cavities. The BLA-MS were prepared by emulsion/solvent evaporation method. The particle size and distribution were assessed by SEM. The crystalline state was investigated by DSC and PXRD. The drug loading (DL), encapsulation efficiency (EE) and cumulative release in vitro were determined by HPLC. The DL and EE were 23.93 ± 0.38 % and 95.73 ± 1.56 % respectively, and the cumulative release was up to 69 days with a stable release curve. The pharmacodynamic results in collagen induced arthritis (CIA) rats showed a noticeable reduction in paw thickness (5.66 ± 0.32 mm), and the decreasing expression level of PGE2, TNF-α and IL-6 which diminished the infiltration of inflammatory cells, thereby alleviating the progression of erosion and repairing the damaged bones (BV/TV (Bone Volume / Total Volume): 81.97 %, BS/BV (Bone Surface / Bone Volume): 6.08 mm−1). In conclusion, intra-articular injection of BLA-MS should have a promising application in the treatment of RA and may achieve clinical transformation in the future.

Graphene oxide quantum dots-loaded sinomenine hydrochloride nanocomplexes for effective treatment of rheumatoid arthritis via inducing macrophage repolarization and arresting abnormal proliferation of fibroblast-like synoviocytes

The characteristic features of the rheumatoid arthritis (RA) microenvironment are synovial inflammation and hyperplasia. Therefore, there is a growing interest in developing a suitable therapeutic strategy for RA that targets the synovial macrophages and fibroblast-like synoviocytes (FLSs). In this study, we used graphene oxide quantum dots (GOQDs) for loading anti-arthritic sinomenine hydrochloride (SIN). By combining with hyaluronic acid (HA)-inserted hybrid membrane (RFM), we successfully constructed a new nanodrug system named HA@RFM@GP@SIN NPs for target therapy of inflammatory articular lesions. Mechanistic studies showed that this nanomedicine system was effective against RA by facilitating the transition of M1 to M2 macrophages and inhibiting the abnormal proliferation of FLSs in vitro. In vivo therapeutic potential investigation demonstrated its effects on macrophage polarization and synovial hyperplasia, ultimately preventing cartilage destruction and bone erosion in the preclinical models of adjuvant-induced arthritis and collagen-induced arthritis in rats. Metabolomics indicated that the anti-arthritic effects of HA@RFM@GP@SIN NPs were mainly associated with the regulation of steroid hormone biosynthesis, ovarian steroidogenesis, tryptophan metabolism, and tyrosine metabolism. More notably, transcriptomic analyses revealed that HA@RFM@GP@SIN NPs suppressed the cell cycle pathway while inducing the cell apoptosis pathway. Furthermore, protein validation revealed that HA@RFM@GP@SIN NPs disrupted the excessive growth of RAFLS by interfering with the PI3K/Akt/SGK/FoxO signaling cascade, resulting in a decline in cyclin B1 expression and the arrest of the G2 phase. Additionally, considering the favorable biocompatibility and biosafety, these multifunctional nanoparticles offer a promising therapeutic approach for patients with RA.Graphical abstract

Fibroblast-like synoviocytes orchestrate daily rhythmic inflammation in arthritis.

Rheumatoid arthritis is a chronic inflammatory disease that shows characteristic diurnal variation in symptom severity, where joint resident fibroblast-like synoviocytes (FLS) act as important mediators of arthritis pathology. We investigate the role of FLS circadian clock function in directing rhythmic joint inflammation in a murine model of inflammatory arthritis. We demonstrate FLS time-of-day-dependent gene expression is attenuated in arthritic joints, except for a subset of disease-modifying genes. The deletion of essential clock gene Bmal1 in FLS reduced susceptibility to collagen-induced arthritis but did not impact symptomatic severity in affected mice. Notably, FLS Bmal1 deletion resulted in loss of diurnal expression of disease-modulating genes across the joint, and elevated production of MMP3, a prognostic marker of joint damage in inflammatory arthritis. This work identifies the FLS circadian clock as an influential driver of daily oscillations in joint inflammation, and a potential regulator of destructive pathology in chronic inflammatory arthritis.

Identification of novel biomarker hsa_circ_0003914 for rheumatoid arthritis from plasma exosomes

Rheumatoid arthritis (RA) is a complex autoimmune disease featuring invasive and infiltrative fibroblast-like synoviocytes (FLS) that lead to joint damage. While current RA pathological mechanisms remain incompletely defined, exosomes have been implicated as having the potential to drive disease progression due to their ability to deliver different types of biomolecules to tissues effected by RA. One potentially disease exacerbating molecule type found in exosomes are Circular RNAs (circRNAs), which are highly stable and have been previously implicated in RA pathogenesis. Here, we examine hsa_circ_0003914, a circRNA found in exosomes located in blood plasma, for a role in RA. Plasma exosomes were isolated and injected into collagen-induced arthritis (CIA) mice, followed by functional experiments to analyze the influence of exosomes on FLS formation. Sequencing revealed the presence of hsa_circ_0003914 in exosomes, so we examined its association with clinical markers in RA. Finally, the role for hsa_circ_0003914 in RA was directly confirmed through in vivo and in vitro experiments. We found that plasma exosomes isolated from RA patients could aggravate the disease of CIA mice, compared to exosomes isolated from healthy control patients. Hsa_circ_0003914 was highly enriched in the exosomes of RA patients. Mechanistically, Hsa_circ_0003914 promoted abnormal cell proliferation, migration, invasion and stimulated the secretion of inflammatory cytokines in FLSs through targeting NF-κB/p65 signaling pathway. Interestingly, knockdown of hsa_circ_0003914 rescued disease phenotypes in CIA mice. Taken together, these data implicate hsa_circ_0003914 as a potential therapeutic target for the prevention and management of RA.

Screening of potentially active compounds against rheumatoid arthritis in the Juan-Bi decoction using systems pharmacology and animal experiments.

Background: The Juan-Bi decoction (JBD) is a classic traditional Chinese medicines (TCMs) prescription for the treatment of rheumatoid arthritis (RA). However, the active compounds of the JBD in RA treatment remain unclear. Aim: The aim of this study is to screen effective compounds in the JBD for RA treatment using systems pharmacology and experimental approaches. Method: Botanical drugs and compounds in the JBD were acquired from multiple public TCM databases. All compounds were initially screened using absorption, distribution, metabolism, excretion, and toxicity (ADMET) and physicochemical properties, and then a target prediction was performed. RA pathological genes were acquired from the DisGeNet database. Potential active compounds were screened by constructing a compound-target-pathogenic gene (C-T-P) network and calculating the cumulative interaction intensity of the compounds on pathogenic genes. The effectiveness of the compounds was verified using lipopolysaccharide (LPS)-induced RAW.264.7 cells and collagen-induced arthritis (CIA) mouse models. Results: We screened 15 potentially active compounds in the JBD for RA treatment. These compounds primarily act on multiple metabolic pathways, immune pathways, and signaling transduction pathways. Furthermore, in vivo and in vitro experiments showed that bornyl acetate (BAC) alleviated joint damage, and inflammatory cells infiltrated and facilitated a smooth cartilage surface via the suppression of the steroid hormone biosynthesis. Conclusion: We screened potential compounds in the JBD for the treatment of RA using systems pharmacology approaches. In particular, BAC had an anti-rheumatic effect, and future studies are required to elucidate the underlying mechanisms.

Inhibition of ferroptosis rescues M2 macrophages and alleviates arthritis by suppressing the HMGB1/TLR4/STAT3 axis in M1 macrophages

Ferroptosis is a type of programmed cell death driven by iron-dependent lipid peroxidation. The TNF-mediated biosynthesis of glutathione has been shown to protect synovial fibroblasts from ferroptosis in the hyperplastic synovium. Ferroptosis induction provides a novel therapeutic approach for rheumatoid arthritis (RA) by reducing the population of synovial fibroblasts. The beginning and maintenance of synovitis in RA are significantly influenced by macrophages, as they generate cytokines that promote inflammation and contribute to the destruction of cartilage and bone. However, the vulnerability of macrophages to ferroptosis in RA remains unclear. In this study, we found that M2 macrophages are more vulnerable to ferroptosis than M1 macrophages in the environment of the arthritis synovium with a high level of iron, leading to an imbalance in the M1/M2 ratio. During ferroptosis, HMGB1 released by M2 macrophages interacts with TLR4 on M1 macrophages, which in turn triggers the activation of STAT3 signaling in M1 macrophages and contributes to the inflammatory response. Knockdown of TLR4 decreased the level of cytokines induced by HMGB1 in M1 macrophages. The ferroptosis inhibitor liproxstatin-1 (Lip-1) started at the presymptomatic stage in collagen-induced arthritis (CIA) model mice, and GPX4 overexpression in M2 macrophages at the onset of collagen antibody-induced arthritis (CAIA) protected M2 macrophages from ferroptotic cell death and significantly prevented the development of joint inflammation and destruction. Thus, our study demonstrated that M2 macrophages are vulnerable to ferroptosis in the microenvironment of the hyperplastic synovium and revealed that the HMGB1/TLR4/STAT3 axis is critical for the ability of ferroptotic M2 macrophages to contribute to the exacerbation of synovial inflammation in RA. Our findings provide novel insight into the progression and treatment of RA.

Morinda officinalis iridoid glycosides alleviate methotrexate-induced liver injury in CIA rats by increasing liver autophagy and improving lipid metabolism homeostasis

Ethnopharmacological relevanceMorinda officinalis How. is a commonly used traditional Chinese herb with the pharmacological properties of tonifying liver and kidney, and enhancing bone and muscle. Iridoid glycosides are the predominant components of this plant, including monotropein, asperuloside, deacetylasperuloside and deacetylasperulosidic acid with their contents reaching more than 2%. Methotrexate (MTX) is the drug of choice for the treatment of rheumatoid arthritis (RA), but liver injury induced by MTX limits its wider use for RA. Morindaofficinalis iridoid glycoside (MOIG) is reported as having anti-RA and hepatoprotective effects, but the exact efficacy on MTX-induced liver injury and the underlying molecular mechanism remain unclear. AimTo elucidate the mitigating effect of MOIG against liver injury in RA rats treated with MTX, and explore the possible mechanism. Materials and methodsThe effect and mechanism of MOIG were investigated in Wistar rats with collagen-induced arthritis (CIA) which were then treated with MTX, and MTX-induced hepatocyte injury in vitro. Network pharmacological and transcriptomic analyses were conducted to predict the possible mechanisms of MOIG in mitigating MTX-induced liver injury, and lipidomic analysis was performed to further verify the regulatory effects of MOIG on lipid metabolism. BRL-3A hepatocytes were used to evaluate the regulatory effects of MOIG against MTX-associated liver injury. ResultsMOIG treatment enhanced the anti-RA effect of MTX, and mitigated oxidative damage, inflammation and apoptosis of liver tissues in CIA rats treated with MTX. Network pharmacological and transcriptomic analyses demonstrated that MOIG attenuated liver injury by regulating autophagy and lipid metabolism. The result of lipidomic analysis showed that MOIG reversed the disturbance of lipid metabolism of the liver tissue in CIA rats after MTX treatment. In addition, MOIG also inhibited the apoptosis, reduced the levels of lactate dehydrogenase (LDH), aspartate aminotransferase (ALT) and alanine aminotransferase (AST), regulated oxidative stress, and increased the formation of autophagosome and translocation of LC3 in the nucleus and expression of autophagy regulatory genes Beclin-1, ATG5, LC3Ⅱ, ATG7 and ATG12 in hepatocytes subjected to MTX damage. ConclusionOur findings demonstrated that MOIG could ameliorate MTX-induced liver injury in the treatment of RA through increasing hepatocyte autophagy and improving lipid metabolism homeostasis.

Wutou decoction alleviates arthritis inflammation in CIA mice by regulating Treg cell stability and Treg/Th17 balance via the JAK2/STAT3 pathway

Ethnopharmacological relevanceWutou Decoction (WTD) is a classic traditional Chinese medicine formula, which has shown clinical efficacy in treating rheumatoid arthritis (RA). The Treg stability and Th17/Treg imbalance is an important immunological mechanism in RA progression. Whether WTD regulates CD4+ T cell subsets has not been thoroughly investigated yet. Aim of the studyThis study aimed to explore the potential role and mechanisms of WTD in regulating the diminished stability of Treg cells and the imbalance of CD4+ T cell subsets via in vivo and in vitro experiments. Materials and methodsFirstly, the therapeutic effects of WTD on the collagen-induced arthritis (CIA) mouse and its potential regulatory function on CD4+ T cell subsets were evaluated in vivo. Animal specimens were collected after 31 days of treatment with WTD. The anti-arthritic and anti-inflammatory effects of WTD were assessed through arthritis scoring, body weight, spleen index, serum IL-6 levels, and micro-PET/CT imaging. Gene enrichment analysis was performed to evaluate the activation T cell-related signaling pathway. Flow cytometry was used to determine the proportions of CD4+ T cell subsets in vitro and in vitro. Additionally, ELISA was used to assess the secretion of IL-10 and TGF-β by Treg cells under inflammatory conditions. The suppressive function of Treg cells on cell proliferation under inflammatory conditions was examined using CFSE labeling. Immunofluorescence staining was performed to detect the phosphorylation levels of STAT3 in CD4+ T cells from mouse spleen tissues. Western blotting was used to evaluate the phosphorylation levels of JAK2/STAT3 in Treg cells. ResultsWTD significantly alleviated joint inflammation in CIA mice. WTD reduced serum IL-6 levels in CIA mice, improved their body weight and spleen index. WTD treatment inhibited the activation of CD4+ T cell subgroup-related signaling in the joint tissues of CIA mice. In vitro and in vitro experiments showed that WTD increased the proportion of Treg cells and decreased the proportion of Th17 cells in CIA mice spleen. Furthermore, WTD promoted the secretion of IL-10 and TGF-β by Treg cells and enhanced the inhibitory capacity of Treg cells on cell proliferation under inflammatory conditions. Immunofluorescence detected decreased STAT3 phosphorylation levels in CD4+ T cells from CIA mice spleen, while western blotting revealed a decrease in JAK2/STAT3 phosphorylation levels in Treg cells in vitro. ConclusionsInhibiting JAK2/STAT3 phosphorylation is a potential mechanism through which WTD improves Treg cell stability, balances CD4+ T cell subsets, and attenuates RA joint inflammation.

RGS1 Enhancer RNA Promotes Gene Transcription by Recruiting Transcription Factor FOXJ3 and Facilitates Osteoclastogenesis Through PLC-IP3R-dependent Ca2+ Response in Rheumatoid Arthritis.

Recent evidence has highlighted the functions of enhancers in modulating transcriptional machinery and affecting the development of human diseases including rheumatoid arthritis (RA). Enhancer RNAs (eRNAs) are RNA molecules transcribed from active enhancer regions. This study investigates the specific function of eRNA in gene transcription and osteoclastogenesis in RA. Regulator of G protein signaling 1 (RGS1)-associated eRNA was highly activated in osteoclasts according to bioinformatics prediction. RGS1 mRNA was increased in mice with collagen-induced arthritis as well as in M-CSF/soluble RANKL-stimulated macrophages (derived from monocytes). This was ascribed to increased RGS1 eRNA activity. Silencing of 5'-eRNA blocked the binding between forkhead box J3 (FOXJ3) and the RGS1 promoter, thus suppressing RGS1 transcription. RGS1 accelerated osteoclastogenesis through PLC-IP3R-dependent Ca2+ response. Knockdown of either FOXJ3 or RGS1 ameliorated arthritis severity, improved pathological changes, and reduced osteoclastogenesis and bone erosion in vivo and in vitro. However, the effects of FOXJ3 silencing were negated by RGS1 overexpression. In conclusion, this study demonstrates that the RGS1 eRNA-driven transcriptional activation of the FOXJ3/RGS1 axis accelerates osteoclastogenesis through PLC-IP3R dependent Ca2+ response in RA. The finding may offer novel insights into the role of eRNA in gene transcription and osteoclastogenesis in RA.

The potential utility of (2S,4R)-4-[18F] fluoroglutamine as a novel metabolic imaging marker for inflammation explored by rat models of arthritis and paw edema.

(2S,4R)-4-[18F]fluoroglutamine ([18F]FGln) is a promising metabolic imaging marker in cancer. Based on the fact that major inflammatory cells are heavily dependent on glutamine metabolism like cancer cells, we explored the potential utility of [18F]FGln as a metabolic imaging marker for inflammation in two rat models: carrageenan-induced paw edema (CIPE) and collagen-induced arthritis (CIA). The CIPE model (n = 4) was generated by injecting 200 μL of 3% carrageenan solution into the left hind paw three hours before the PET. The CIA model (n = 4) was generated by injecting 200 μg of collagen emulsion subcutaneously at the tail base 3-4 weeks before the PET. A qualitative scoring system was used to assess the severity of paw inflammation. After a CT scan, 15.7 ± 4.9 MBq of [18F]FGln was injected via the tail vein, followed by a dynamic micro-PET scan for 90 minutes under anesthesia with isoflurane. The standard uptake value of [18F]FGln was measured by placing a volume of interest in each paw. The non-injected right hind paws of the CIPE model rats served as controls for both models. The paws with CIA were pathologically examined after PET. In CIPE models, uptake in the injected paw was higher compared to the non-injected paw by 52-83%. In CIA models, uptake in the paws with severe inflammation was higher than the averaged controls by 54-173%, while that with mild and no inflammation was slightly higher (33%) and lower (-7%), respectively. Combined overall, the [18F]FGln uptake in CIA showed a significant positive correlation with inflammation severity (r = 0.88, P = 0.009). The pathological findings confirmed profound inflammation in CIA. [18F]FGln uptake was increased in both acute and chronic inflammation, and the uptake level was significantly correlated with the severity, suggesting its potential utility as a novel metabolic imaging marker for inflammation.