Collagen-induced Arthritis Mice Research Articles

Dantrolene, a stabilizer of the ryanodine receptor, prevents collagen-induced arthritis

Dantrolene inhibits Ca2+ leakage from destabilized ryanodine receptors and therefore may serve as a therapeutic agent against endoplasmic reticulum stress-associated diseases. However, its effectiveness in treating autoimmune diseases remains unclear. Here, we investigated the effect of dantrolene on collagen-induced arthritis (CIA) in mice. Oral administration of dantrolene resulted in significantly lower arthritic scores in both male and female CIA mice than in the control mice. Micro-computed tomographic and histological analyses showed that dantrolene suppressed bone and chondral destruction. The serum levels of anti-type II collagen (CII) IgG were positively correlated with the arthritic scores (r = 0.704, p < 0.01). In addition, the serum levels of anti-CII IgG were significantly lower in the dantrolene group than in the control group (p < 0.05). These results demonstrate that oral administration of dantrolene to CIA mice inhibits the production of serum anti-CII IgG and consequently prevents arthritis. Therefore, dantrolene may be a potential anti-rheumatic drug.

Chemo-photothermal therapeutic effect of chitosan-gelatin hydrogels containing methotrexate and melanin on a collagen-induced arthritis mouse model

Heat stimulation can promote osteoblast differentiation and bone formation. Combining photothermal therapy and chemotherapy is an effective strategy for treating rheumatoid arthritis (RA). Herein, we prepared chitosan/gelatin/β-glycerophosphate-melanin-methotrexate (CMM) hydrogel that could be used to perform simultaneous chemotherapy and photothermal therapy for patients with RA. The CMM solution was successfully converted to a gel state at body temperature. Due to intrinsic photothermal properties of melanin, CMM hydrogel exhibited effective temperature increase both in vitro and in vivo with increasing time of near-infrared (NIR) laser irradiation. After NIR laser irradiation, 50 % of methotrexate was rapidly released from the hydrogel within 3 h. Its release rate showed an instantaneous increase with additional NIR laser irradiation. After CMM hydrogel was injected directly into the paw joint of each collagen-induced arthritis (CIA) mouse followed by irradiation with a NIR laser (808 nm, 0.5 W/cm2, 3 min), swelling and redness at the inflamed area were significantly alleviated at 14 days after treatment. Micro-CT analysis confirmed that treated joints of mice were similar to normal joints. Hence, CMM hydrogel could be used as an attractive RA therapeutic agent for simultaneous chemo-photothermal therapy.

Novel isatin-based hybrids as potential anti-rheumatoid arthritis drug candidates: Synthesis and biological evaluation

Rheumatoid arthritis (RA) is an autoimmune disease accompanied with serious symptoms, such as joint destruction and chronic synovitis. Though many anti-RA drugs could improve the outcome of RA patients to a certain extent, about 40% inefficient rate, severe side effects, and high costs have become urgent problems. Therefore, exploring new alternative drugs for RA therapy is still an urgent need so far. Isatin is an important structural motif found in numerous biologically active compounds and therapeutic agents. Herein, we aim to synthesize several novel isatin analogues for RA therapy and further explore the mechanism of the most potential anti-RA drug candidate in suppressing the pathological progress of RA in vitro and in vivo. We found that the most therapeutic potential compound, a novel small molecule isatin-honokiol hybrid named CT5-2 inhibited the viability of RA-fibroblast-like synoviocytes (FLSs), an effector cell of synovial hyperplasia in the RA synovial tissue with IC50 ranging from 8.54 to 10.66 μM. In addition, CT5-2 reduced the DNA replication and triggered cell cycle arrest and apoptosis of RA-FLSs. Moreover, differential analyses of RNA-sequencing and the mechanistic studies demonstrated that CDCA7 is a key gene correlated with RA progression, and CT5-2 could inhibit the c-Myc/CDCA7/p65 pathway to regulate CDK1, Bcl-2, and vimentin in RA-FLSs. Furthermore, CT5-2 relieved collagen-induced arthritis (CIA) and reduced the level of CDCA7, CDK1, Bcl-2, and vimentin of synovial tissue in CIA mice. Taken together, the novel small molecule isatin-honokiol hybrid CT5-2 exhibits a potential anti-RA drug candidate that inhibits proliferation and triggers cell cycle arrest and apoptosis of RA-FLSs by regulating the c-Myc/CDCA7/p65 pathway. Our study lays a good foundation for further clinical research and structuralmodification of CT5-2.

Anti-Arthritogenic Property of Interleukin 10-Expressing Human Amniotic MSCs Generated by Gene Editing in Collagen-Induced Arthritis

Although stem cells are promising tools for the treatment of arthritis, their therapeutic effects remain controversial. In this study, we investigated the therapeutic properties of interleukin (IL)-10-overexpressing human amniotic mesenchymal stem cells (AMMs) generated via gene editing in a collagen-induced mouse model. IL-10 was inserted into the genomic loci of AMMs via transcription activator-like effector nucleases. In vitro immunomodulatory effects of IL-10-overexpressing AMMs (AMM/I) were evaluated and their anti-arthritogenic properties were determined in collagen-induced arthritis (CIA) mice. Transplantation of AMM/I attenuates CIA progression. In addition, the regulatory T cell population was increased, while T helper-17 cell activation was suppressed by AMM/I administration in CIA mice. Consistently, AMM/I injection increased proteoglycan expression, while reducing inflammation and the expression levels of the pro-inflammatory factors, IL-1 β, IL-6, monocyte chemoattractant protein-1, and tumor necrosis factor- α, in joint tissues. In conclusion, use of IL-10-edited human AMM/I may be a novel therapeutic strategy for the treatment of arthritis.

Immune-regulating strategy against rheumatoid arthritis by inducing tolerogenic dendritic cells with modified zinc peroxide nanoparticles

In hypoxic dendritic cells (DCs), a low level of Zn2+ can induce the activation of immunogenic DCs (igDCs), thereby triggering an active T-cell response to propel the immune progression of rheumatoid arthritis (RA). This finding indicates the crucial roles of zinc and oxygen homeostasis in DCs during the pathogenesis of RA. However, very few studies have focused on the modulation of zinc and oxygen homeostasis in DCs during RA treatment. Proposed herein is a DC-targeting immune-regulating strategy to induce igDCs into tolerogenic DCs (tDCs) and inhibit subsequent T-cell activation, referred to as ZnO2/Catalase@liposome-Mannose nanoparticles (ZnCM NPs). ZnCM NPs displayed targeted intracellular delivery of Zn2+ and O2 towards igDCs in a pH-responsive manner. After inactivating OTUB1 deubiquitination, the ZnCM NPs promoted CCL5 degradation via NF-κB signalling, thereby inducing the igDC-tDC transition to further inhibit CD4+ T-cell homeostasis. In collagen-induced arthritis (CIA) mice, this nanoimmunoplatform showed significant accumulation in the spleen, where immature DCs (imDCs) differentiated into igDCs. Splenic tDCs were induced to alleviate ankle swelling, improve walking posture and safely inhibit ankle/spleen inflammation. Our work pioneers the combination of DC-targeting nanoplatforms with RA treatments and highlights the significance of zinc and oxygen homeostasis for the immunoregulation of RA by inducing tDCs with modified ZnO2 NPs, which provides novel insight into ion homeostasis regulation for the treatment of immune diseases with a larger variety of distinct metal or nonmetal ions.Graphical

Multi-omics profiling of collagen-induced arthritis mouse model reveals early metabolic dysregulation via SIRT1 axis

Rheumatoid arthritis (RA) is characterized by joint infiltration of immune cells and synovial inflammation which leads to progressive disability. Current treatments improve the disease outcome, but the unmet medical need is still high. New discoveries over the last decade have revealed the major impact of cellular metabolism on immune cell functions. So far, a comprehensive understanding of metabolic changes during disease development, especially in the diseased microenvironment, is still limited. Therefore, we studied the longitudinal metabolic changes during the development of murine arthritis by integrating metabolomics and transcriptomics data. We identified an early change in macrophage pathways which was accompanied by oxidative stress, a drop in NAD+ level and induction of glucose transporters. We discovered inhibition of SIRT1, a NAD-dependent histone deacetylase and confirmed its dysregulation in human macrophages and synovial tissues of RA patients. Mining this database should enable the discovery of novel metabolic targets and therapy opportunities in RA.

Chinese Herbal Formula Huayu-Qiangshen-Tongbi Decoction Attenuates Rheumatoid Arthritis through Upregulating miR-125b to Suppress NF-κB-Induced Inflammation by Targeting CK2.

The Huayu-Qiangshen-Tongbi (HQT) decoction, a Chinese medical formula, has been identified to show a potent therapeutic effect on rheumatoid arthritis (RA). However, the specific molecular mechanism of HQT in RA has not been well studied. In the present study, LPS-treated human rheumatoid fibroblast-like synoviocyte (FLS) MH7A cells and collagen-induced arthritis (CIA) mice were utilized as in vitro and in vivo models. Our results demonstrated that HQT could efficiently inhibit RA-induced inflammation by reducing the production of cytokines including tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). Moreover, HQT significantly upregulated the expression of miR-125b. Besides, analysis of bioinformatics suggested casein kinase 2 (CK2) was a potential target of miR-125b. Luciferase reporter assay was performed and revealed that miR-125b suppressed CK2 expression in MH7A cells. Furthermore, miR-125b inhibited LPS-induced NF-kappa-B (NF-κB) activation, which is a downstream target of CK2. In addition, the NF-κB inhibitor ammonium pyrrolidinedithiocarbamate (PDTC) and NF-kappa-B inhibitor alpha (IkB-α) enhanced the inhibitory effect of miR-125b on the expression of TNF-α, IL-1β, and IL-6. Taken together, our study revealed that HQT could attenuate RA through upregulating miR-125b to suppress NF-κB-induced inflammation by targeting CK2. The findings of this study should facilitate investigating the mechanism of HQT on RA and discovering novel therapeutic targets for RA.

The mixture of Agrimonia pilosa Ledeb. and Salvia miltiorrhiza Bunge. extract produces analgesic and anti-inflammatory effects in a collagen-induced arthritis mouse model

ABSTRACT Pain and inflammation typically manifest in patients with arthritis. It is now widely known that Agrimonia pilosa Ledeb (AP) and Salvia miltiorrhiza Bunge (SM) exert anti-inflammatory and antinociceptive effects. We have previously reported that the mixture extract (ME) from AP and SM produces antinociceptive and anti-inflammatory effects in gout arthritis and monoiodoacetate (MIA)-induced arthritis models. In the present study, we assessed the antinociceptive and anti-inflammatory effects on the collagen-induced arthritis (CIA) model. The antinociceptive effects in mice were measured using the von Frey test. ME administered once or for one week (once per day) once, and one-week reduced the pain in a dose-dependent manner (from 50 to 100 mg/kg) in the CIA-induced osteoarthritis (OA) model. ME treatment also reduced tumor necrosis factor (TNF)-α and C-reactive protein (CRP) levels in plasma and ankle tissues. Furthermore, COX-1, COX-2, NF-κB, TNF-α, and IL-6 expressions were attenuated after ME treatment. In most experiments, the antinociceptive and anti-inflammatory effects induced by ME treatment were almost equal to or slightly better than those induced by Perna canaliculus (PC) treatment, which was used as a positive control. Our results suggest that ME possesses antinociceptive and anti-inflammatory effects, indicating its potential as a therapeutic agent for arthritis treatment.

Inflammatory Cytokine-Neutralizing Antibody Treatment Prevented Increases in Follicular Helper T Cells and Follicular Regulatory T Cells in a Mouse Model of Arthritis.

BackgroundFollicular T helper (TFH) and follicular regulatory T (TFR) cells play important roles in humoral immunity. Nevertheless, their significance in rheumatoid arthritis (RA) pathogenesis has not been fully elucidated. As an important treatment strategy, the effect of inflammatory factor-neutralizing antibodies on TFH and TFR in RA remains unclear.MethodsWe used the collagen-induced arthritis (CIA) mouse model to illustrate the quantity and functional changes in TFH and TFR cells. The changes of plasmablast, TFH and TFR cells in the spleen and peripheral blood of CIA mice were analyzed by flow cytometry. The levels of TFH and TFR and their functional subsets in the spleen after anti-inflammatory antibody treatment were analyzed and compared. The functional changes of TFH and TFR in CIA mice before and after treatment were detected by in vitro culture experiments.ResultsPlasmablast levels were increased in CIA spleen and peripheral blood and both TFH and TFR cell levels were upregulated. TFH and TFR cells were decreased significantly after the anti-inflammatory antibody treatment. TIGIT+ and TIGIT+CD226− TFH cells in CIA mouse spleen were elevated and PD-1 and ICOS expression on spleen TFH and TFR cells was increased. Both the ability of TFH cells to secrete IL-21 and aid B cells and the ability of TFR cells to secrete IL-10 and inhibit TFH cells were enhanced in the CIA mice. After antibody treatment, the cell subsets and functions were recovered.ConclusionGerminal center TFH and TFR cells were increased and their functions were enhanced. With inflammatory factor-neutralizing antibody treatment, TFH and TFR subsets and their functions returned to normal. These findings provide important information on the dynamics of humoral immune-related cell subsets in RA and the effects of treatment on them.

A novel mechanism for macrophage pyroptosis in rheumatoid arthritis induced by Pol β deficiency

Rheumatoid arthritis (RA) is a chronic and inflammatory autoimmune disease. Macrophage pyroptosis, a proinflammatory form of cell death, is critically important in RA; however, the detailed mechanism underlying pyroptosis induction is not yet well understood. Here, we report that DNA polymerase β (Pol β), a key enzyme in base excision repair, plays a pivotal role in RA pathogenesis. Our data shows that Pol β expression is significantly decreased in peripheral blood mononuclear cells (PBMCs) from active RA patients and collagen-induced arthritis (CIA) mice, and Pol β deficiency increases the incidence of RA, macrophage infiltration, and bone destruction in CIA mouse models. In vitro, experiments showed that Pol β deficiency exacerbated macrophage pyroptosis induced by LPS plus ATP, while overexpression of Pol β inhibited macrophage pyroptosis. Further characterization revealed that Pol β knockout resulted in DNA damage accumulation and cytosolic dsDNA leakage, which activated the cGAS-STING-NF-κB signaling pathway and upregulated the expression of NLRP3, IL-1 β, and IL-18. In conclusion, our findings clarify the influence of Pol β on the development of RA and provide a detailed explanation for the STING-NF-κB pathway to induce macrophage pyroptosis.

Age-associated B cells contribute to the pathogenesis of rheumatoid arthritis by inducing activation of fibroblast-like synoviocytes via TNF-α-mediated ERK1/2 and JAK-STAT1 pathways

ObjectivesAge-associated B cells (ABCs) are a recently identified B cell subset, whose expansion has been increasingly linked to the pathogenesis of autoimmune disorders. This study aimed to investigate whether ABCs...

Preclinical evaluation and pilot clinical study of [18F]AlF-NOTA-FAPI-04 for PET imaging of rheumatoid arthritis.

Fibroblast-like synoviocytes (FLSs) are key effector cells in the inflamed joints of patients with rheumatoid arthritis (RA). Previous studies have suggested that fibroblast activation protein (FAP) is highly expressed in RA-derived FLSs and is a specific marker of activated RA FLSs. In this study, we developed aluminum-[18F]-labeled 1,4,7-triazacyclononane-N,N',N″-triacetic acid-conjugated FAP inhibitor 04 ([18F]AlF-NOTA-FAPI-04) to image RA-FLSs in vitro and arthritic joints in collagen-induced arthritis (CIA) mice and RA patients. RA FLSs and NIH3T3 cells transfected with FAP were used to perform in vitro-binding studies. Biodistribution was conducted in normal DBA1 mice. Collagen-induced arthritis (CIA) models with different arthritis scores were subjected to [18F]AlF-NOTA-FAPI-04 and 18F-FDG PET imaging. Histological examinations were performed to evaluate FAP expression and Cy3 dye-labeled FAPI-04(Cy3-FAPI-04) uptake. Blocking studies with excess unlabeled FAPI-04 in CIA mice and NIH3T3 xenografts in immunocompromised mice were used to evaluate the binding specificity of [18F]AlF-NOTA-FAPI-04. Additionally, [18F]AlF-NOTA-FAPI-04 PET imaging was performed on two RA patients. The binding of [18F]AlF-NOTA-FAPI-04 increased significantly in RA FLSs and NIH3T3 cells overexpressing FAP compared to their parental controls (FAP-GFP-NIH3T3 vs. GFP-NIH3T3, 2.40 ± 0.078 vs. 0.297 ± 0.05% AD/105 cells; RA FLSs vs. OA FLSs, 1.54 ± 0.064 vs. 0.343 ± 0.056% AD/105 cells). Compared to 18F-FDG imaging, [18F]AlF-NOTA-FAPI-04 showed high uptake in inflamed joints in the early stage of arthritis, which was positively correlated with the arthritic scores (Pearson r=0.834, P<0.001). In addition, the binding of [18F]AlF-NOTA-FAPI-04 to cells with high FAP expression and the uptake of [18F]AlF-NOTA-FAPI-04 in arthritic joints both could be blocked by excessive unlabeled FAPI-04. Fluorescent staining showed that the intensity of Cy3-FAPI-04 binding to FAP increased accordingly as the expression of FAP protein increased in cells and tissue sections. Furthermore, the uptake of [18F]AlF-NOTA-FAPI-04 in FAP-GFP-NIH3T3 xenografts was significantly higher than that in GFP-NIH3T3 xenograft (35.44 ± 4.27 vs 7.92 ± 1.83% ID/mL). Finally, [18F]AlF-NOTA-FAPI-04 PET/CT imaging in RA patients revealed nonphysiologically high tracer uptake in the synovium of arthritic joints. [18F]AlF-NOTA-FAPI-04 is a promising radiotracer for imaging RA FLSs and could potentially complement the current noninvasive diagnostic parameters.

Triptolide inhibits Th17 differentiation via controlling PKM2-mediated glycolysis in rheumatoid arthritis

Context Rheumatoid arthritis (RA) is an autoimmune disease with the aberrant differentiation of T helper 17 (Th17) cells. Pyruvate kinase M2 (PKM2), a key enzyme of glycolysis, was associated with Th17 cell differentiation. Aim To investigate the potential therapeutic effects of triptolide (TP) in collagen-induced arthritis (CIA) and Th17 cell differentiation, and elucidated the underlying mechanisms. Methods PKM2 expression and IL-17A production in peripheral blood of RA patients were detected by RT-qPCR or ELISA. Flow cytometry and ELISA were employed to assess the effect of Th17 cell differentiation by TP. PKM2 expression and other glycolysis-related factors were detected using RT-qPCR and Western Blot. PKM2 specific inhibitor Compound 3 K was used to verify the mechanisms. Male DBA/1J mice were divided into control, model, and TP (60 μg/kg) groups to assess the anti-arthritis effect, Th17 cell differentiation and PKM2 expression. Results PKM2 expression positively correlated with IL-17A production in RA patients. PKM2 expression was increased upon Th17 cell differentiation. Down-regulating PKM2 expression could strongly reduce Th17 cell differentiation. Molecular docking analysis predicted that TP targeted PKM2. TP treatment significantly reduced Th17 cell differentiation, PKM2 expression, pyruvate, and lactate production. In addition, compared with down-regulating PKM2 alone (Compound 3 K treatment), co-treatment with TP and Compound 3 K further significantly decreased PKM2-mediated glycolysis and Th17 cell differentiation. In CIA mice, TP repressed the PKM2-mediated glycolysis and attenuated joint inflammation. Conclusion TP inhibited Th17 cell differentiation through the inhibition of PKM2-mediated glycolysis. We highlight a novel strategy for the use of TP in RA treatment.

Antcin K inhibits VCAM-1-dependent monocyte adhesion in human rheumatoid arthritis synovial fibroblasts.

BackgroundAntcin K, an extract of Antrodia cinnamomea (a medicinal mushroom endemic to Taiwan commonly used in Chinese medicine preparations), inhibits proinflammatory cytokine production and angiogenesis in human rheumatoid arthritis synovial fibroblasts (RASFs), major players in RA disease. Antcin K also inhibits disease activity in mice with collagen-induced arthritis (CIA). Up until now, the effects of Antcin K upon cell adhesion molecules (CAMs) were unknown.MethodsRA and healthy synovial tissue samples (n = 10 in each group) were retrieved from the Gene Expression Omnibus (GEO) database (accession code: GDS5401) to compare CAM and monocyte marker expressions. In addition, synovial tissue samples from six RA patients and six patients undergoing arthroscopy for trauma/joint derangement (healthy controls) were subjected to immunohistochemical (IHC) analysis. mRNA and protein expression levels were analyzed in RASFs using RT-qPCR (Reverse transcription-quantitative polymerase chain reaction) and Western blot. RASFs were incubated with Antcin K and examined for monocyte adherence by fluorescence microscopy. Ankle joint tissue specimens from a CIA mouse model and healthy controls were stained with hematoxylin and eosin (H&E) and Safranin-O/Fast Green to examine histological changes and evidence of bone loss. IHC analysis determined levels of vascular cell adhesion molecule 1 (VCAM-1) and CD11b in CIA ankle tissue and clinical synovial tissue.ResultsLevels of VCAM-1 expression were higher in the GEO database specimens and the study’s clinical samples of RA synovial tissue compared with the healthy specimens. Antcin K dose-dependently inhibited VCAM-1 expression and monocyte adhesion in RASFs. Antcin K also significantly inhibited levels of VCAM-1 and monocyte CD11b expression in CIA tissue. These effects appeared to be mediated by MEK1/2-ERK, p38, and AP-1 signaling.ConclusionsAntcin K seems promising for the treatment of RA and deserves further investigations.

Fecal microbiota transplantation from patients with rheumatoid arthritis causes depression-like behaviors in mice through abnormal T cells activation

Depression is common in patients with rheumatoid arthritis (RA); however, the precise mechanisms underlying a link between depression and RA remain unclear. Accumulating evidence suggests the role of gut–microbiota–brain axis in depression. In this study, we investigated whether collagen-induced arthritis (CIA) mice produce depression-like behaviors and abnormal composition of gut microbiota. Furthermore, we investigated whether fecal microbiota transplantation (FMT) from RA patients causes depression-like phenotypes in antibiotic cocktail (ABX)-treated mice. CIA mice displayed depression-like behaviors, increased blood levels of pro-inflammatory cytokine interleukin-6 (IL-6), decreased expression of synaptic proteins in the prefrontal cortex (PFC), and abnormal composition of gut microbiota. Furthermore, FMT from RA patients caused depression-like phenotypes, alterations of gut microbiota composition, increased levels of IL-6 and tumor necrosis factor-α (TNF-α), and downregulation of synaptic proteins in the PFC compared to FMT from healthy controls. There were correlations between relative abundance of microbiota and plasma cytokines, expression of synaptic proteins in the PFC or depression-like behaviors. Interestingly, FMT from RA patients induced T cells differentiation in Peyer’s patches and spleen. Reduced percentage of Treg cells with an increase of Th1/Th2 index was observed in the mice after FMT from RA patients. These findings suggest that CIA mice exhibit depression-like behaviors, systemic inflammation, and abnormal composition of gut microbiota, and that FMT from RA patients produces depression-like behaviors in ABX-treated mice via T cells differentiation. Therefore, abnormalities in gut microbiota in RA patients may contribute to depression via gut–microbiota–brain axis.

Emodin Alleviates Arthritis Pain through Reducing Spinal Inflammation and Oxidative Stress.

Chronic pain is the predominant problem for rheumatoid arthritis patients, and negatively affects quality of life. Arthritis pain management remains largely inadequate, and developing new treatment strategies are urgently needed. Spinal inflammation and oxidative stress contribute to arthritis pain and represent ideal targets for the treatment of arthritis pain. In the present study, collagen-induced arthritis (CIA) mouse model was established by intradermally injection of type II collagen (CII) in complete Freund's adjuvant (CFA) solution, and exhibited as paw and ankle swelling, pain hypersensitivity and motor disability. In spinal cord, CIA inducement triggered spinal inflammatory reaction presenting with inflammatory cells infiltration, increased IL-1β expression, and up-regulated NLRP3 and cleaved caspase-1 levels, elevated spinal oxidative level presenting as decreased Nrf2 expression and SOD activity. To explore potential therapeutic options for arthritis pain, emodin was intraperitoneally injected for three days on CIA mice. Emodin treatment statistically elevated mechanical pain sensitivity, suppressed spontaneous pain, recovered motor coordination, decreased spinal inflammation scores and expression levels of IL-1β, increased spinal Nrf2 expression and SOD activity. Further, AutoDock data showed that emodin bind to AMPK through two electrovalent bonds. And emodin treatment increased the phosphorylated AMPK at threonine 172. In summary, emodin treatment activates AMPK, suppresses NLRP3 inflammasome response, elevates antioxidant response, inhibits spinal inflammatory reaction and alleviates arthritis pain.

AB0082 ANTI-INFLAMMATORY EFFECTS OF TOTAL SAPONINS OF PANAX JAPONICUS ON RHEUMATOID ARTHRITIS

BackgroundRheumatoid arthritis (RA) is a common autoimmune disease with inflammation[1]. Total saponins of Panax japonicus (TSPJs) are effective components extracted from Panax japonicus[2]. They are known to exhibit anti-inflammatory and immunosuppressoive properties, but their effect of anti-inflammation in collagen-induced arthritis (CIA) remains unclear.ObjectivesTo investigate the anti-inflammatory targets of TSPJ predicted by bioinformatics and the verification in CIA mice.MethodsThe targets of RA are obtained in the GeneCards database. we used Cytoscape 3.7.2 software to construct a protein-protein interactions (PPI) network and obtain the hub genes. There are four effective components of TSPJ: Araloside A, chikusetsusaponin IVa, ginsenoside Rg2, and ginsenoside Ro. Through molecular docking between the screened hub genes and the four effective components of TSPJ, the possibility of TSPJ treating CIA mice can be predicted. The collagen II (CII) and complete Freund’s adjuvant (CFA) were used to induce the CIA model. After establishing the model, 32 DBA1/J mice were divided into C group (n=8), M group (n=8), L group(n=8), and H group(n=8). The L and H groups were gavaged with TSPJ at 30 mg/kg or 150 mg/kg, and the C and M groups were gavaged with normal saline. The thickness of the hind paw, number of swollen joints, and arthritis index were evaluated. After 11 days of treatment, all the mice were sacrificed after anesthesia. Sera were collected to centrifuge tubes and the levels of inflammatory factor were determined by the ELISA kit following the instruction.ResultsA gene list that enriches 263 genes was obtained by searching RA from the GeneCards database. The hub genes of the top 3 obtained from Cytoscape 3.7.2 software were tumor necrosis factor (TNF), interleukin-1β (IL-1β), and interleukin-6 (IL-6). In addition, interleukin-17A (IL-17A), a classical inflammatory index in the top 10, was selected and included in the predicted target. The results of molecular docking between the predicted target and the components of TSPJ showed that the combined pose has good stability. The numerical value of hind paw thickness, swollen joint counts, and arthritis index in the intervention groups were lower than those in the M group, suggesting TSPJ played a critical role in improving pathological changes. Compared to those of the C group, the serum levels of TNF-α, IL-1β, IL-6, and IL-17A were increased in the M group. Compared to those of the M group, the levels of TNF-α, IL-1β, IL-6, and IL-17A in the L and H groups were decreased. Compared to those of the L group, the levels of TNF-α, IL-1β, IL-6, and IL-17A in the H group were decreased. The results suggested that TSPJ may decrease the levels of TNF-α, IL-1β, IL-6, and IL-17A in CIA mice. These results suggest that TSPJ may inhibit the inflammatory effects of CIA mice.ConclusionCurrent study demonstrated a novel inhibitory effect of TSPJ on inflammation in CIA mice, and TSPJ can act on the targets predicted by bioinformatics of CIA mice, suggesting the potential of TSPJ as a therapeutic agent for RA and providing new ideas for the clinical treatment of RA.

Alamandine, a new member of the renin-angiotensin system (RAS), attenuates collagen-induced arthritis in mice via inhibiting cytokine secretion in synovial fibroblasts

Alamandine is a novel component of the renin-angiotensin system (RAS) as well as an important biologically active peptide. It has predominantly been studied in cardiovascular context. However, its role in rheumatoid arthritis (RA) remains unknown. Here we illustrated its effects on inflammatory cytokines production by synovial fibroblasts from RA and pathological changes in collagen-induced arthritis (CIA) mice. Alamandine (0.1, 1 and 10 µg/ml) did not affect the survival of the synovial fibroblasts, but decreased the migration and proinflammatory cytokines expression in TNF-α (10 ng/ml) stimulated cells in vitro. Additionally, alamandine selectively decreased phosphorylated-JNK expression induced by TNF-a stimulation in RA FLS. DBA/1 J mice were induced arthritis by a primary injection with an emulsion of bovine type II collagen (CII) and complete Freund’s adjuvant (day 0) and a booster injection of CII in incomplete Freund’s adjuvant (day 21). Mice were then given alamandine intraperitoneally in saline (50 μg/kg/day) from days 21–42. Histology and multiplex immunobead assay showed that alamandine treatment inhibited the development of arthritis and reduced the joint damage. This effect was accompanied by the reduced inflammatory cytokines (IL-6, IL-23, IFN-γ) mRNA expression in local joints, the decreased TNF-α, IL-6, IL-17 and the increased IL-10 levels in the serum from alamandine administrated CIA mice. In conclusion, alamandine attenuates the development of arthritis by suppressing inflammatory cytokines expression in RA synovial fibroblasts via MAPK signaling pathway, suggesting a potential therapeutic role for RA.

MicroRNA-133 suppresses cell viability and migration of rheumatoid arthritis fibroblast-like synoviocytes by down-regulation of MET, EGFR, and FSCN1 expression.

Aberrant proliferation and migration of fibroblast-like synoviocytes (FLS) are major characteristics of rheumatoid arthritis (RA). MicroRNA-133 (miR-133) is a tumor-suppressive miRNA that targets various genes responsive for cell proliferation and migration. The aim of this study was to examine the effect of miR-133 on RA FLS. A high throughput miRNA microarray was performed in synovium from mice with collagen-induced arthritis (CIA). Expression levels of miR-133 and the putative targets were determined in synovium and FLS from patients with RA and mice with CIA. Overexpression of miR-133 in RA FLS was performed by lentiviral vector-mediated transfer of precursor miRNA (pre-miR). The expression of miR-133a/b was decreased in the joint tissue and FLS of CIA mice, as determined by miRNA array and qRT-PCR. Down-regulation of miR-133a/b expression could also be observed in synovium and FLS from patients with RA. Overexpression of miR-133 reduced cell viability and migration of RA FLS, with decreased levels of FSCN1, EGFR, and MET. Our findings demonstrated the inhibitory effects of miR-133 on FLS viability and migration, and might contribute to the pharmacologic development of miR-133 therapeutics in patients with RA.

Extracellular vesicle-guided in situ reprogramming of synovial macrophages for the treatment of rheumatoid arthritis

Activation state of synovial macrophages is significantly correlated with disease activity and severity of rheumatoid arthritis (RA) and provides valuable clues for RA treatment. Classically activated M1 macrophages in inflamed synovial joints secrete high levels of pro-inflammatory cytokines and chemokines, resulting in bone erosion and cartilage degradation. Herein, we propose extracellular vesicle (EV)-guided in situ macrophage reprogramming toward anti-inflammatory M2 macrophages as a novel RA treatment modality based on the immunotherapeutic concept of reestablishing M1-M2 macrophage equilibrium in synovial tissue. M2 macrophage-derived EVs (M2-EVs) were able to convert activated M1 into reprogrammed M2 (RM2) macrophages with extremely high efficiency (>90%), producing a distinct protein expression pattern characteristic of anti-inflammatory M2 macrophages. In particular, M2-EVs were enriched for proteins known to be involved in the generation and migration of M2 macrophages as well as macrophage reprogramming factors, allowing for rapid and efficient driving of macrophage polarization toward M2 phenotype. After administration of M2-EVs into the joint of a collagen-induced arthritis mouse model, the synovial macrophage polarization was significantly shifted from M1 to M2 phenotype, a process that benefited greatly from the long residence time (>3 days) of M2-EVs in the joint. This superb in situ macrophage-reprogramming ability of EVs resulted in decreased joint swelling, arthritic index score and synovial inflammation, with corresponding reductions in bone erosion and articular cartilage damage and no systemic toxicity. The anti-RA effects of M2-EVs were comparable to those of the conventional disease-modifying antirheumatic drug, Methotrexate, which causes a range of toxic adverse effects, including gastrointestinal mucosal injury. Overall, our EV-guided reprogramming strategy for in situ tuning of macrophage responses holds great promise for the development of anti-inflammatory therapeutics for the treatment of various inflammatory diseases in addition to RA.