CIA Mice Research Articles

Aberrant overexpression of the autoantigen protein vimentin promotes Th17 cell differentiation and autoimmune arthritis via activation of STAT3 signaling

Vimentin contributes to the positioning and function of organelles, cell migration, adhesion, and division. However, secreted vimentin accumulates on the cell surface (Mor-Vaknin et al., 2003; Ramos et al., 2020 [1,2]) where it acts as a coreceptor for viral infection and as an autoantigen in inflammatory and autoimmune diseases. The roles of vimentin in Th17 cells were examined in mice with knockdown of vimentin. We also examined whether STAT3 is required for vimentin expression.Vimentin expression was significantly increased in Th17 cells through STAT3 activation, and vimentin+ IL-17+ T cells were markedly increased in the joint and spleen tissues of CIA mice. The arthritis score and expression levels of proinflammatory cytokines were significantly decreased in CIA mice treated with vimentin shRNA vector.In this study, we demonstrated that vimentin is significantly expressed in Th17 cells through STAT3 activation. Our results provide new insights into the role of vimentin in Th17 cells and the complex pathogenesis of RA.

Kunduan Yimu Decoction affected Th17/Treg balance through microRNA-124 to improve rheumatoid arthritis pathology

BackgroundRheumatoid arthritis (RA) is an autoimmune condition characterized by inflammation and the deterioration of joints. Current treatments often have side effects, highlighting the need for safer options. This study investigates the therapeutic effects of Kunduan Yimu Decoction (KDYMD) on RA, focusing on the role of miR-124 in regulating Th17/Treg differentiation. MethodsPBMCs from RA patients were analyzed before and after KDYMD treatment. RT-qPCR was used to measure the miR-124 expressions. Flow cytometry was used to assess the ratios of Th17 to Treg cells. ELISA was used to quantify the cytokine concentrations. The effects of KDYMD on JAK2/STAT3 signaling were evaluated by western blot analysis. A CIA mouse model was used to validate the in vivo effects of KDYMD. ResultsMiR-124 expression was significantly upregulated in PBMCs of RA patients after KDYMD treatment. This upregulation was associated with increased Tip60 and Foxp3 expression and decreased RORγt expression. In the cytokine analysis, IL-1, IL-6, and IL-17A were decreased, and IL-10 and TGF- were increased after treatment. Flow cytometry showed a restoration of the Th17/Treg balance, with a decrease in Th17 and an increase in Treg cells. In vivo, KDYMD treatment ameliorated ankle swelling and arthritis index in CIA mice, comparable to methotrexate (MTX). In addition, KDYMD modulated JAK2/STAT3 signaling and enhanced anti-inflammatory responses. ConclusionsKDYMD exerts significant anti-inflammatory effects in RA by upregulating miR-124, which in turn regulates Th17/Treg differentiation and modulates JAK2/STAT3 signaling. A novel mechanism involving miR-124 and immune cell balance suggests KDYMD could be a promising therapeutic agent for RA.

Columbianadin ameliorates rheumatoid arthritis by attenuating synoviocyte hyperplasia through targeted vimentin to inhibit the VAV2/Rac-1 signaling pathway

IntroductionRheumatoid arthritis (RA) is an autoimmune disease pathologically characterized by synovial inflammation. The abnormal activation of synoviocytes seems to accompany the progression of RA. The role and exact molecular mechanism in RA of columbianadin (CBN) which is a natural coumarin is still unclear. ObjectivesThe present research aimed to investigate the effect of vimentin on the abnormal growth characteristics of RA synoviocytes and the targeted regulatory role of CBN. MethodsCell migration and invasion were detected using the wound healing and transwell method. Mechanistically, the direct molecular targets of CBN were screened and identified by activity-based protein profiling. The expression of relevant proteins and mRNA in cells and mouse synovium was detected by western blotting and qRT-PCR. Changes in the degree of paw swelling and body weight of mice were recorded. H&E staining, toluidine blue staining, and micro-CT were used to visualize the degree of pathological damage in the ankle joints of mice. Small interfering RNA and plasmid overexpression of vimentin were used to observe their effects on MH7A cell proliferation, migration, apoptosis, and downstream molecular signaling. ResultsThe TNF-α-induced proliferation and migration of MH7A cells could be significantly repressed by CBN (25,50 μM), and the expression of apoptosis and autophagy-associated proteins could be modulated. Furthermore, CBN could directly bind to vimentin and inhibit its expression and function in synoviocytes, thereby ameliorating foot and paw swelling and joint damage in CIA mice. Silencing and overexpression of vimentin might be involved in developing RA synovial hyperplasia and invasive cartilage by activating VAV2 phosphorylation-mediated expression of Rac-1, which affects abnormal growth characteristics, such as synoviocyte invasion and migration. ConclusionCBN-targeted vimentin restrains the overactivation of RA synoviocytes thereby delaying the pathological process in CIA mice, which provides valuable targets and insights for understanding the pathological mechanisms of RA synovial hyperplasia.

Fetal microchimerism cells suppress arthritis progression by inducing CD14+ IL-10+ cells in pregnant experimental mice.

Fetal microchimerism occurs in the mother after a pregnancy. To investigate the role of fetal microchimerism cells (FMCs) in rheumatoid arthritis, we analyzed the population of fetal cells in pregnant experimental arthritis mice. We used EGFP+ fetuses, which were mated with either healthy female mice or CIA mice, and male C57BL/6J-Tg (Pgk1-EGFP)03Narl mice, to detect the population of FMCs in maternal circulation. The disease progression was determined by measuring the clinical score and histological stains during pregnancy. The fetal cells have been analyzed if expressing EGFP, CD45, and Scal by flow cytometry. We also detected the expression of CD14+ IL-10+ cells invivo and invitro. Our data showed that the pregnancy ameliorated the arthritis progression of CIA mice. The IHC stains showed the CD45 -Sca-1+ EGFP+ FMCs were expressed in the bone marrow and peripheral blood mononuclear cells (PBMC) at 14 gestation days. However, Treg and Tc cell populations showed no significant change in the bone marrow. The data showed the H2Kb + fetal cells induced CD14+ IL10+ cell populations increased in the bone marrow invitro and invivo. Our investigations demonstrated that the FMCs protected the CIA mice from cartilage damage and triggered an immunosuppressive response in them by increasing the number of CD14+ IL10+ cells. In conclusion, the FMCs could potentially exhibit protective properties within the context of inflammatory arthritis that arises during pregnancy.

Role and mechanism of IRF9 in promoting the progression of rheumatoid arthritis by regulating macrophage polarization via PSMA5

AimTo explore the mechanisms of IRF9 in the progression of rheumatoid arthritis(RA), and the effects of IRF9 on M1/M2 polarization. MethodsRA dataset (GSE55457) was downloaded from GEO. Correlation analysis between IRF9 and its downstream target protein PSMA5 was performed using bioinformatics analysis. The M1/M2 cell ratio of peripheral blood mononuclear cells which from 20 healthy specimen and 40 RA patients was determined. The expression of IRF9 and PSMA5 was detected using qPCR and Western blot. Then, knockdown IRF9 in RAW264.7 cell line (sh-IRF9 RAW264.7) was constructed. The effect of sh-IRF9 RAW264.7 on RA was explored by constructing a CIA mouse model. ResultsIRF9 is upregulated in RA and is of good early screening effect. The results of pathway analysis showed that IRF9 targets and regulates the PSMA5 signaling pathway. IRF9 and PSMA5 were significantly elevated in RA patients, M1/M2 ratio was also increased. The effects of IRF9 on RAW264.7 macrophages were deeply explored in vitro, revealing that knockdown of IRF9 suppressed PSMA5, M1/M2 ratio and the secretion of pro-inflammatory factor in RAW264.7. In mouse in vivo experiments, sh-IRF9 RAW264.7 cells were found to modulate RA by downregulating PSMA5, modulating the M1/M2 ratio through enhancing the anti-inflammatory factor, and suppressing the pro-inflammatory factor. ConclusionIRF9 promoted the progression of RA via regulating macrophage polarization through PSMA5.

Anti-inflammatory effect of the combined treatment of LMT-28 and kaempferol in a collagen-induced arthritis mouse model.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation and swelling. Several studies have demonstrated that RA fibroblast-like synovial cells (RA-FLS) play an important role in RA pathogenesis. Activated RA-FLS contribute to synovial inflammation by secreting inflammatory cytokines including interleukin (IL)-1β, IL-6 and tumor necrosis factor-α. LMT-28 is derivative of oxazolidone and exerts anti-inflammatory effects on RA via IL-6 signaling pathway regulation. LMT-28 also regulates T cell differentiation in RA condition. However, the effect of LMT-28 on the migration and invasion of RA-FLS remains unknown. Kaempferol has been reported to have pharmacological effects on various diseases, such as inflammatory diseases, autoimmune diseases, and cancer. Additionally, kaempferol has been reported to inhibit RA-FLS migration and invasion, but it is not known about the therapeutic mechanism including molecular mechanism such as receptor. The present study aimed to investigate the synergistic effects of the combined treatment of LMT-28 and kaempferol on RA-FLS activation and RA pathogenesis in mouse model. LMT-28 and kaempferol co-administration inhibited RA disease severity and histological collapse in the joint tissues of CIA mice, as well as downregulated the levels of pro-inflammatory cytokines in mouse serum. Additionally, the combined treatment inhibited excessive differentiation of T helper 17 cells and osteoclasts. Furthermore, compared with single treatments, combined treatment showed enhanced inhibitory effects on the hyperactivation of IL-6-induced signaling pathway in RA-FLS. Combined treatment also inhibited RA-FLS cell proliferation, migration, and invasion and suppressed the expression of matrix metalloproteinase in RA-FLS. Furthermore, we confirmed that the combined treatment inhibited chondrocyte proliferation, migration, and invasion. In conclusion, our results suggest that the combined treatment of LMT-28 and kaempferol exerts a synergistic effect on the RA development via the regulation of IL-6-induced hyperactivation of RA-FLS. Furthermore, this study suggests that combination therapies can be an effective therapeutic option for arthritis.

WTAP/YTHDF1-mediated m6A modification amplifies IFN-γ-induced immunosuppressive properties of human MSCs

IntroductionThe immunosuppressive capacity of mesenchymal stem cells (MSCs) is dependent on the “license” of several pro-inflammatory factors to express immunosuppressive molecular profiles, which determines the therapeutic efficacy of MSCs in immune-mediated inflammatory diseases. Of those, interferon-γ (IFN-γ) is a key inducer for the expression of immunosuppressive molecular profiles; however, the mechanism underlying this effect is unknown. ObjectivesTo elucidate the regulation mechanism and biological functions of N6-methyladenosine (m6A) modification in the immunosuppressive functions by the IFN-γ-licensing MSCs. MethodsEpitranscriptomic microarray analysis and MeRIP-qPCR assay were performed to identify the regulatory effect of WTAP in the IFN-γ-licensing MSCs. RIP-qPCR, western blot, qRT-PCR and RNA stability assays were used to determine the regulation of WTAP/m6A/YTHDF1 signaling axis in the expression of immunosuppressive molecules. Further, functional capacity of T cells was tested using flow cytometry, and both DSS-induced colitis mice and CIA mice were constructed to clarify the effect of WTAP and YTHDF1 in MSC-mediated immunosuppression. ResultsWe identified that IFN-γ increased the m6A methylation levels of immunosuppressive molecules, while WTAP deficiency abolished the IFN-γ-induced promotion of m6A modification. IFN-γ activated ERK signaling, which induced WTAP phosphorylation. Additionally, the stabilization of WTAP post-transcriptionally increased the mRNA expression of immunosuppressive molecules (IDO1, PD-L1, ICAM1, and VCAM1) in an m6A-YTHDF1-dependent manner; this effect further impacted the immunosuppressive capacity of IFN-γ licensing MSCs on activated T cells. Notably, WTAP/YTHDF1 overexpression enhanced the therapeutic efficacy of IFN-γ licensing MSCs and restructures the ecology of inflammation in both colitis and arthritis models. ConclusionOur results showed that m6A modification of IDO1, PD-L1, ICAM1, and VCAM1 mRNA mediated by WTAP-YTHDF1 is involved in the regulation of IFN-γ licensing MSCs immunosuppressive abilities, and shed a light to enhance the clinical therapeutic potential of IFN-γ-licensing MSCs.

Wogonin inhibits the migration and invasion of fibroblast-like synoviocytes by targeting PI3K/AKT/NF-κB pathway in rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is currently an autoimmune inflammatory disease with an unclear pathogenesis. Fibroblast-like synoviocytes (FLSs) have tumor-like properties, and their activation and secretion of pro-inflammatory factors are important factors in joint destruction. Wogonin (5,7-dihydroxy-8-methoxyflavone), a natural flavonoid isolated from Scutellaria baicalensis root, has been shown to have significant anti-inflammatory, anti-oxidative stress, and anti-tumor effects in a variety of diseases. However, the role of wogonin in RA has not yet been demonstrated. PurposeTo investigate the inhibitory effect of wogonin on the invasive behavior of fibroblast-like synoviocytes and to explore the mechanism of action of wogonin in RA. MethodsCCK-8, EdU, cell migration and invasion, immunofluorescence staining, RT-qPCR, and protein blot analysis were used to study the inhibitory effects of wogonin on migration, invasion, and pro-inflammatory cytokine overexpression in the immortalized rheumatoid synovial cell line MH7A. The therapeutic effects of wogonin were validated in vivo using arthritis scores and histopathological evaluation of collagen-induced arthritis mice. ResultsWogonin inhibited the migration and invasion of MH7A cells, reduced the production of TNF-α, IL-1β, IL-6, MMP-3 and MMP-9, and increased the expression of IL-10. Moreover, wogonin also inhibited the myofibrillar differentiation of MH7A cells, increased the expression of E-cadherin (E-Cad) and decreased the expression of α-smooth muscle actin (α-SMA). In addition, wogonin treatment effectively ameliorated joint destruction in CIA mice. Further molecular mechanism studies showed that wogonin treatment significantly inhibited the activation of PI3K/AKT/NF-κB signaling pathway in TNF-α-induced arthritic FLSs. ConclusionWogonin effectively inhibits migration, invasion and pro-inflammatory cytokine production of RA fibroblast-like synoviocytes through the PI3K/AKT/NF-κB pathway, and thus wogonin, as a natural flavonoid, has great potential for treating RA.

Novel mechanistic study of HDAC6 regulation of rheumatoid arthritis via CMA: exploring potential therapeutic targets.

Rheumatoid arthritis (RA) is a systemic autoimmune disease. Its pathogenesis has not yet been clarified, so it is urgent to explore therapeutic targets. Here, we clarified the role of HDAC6 in the mechanism of action of RA through mediating chaperone-mediated autophagy (CMA) to provide a clinical treatment of RA. We used rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and collagen-induced arthritis mice (CIA mice) as models of RA and pharmacological inhibitors as well as genetic interference with adeno-associated viruses to reduce the expression of HDAC6. We explored the influence of CAY10603 on RA-FLS proliferation and inflammation, as well as the expression of proteins related to the CMA signaling pathway. CIA model was constructed using DBA/1J mice. Arthritis symptoms in CIA mice were evaluated, and the expression and localization of CMA-related proteins in mouse ankle joints were examined. CAY10603 inhibited proliferation as well as the level of the molecular chaperone autophagy in RA-FLS. HDAC6 shRNA significantly reduced the clinical signs of arthritis in CIA mice, as did the expression of HDAC6 in the serum and ankle synovial tissues of CIA mice. Finally, it significantly inhibited the level of Hsc70 and LAMP-2A, which are involved in the CMA signaling pathway, in ankle joint tissues. Downregulation of HDAC6 may inhibit CMA and thereby ameliorate RA.

Cytoprotective role of human dental pulp stem cell-conditioned medium in chemotherapy-induced alopecia

BackgroundChemotherapy-induced alopecia (CIA) is a distressing adverse effect of chemotherapy, with an estimated incidence of 65% and limited treatment options. Cyclophosphamide (CYP) is a common alopecia-inducing chemotherapy agent. Human dental pulp stem cells (DPSCs) secrete several paracrine factors that up-regulate hair growth. Conditioned medium (CM) collected from DPSCs (DPSC-CM) promotes hair growth; culturing mesenchymal stem cells under hypoxic conditions can enhance this effect.MethodsThe effect of DPSC-CM cultured under normoxic (N-) and hypoxic (H-) conditions against CYP-mediated cytotoxicity in keratinocytes was examined using cell viability assay, lactate dehydrogenase (LDH) cytotoxicity assay, and apoptosis detection. The damage-response pathway was determined in a well-established CIA mouse model by analyzing macroscopic effects, histology, and apoptosis. Reverse transcription-quantitative PCR and Caspase-3/7 activity assay were used to investigate the impact of DPSC-CM on the molecular damage-response pathways in CYP-treated mice. The effect of post-CIA DPSC-CM application on post-CIA hair regrowth was analyzed by macroscopic effects and microstructure observation of the hair surface. Furthermore, to investigate the safety of DPSC-CM as a viable treatment option, the effect of DPSC-CM on carcinoma cell lines was examined by cell viability assay and a subcutaneous tumor model.ResultsIn the cell viability assay, DPSC-CM was observed to increase the number of keratinocytes over varying CYP concentrations. Furthermore, it reduced the LDH activity level and suppressed apoptosis in CYP-treated keratinocytes. DPSC-CM exhibited the cytoprotective role in vivo via the dystrophic anagen damage-response pathway. While both N-CM and H-CM downregulated the Caspase-3/7 activity level, H-CM downregulated Caspase-3 mRNA expression. The proportion of post-CIA H-CM-treated mice with > 90% normal hair was nearly twice that of vehicle- or N-CM-treated mice between days 50 and 59 post-depilation, suggesting that post-CIA H-CM application may accelerate hair regrowth and improve hair quality. Furthermore, DPSC-CM suppressed proliferation in vitro in certain carcinoma cell lines and did not promote the squamous cell carcinoma (SCC-VII) tumor growth rate in mice.ConclusionsThe potentiality of DPSC-CM and H-CM as a promising cytoprotective agent and hair regrowth stimulant, respectively, for CIA needs in-depth exploration.

Apoptotic extracellular vesicles restore homeostasis of the articular microenvironment for the treatment of rheumatoid arthritis

Rheumatoid arthritis (RA) is a severe autoimmune disease with symptoms including synovial inflammation, cartilage erosion, and bone loss in RA lesions, which eventually lead to joint deformity and function loss. Most current treatments fail to achieve satisfying therapeutic outcomes with some adverse effects. Extracellular vesicles derived from apoptotic cells (apoEVs) have emerged as important mediators in intercellular communication regulating diverse physiological and pathological processes. In this study, we investigated the therapeutic efficacy of macrophage-derived and osteoclast-derived apoEVs (Mφ-apoEVs and OC-apoEVs) on RA. The in vitro results showed that both Mφ-apoEVs and OC-apoEVs induced macrophage repolarization toward the anti-inflammatory M2 phenotype, promoted chondrocyte functions and chondrogenesis, and inhibited osteoclast formation and maturation. In addition, OC-apoEVs promoted osteogenic differentiation. The in vivo study on the CIA mouse model further demonstrated that apoEVs could couple various functions and exert synergistic effects on the joint with RA, as evidenced by the regression of synovial inflammation, the reversal of cartilage damage and bone erosion, and the preservation of joint structure. These findings demonstrated that Mφ-apoEVs and OC-apoEVs contributed to restoring the homeostasis of the overall microenvironment in the RA joint and highlighted their potential application as a promising alternative to treat RA.

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.

The Role of Torkinib in Inhibiting the Proliferation and Migration of Rheumatoid Arthritis Fibroblast-Like Synoviocytes

This study aims to investigate the potential of an inhibitor of the mammalian target of rapamycin (Torkinib) as a therapeutic drug targeting fibroblast-like synoviocytes (FLS) presenting it as a promising alternative for the treatment of rheumatoid arthritis (RA). FLS were isolated and cultured from RA patients for cellular experiments. The collagen-induced arthritis model in mice was constructed using type II collagen emulsion for animal experiments. Cell viability was assessed using cell counting kit-8 and 5-ethynyl-2′-deoxyuridine staining assays, and flow cytometry was utilized to detect cell apoptosis. The scratch assay assessed cell migratory capacity, while the transwell assay evaluated cell invasive potential. Electron microscopy and monodansylcadaverine staining were employed for autophagy detection, and western blot analysis assisted by nanomagnetic beads examined the expression of relevant proteins. In animal experiments, arthritis development was evaluated using clinical scores and hematoxylin and eosin staining. As a result, Torkinib effectively inhibits the proliferation of RA-FLS with a 50% inhibitory concentration of 8.53 nM. Furthermore, Torkinib induces apoptosis in FLS and activates cellular autophagy while suppressing cell migration and invasion by inhibiting the mammalian target of the rapamycin (mTOR) signaling pathway. Additionally, we observed an antagonistic effect between Torkinib and 3-methyladenine. In summary, Torkinib demonstrates its potential in alleviating RA symptoms in CIA mice and enhancing disease progression.

Irisin mitigates rheumatoid arthritis by suppressing mitochondrial fission via inhibiting YAP-Drp1 signaling pathway

BackgroundIrisin is a hormone-like factor secreted by muscle cells and produced by cleavage of the membrane protein fibronectin type III domain protein 5 (FNDC5), which exerts anti-inflammatory and anti-proliferative effects. However, the effects and the underlying mechanisms of irisin in rheumatoid arthritis (RA) are still unclear. MethodCollagen-induced arthritis (CIA) model was induced in DBA/1 mice and then treated with irisin. Arthritis index, paw thickness, weight, number of affected paws, serum inflammatory factors and related pathological tests were measured. RA fibroblast-like synoviocytes (RA-FLSs) were pretreated with IL-1β and irisin, and the migration, proliferation, invasion, oxidative stress and mitochondrial related function of RA-FLSs were detected. ResultsIrisin significantly improved arthritis symptoms in CIA mice, as indicated by reduced arthritis index, alleviated paw thickness, decreased the number of affected paws and inhibited release of inflammatory factors. Irisin alleviated joint destruction, FLSs proliferation and the expression of YES-associated protein (YAP) and mitochondrial dynamic related protein 1 (Drp1) in the FLSs of CIA mice. In vitro experiment, irisin inhibited the proliferation, migration and invasion of RA-FLSs and improved oxidative stress induced by IL-1β, thereby restraining the pathogenic transformation of RA-FLSs. Mechanically, irisin suppressed the nuclear translocation of YAP, in turn, could reduce the synthesis of Drp1 protein and inhibit the mitochondrial fission of RA-FLSs, which was reversed by YAP agonists. Therefore, irisin has a protective effect on RA. ConclusionIrisin inhibits the proliferation, migration, invasion and inflammatory response of RA-FLSs by inhibiting the YAP-Drp1 signaling pathway, which implies a potential therapeutic effect on RA.

BMP4 up-regulated by 630 nm LED irradiation is associated with the amelioration of rheumatoid arthritis

Rheumatoid arthritis (RA) is caused by inflammatory response of joints with cartilage and damage of synovium and bone erosion. In our previous studies, it has showed that irradiation of 630 nm LED reduce inflammation of synovial fibroblasts and cartilage and bone destruction in RA. However, the key genes and mechanism in ameliorating RA by irradiation of 630 nm LED remains unknown. In this study, human fibroblast-like synoviocytes (FLS) cell line MH7A and primary human RA-FLSs were treated with TNF-α and 630 nm LED irradiation with the different energy density. The mRNA sequencing was performed to screen the differentially expressed genes (DEGs). In all datasets, 10 DEGs were identified through screening. The protein interaction network analysis showed that 8 out of the 10 DEGs interacted with each other including IL-6, CXCL2, CXCL3, MAF, PGF, IL-1RL1, RRAD and BMP4. This study focused on BMP4, which is identified as important morphogens in regulating the development and homeostasis. CCK-8 assay results showed that 630 nm LED irradiation did not affect the cell viability. The qPCR and ELISA results showed that TNF-α stimulation inhibited BMP4 mRNA and protein level and irradiation of 630 nm LED increased the BMP4 mRNA and protein level in MH7A cells. In CIA and transgenic hTNF-α mice models, H&E staining showed that irradiation of 630 nm LED decreased the histological scores assessed from inflammation and bone erosion, while BMP4 expression level was up-regulated after 630 nm LED irradiation. Pearson correlation analysis shown that BMP4 protein expression was negatively correlated with the histological score of CIA mice and transgenic hTNF-α mice. These results indicated that BMP4 increased by irradiation of 630 nm LED was associated with the amelioration of RA, which suggested that BMP4 may be a potential targeting gene for photobiomodulation.

Gut–Joint Axis: Impact of Bifidobacterial Cell Wall Lipoproteins on Arthritis Development

Gut microbiota affect progression of rheumatoid arthritis (RA). The present study aims at investigating the protective potential of Bifidobacterium longum cell wall lipoproteins (Lpps) shown to modulate the intestinal microbiome and prevent osteoarthritis. Arthritis was induced by collagen (CIA) or anti-collagen antibodies (CAIA) injection. Intake of 0.5 mg of Lpps/L, but not 0.25 and 1 mg of Lpps/L, significantly alleviated RA symptoms in CIA DBA/1OOaHsd mice. The arthritis index (AI) was also reduced in CAIA mice. In the CIA-protected group, colon Ligilactobacillus murinus, caecal Lactobacillus johnsonii and spleen weight correlated with AI, whereas the reverse was observed with splenic CD11c+ dendritic cells (cDCs). The unprotected CIA Lpps group harbored higher cecal and colon E. coli and lower caecal L. murinus. Lpps administration to CAIA mice after arthritis induction led to lower colon E. plexicaudatum counts. Splenocytes from CIA-protected mice triggered by LPS secreted higher Il-10 than control ones. However, a higher IL-10 response was not elicited in gnotobiotic RA mice splenocytes with lower cDCs’ recruitment. Labeled bacteria with the Lpps signal were detected in CIA mice bone marrow (BM) cDCs 5 and 16 h post-gavage but not in Peyer’s patches and the spleen. In vitro uptake of Lpps by primary BM and thymus cells was observed within 24 h. An FACS analysis detected the Lpps signal in the plasmacytoid cell compartment but not in cDCs. In conclusion, Lpps dosing is critical for preventing arthritis progression and appropriately modulating the microbiome. Our results also highlight the possible triggering of the immune system by Lpps.

Anti-inflammatory and anti-arthritic potential of methotrexate in combination with BA-25, an amino analogue of β-boswellic acid in the treatment of rheumatoid arthritis

β- boswellic acid, a pentacyclic triterpene derived from Boswellia serrata is extensively known for its anti-inflammatory potential. BA-25 (3-α-o-acetoxy-4β-amino-11-oxo-24-norurs-12-ene) is an amino analogue of β-boswellic acid that has shown anti-inflammatory potential in LPS-induced macrophages and animal models. The present study aims at investigation of the combination of BA-25 with the conventional gold standard DMARD methotrexate (MTX) for its anti-inflammatory and anti-arthritic potential using in vitro and in vivo experimental models. The anti-inflammatory potential of MTX versus the combination (BA-25 + MTX) was investigated for inhibition of NO, ROS and pro-inflammatory cytokines including TNF-α and IL-6 using ELISA in LPS-stimulated RAW-264.7 cells. The results demonstrated significant reduction in NO, ROS, TNF- α and IL-6 production with the combination treatment in comparison to MTX alone. The cytokine inhibition potential of the combination was further validated in-vivo using balb/c wherein the combination restored LPS-induced increase in pro-inflammatory cytokines. The toxicological aspect of the in vivo doses of the combination was also investigated in mice after dosing for 28 days wherein the results suggested no significant change in the hematological parameters and serum biochemical parameters in the combination versus the vehicle group. The effect of BA-25 was also investigated on MTX-induced increase in liver function tests and the expression of Bax and blc2. The results demonstrated decrease in the production of liver enzymes with BA-25 administration along with downregulating the expression of apoptotic protein Bax while increasing the expression of anti-apoptotic protein Bcl2. Furthermore, pharmacokinetic studies of BA-25 were conducted in Balb/c mice wherein the compound showed rapid absorption, high volume of distribution and a t1/2 of 13.08. Finally the anti-arthritic effect of the combination of MTX + BA-25 vs MTX alone was investigated using CIA model in DBA/1 mice wherein the treatment with the combination resulted in significant reduction in paw inflammation, IL-6 and IL-1β levels. Furthermore, the western blot analysis demonstrated considerable decrease in the expression of p-NF-κB p65 and p-IκB in the ankle-joint tissue of the CIA mice treated with the combination therapy. The results insinuated increased anti-inflammatory and anti-arthritic potential of the combination of MTX with BA-25 as evident from in to vitro and in-vivo studies.

IL-1β stimulated human umbilical cord mesenchymal stem cells ameliorate rheumatoid arthritis via inducing apoptosis of fibroblast-like synoviocytes

Rheumatoid arthritis (RA) is characterized by synovial proliferation and lymphocyte accumulation leading to progressive damage of the periarticular bone and the articular cartilage. The hyperplasia of the synovial intima lining mainly consists of fibroblast-like synoviocytes-rheumatoid arthritis (HFLS-RA) which exhibit apoptosis-resistance, hyper-proliferation, and high invasiveness. The therapeutic efficacy of mesenchymal stem cells (MSCs) treatment in RA has been shown to be due to its immuno-regulatory ability. However, the exact factors and mechanisms involved in MSCs treatment in RA remain unclear. In this study, TRAIL receptor-Death receptor 4 (DR4), DR5, and LFA-1 ligand-intercellular adhesion molecule-1 (ICAM-1) were upregulated in IL-1β-stimulated HFLS-RA. We demonstrated that the total cell number of IL-1β-stimulated hUCMSCs adhering to IL-1β-stimulated HFLA-RA increased via LFA-1/ICAM-1 interaction. Direct co-culture of IL-1β-stimulated hUCMSCs with IL-1β-stimulated HFLS-RA increased the apoptosis of HFLS-RA. RA symptoms in the CIA mouse model improved after administration of IL-1β-stimulated hUCMSCs. In conclusion, IL-1β-stimulated hUCMSCs adhering to HFLS-RA occurred via LFA-1/ICAM-1 interaction, apoptosis of HFLS-RA was induced via TRAIL/DR4, DR5 contact, and RA symptoms and inflammation were significantly improved in a CIA mouse model. The results of this study suggest that IL-1β-stimulated hUCMSCs have therapeutic potential in RA treatment.

Clinical features and immune mechanisms directly linked to the altered liver function in patients with rheumatoid arthritis

BackgroundThe aim of this study was to explore the impact of arthritis on liver function using different approaches in vivo and in vitro. MethodsA cross-sectional study was performed on 330 non-obese/non-T2DM subjects: 180 RA patients, 50 NAFLD non-RA patients, and 100 healthy donors (HDs). A longitudinal study was conducted on 50 RA patients treated with methotrexate for six months. Clinical and laboratory parameters and markers of liver disease were collected. Mechanistic studies were carried out in both the CIA mouse model and hepatocytes treated with anti-citrullinated protein antibodies (ACPAs). ResultsRA patients have an increased risk of suffering from liver disease independent of obesity or T2DM. This risk was associated with factors such as insulin resistance, autoantibodies, inflammation, and component C3. Methotrexate treatment for six months was associated with liver abnormalities in those newly-diagnosed patients having CV risk factors. ACPAs induced a defective hepatocyte function, promoting IR and inflammation. The induction of arthritis in mice caused the infiltration of immune cells in the liver and increased inflammatory, apoptotic, and fibrotic processes. ConclusionRA patients may experience mild to moderate liver inflammation due to the infiltration of T, B cells, and macrophages, and the action of ACPAs. This is independent of obesity or diabetes and linked to systemic inflammation, and disease activity levels. The negative effects of methotrexate on liver function could be restricted to the concomitant presence of cardiovascular risk factors.

Koumine alleviates rheumatoid arthritis by regulating macrophage polarization.

The imbalance between M1-and M2-polarized macrophages is one of the major pathophysiological changes in RA. Therefore, targeted macrophage polarization may be an effective therapy for RA. Koumine, an alkaloid monomer with the highest content and low toxicity in Gelsemium elegans Benth., has the effect of treating RA by playing an immunomodulatory role by influencing various immune cells. However, whether koumine affects macrophage polarization in RA and the associated molecular mechanisms remain unknown. To investigate the mechanism of the anti-RA effect of koumine on macrophage polarization. The effect of koumine on macrophage polarization was investigated in vivo and in vitro. We first explored the effects of koumine on AIA rats and detected the levels of M1/M2 macrophage polarization markers in the spleen by western blotting. Then, we explored the regulatory effect of koumine on M1/M2 macrophage polarization and the effect on the PI3K/AKT signaling pathway in vitro. Finally, we verified the effects of koumine on macrophage polarization in CIA mice. We found that koumine alleviated symptoms, including relieving pain, reducing joint redness and swelling in AIA rats and restoring the M1/M2 macrophage balance in vivo. Interestingly, koumine had an inhibitory effect on both M1 and M2 macrophage polarization in vitro, but it had a stronger inhibitory effect on M1 macrophage. In a mixed polarization experiment, koumine mainly inhibited M1 macrophage polarization and had an inhibitory effect on the PI3K/AKT signaling pathway. Finally, we found that koumine had therapeutic effects on CIA mice, regulated macrophage polarization and inhibited the PI3K/AKT signaling pathway. Our results reveal that koumine regulates macrophage polarization through the PI3K/AKT signaling pathway. This may be one of the important mechanisms of its anti-RA effect, which provides a theoretical and scientific basis for the possible clinical application of koumine.