Geniposide prevents tumor growth by inhibiting colonic interleukin-1ß and monocyte chemoattractant protein-1 via down-regulated expression of cyclooxygenase-2 and thymocyte selection-associated high mobility box proteins TOX/TOX2 in azoxymethane/dextran sulfate sodium-treated mice.

Colon cancer was the second leading cause of cancer-related deaths in Japan in 2019. The effects of geniposide isolated from Gardenia jasminoides fructus (Rubiaceae) on the azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced growth of colon tumors and changes in interleukin (IL)-1 ß, monocyte chemoattractant protein (MCP)-1, IL-10, and programmed cell death-1 (PD-1) levels in the colon were investigated. The intraperitoneal administration of AOM (10 mg/kg) on days 0 and 27 induced colorectal carcinogenesis. Free access to 1% (w/v) DSS drinking water was given to mice on days 7-15, 32-33, and 35-38. Geniposide (30 and 100 mg/kg) was orally administered on days 1-16, discontinued for 11 days (days 16 to 26), and then administered again on days 27-41. Colonic levels of cytokines, chemokine, and PD-1 were measured using by enzyme-linked immunosorbent assay (ELISA). Increases in colorectal tumor numbers and areas were significantly inhibited by geniposide. In addition, geniposide (100 mg/kg) reduced colonic levels of IL-1 ß, MCP-1, PD-1 and IL-10 by 67.4, 57.2, 100%, and 100% respectively. Cyclooxygenase (COX)-2- and thymocyte selection high mobility group box proteins (TOX/TOX2)-positive cell numbers were significantly reduced by geniposide. Geniposide (30 and 100 mg/kg) decreased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) expressions in immunohistochemical analysis by 64.2 and 98.2%, respectively. Thus, the inhibitory effects of geniposide on colon tumor growth may be associated with reductions in the colonic levels of IL-1 ß, MCP-1, IL-10, and PD-1 via the down-regulated expression of COX-2 and TOX/TOX2 through the inhibition of Phospho-STAT3 expression (in vivo and in vitro).

Anethole ameliorates inflammation induced by monosodium urate in an acute gouty arthritis model via inhibiting TLRs/MyD88 pathway.

OBJECTIVE: To assess the effects of anethole on monosodium urate (MSU)-induced inflammatory response, investigate its role in acute gouty arthritis (AGA), and verify its molecular mechanism. METHODS: Hematoxylin and eosin staining assay and time-dependent detection of degree of ankle swelling were performed to assess the effects of anethole on joint injury in MSU-induced AGA mice. Enzyme-linked-immunosorbent serologic assay was performed to demonstrate the production levels of inflammatory factors (interleukin 1ß [IL-1ß], interleukin 6 [IL-6], interleukin 8 [IL-8], tumor necrosis factor α [TNF-α], and monocyte chemo-attractant protein-1 [MCP-1]) in MSU-induced AGA mice. Western blot assays were used to confirm the effects of anethole on oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activity and the activation of toll-like receptors (TLRs)-myeloid differentiation factor 88 (MyD88) pathway in MSU-induced AGA mice. RESULTS: We observed that a significant joint injury occurred in MSU-induced AGA mice. Anethole could alleviate the pathological injury of the synovium in MSU-induced AGA mice and suppressed ankle swelling. In addition, we observed that anethole could inhibit MSU-induced inflammatory response and inflammasome activation in MSU-induced AGA mice. Moreover, we discovered that anethole enabled to inhibit the activation of TLRs/MyD88 pathway in MSU-induced AGA mice. Our findings further confirmed that anethole contributed to the inhibitory effects on progression in MSU-induced AGA mice. CONCLUSION: It confirmed that anethole ameliorated the MSU-induced inflammatory response in AGA mice in vivo via inhibiting TLRs-MyD88 pathway.

Effect of baricitinib in regulating programmed death 1 and ligand programmed cell death ligand 1 through JAK/STAT pathway in psoriasis.

OBJECTIVES: Psoriasis is a chronic infectious skin disease triggered by an autoimmune process involving T-cell-mediated hyper-proliferation of keratinocytes. The objective of this study is to assess the modulation of programmed death 1 (PD-1) and its ligand programmed cell death ligand 1 (PD-L1) through JAK/STAT pathway during the development of a psoriasis-like disease by both in vitro and in vivo model. Baricitinib, a known inhibitor of JAK1 and JAK2, was used to study the impact on PD-1 and PD-L1. MATERIALS AND METHODS: Human peripheral blood mononuclear cells (PBMC) were stimulated with either anti-CD3/CD28 or PMA/Ionomycin, to modulate level of PD-1 and PD-L1 under psoriasis-like condition. Interferon-gamma (IFNγ) was used to treat HaCaT cells to mimic the diseased keratinocytes found in Psoriatic patients. Psoriasis was induced with Imiquimod (IMQ) in animal model to study the cross-talk between different cell types and pathways. RESULTS: Expression levels of PD-1 and PD-L1 in PBMC, and secretion of cytokines, namely tumor necrosis factor-α (TNFα), IFNγ, interleukin (IL)-6, and IL-1 ß, were down-regulated on treatment with baricitinib. Further, in IFNγ-treated HaCaT cells (keratinocytes) mRNA levels of KRT-17 and PD-L1 were up-regulated.). Interestingly, in IFNγ-treated HaCat cells baricitinib decreased the levels of inflammatory cytokines such as IL-1 ß, IL-6, and TNFα along with KRT-17 and PD-L1. On IFNγ-treatment. Data from both PBMC and HaCaT suggest an anti-inflammatory role for this compound. Accordingly, baricitinib was able to alleviate disease symptom in IMQ induce mice model of psoriasis. As a consequence of baricitinib treatment down-regulation of p-STAT3, PD- and PD-L1 expression levels were observed. CONCLUSION: This study demonstrates a crosstalk between JAK/STAT and PD-1/PD-L1 pathways. It also demonstrates that cytokines such as IFNγ and IL-17 are down-regulated by baricitinib. We believe decreased expressions of PD-1 and PD-L1 may be a consequence of baricitinib-induced down-regulation of IFNγ and IL-17. More importantly, our data from the acute model of psoriasis indicates that PD-L1 behaves as a T-cell-associated T-cell-associated surrogate activation marker rather than immunosuppressive marker in early phase of psoriasis. Therefore it does not exhibit a causal relationship to disease.

microRNA-4701-5p protects against interleukin-1ß induced human chondrocyte CHON-001 cells injury via modulating HMGA1.

BACKGROUND: miRNA-4701-5p has been reported to be a vital regulator in many diseases, including rheumatoid arthritis, and miRNA-4701-5p is evidenced to be participated in synovial invasion and joint destruction. In our report, we investigated the roles of miRNA-4701-5p in osteoarthritis (OA) and analyzed the molecular mechanism. METHODS: Interleukin-1ß (IL-1ß) was applied for stimulating human chondrocyte CHON-001 cells to establish an OA injury model. mRNA levels and protein expression were measured using qRT-PCR and western blot assay, respectively. The proliferation ability and cytotoxicity of CHON-001 cells were checked using MTT assay and lactate dehydrogenase activity. The inflammation of chondrocytes was accessed by the secretion levels of interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor (TNF)-α. The apoptosis of chondrocytes was determined by flow cytometry assay. Bioinformatics software Starbase v2.0 analyzed the functional binding sites between miRNA-4701-5p and HMGA1 and the interaction was further confirmed using dual luciferase reporter analysis. RESULTS: miRNA-4701-5p was down-regulated in the IL-1ß-stimulated chondrocytes and HMGA1 directly targeted miRNA-4701-5p. Up-regulation of miRNA-4701-5p could alleviate IL-1ß-treated CHON-001 cells inflammation and apoptosis, and reversed the cell proliferation decrease and cytotoxicity increase after IL-1ß treatment. Nevertheless, all the roles of miRNA-4701-5p overexpression in CHON-001 cells could be reversed by HMGA1 up-regulation. CONCLUSIONS: miRNA-4701-5p could alleviate the inflammatory injury of IL-1ß-treated CHON-001 cells via down-regulating HMGA1, indicating that miRNA-4701-5p/HMGA1 is a promising therapeutic target for OA.

The protective effects of dezocine on interleukin-1ß-induced inflammation, oxidative stress and apoptosis of human nucleus pulposus cells and the possible mechanisms.

Intervertebral disc degeneration (IDD) is a natural problem linked to the inflammation. We aimed to investigate the role of dezocine (DEZ) in the development of IDD. Human nucleus pulposus cells (HNPCs) induced by interleukin (IL)-1ß was used as a cellular model of IDD. After treatment with DEZ, HNPCs viability was evaluated with a CCK-8 assay. Then, the levels of inflammatory factors, including IL-6 and tumor necrosis factor-α (TNF-α), and oxidative stress-related markers, including reactive oxygen species (ROS), malondialdehyde (MDA) and reduced glutathione (GSH), were tested by RT-qPCR or kits. TUNEL staining was employed to detect cell apoptosis and Western blot was used to determine the expression of proteins related to inflammation, oxidative stress, apoptosis, endoplasmic reticulum stress (ERS) and MAPK signaling. Afterward, PMA, a MAPK signaling pathway agonist, was adopted for exploring the regulatory effects of DEZ on MAPK pathway. Results indicated that DEZ enhanced cell viability of HNPCs after IL-1ß exposure. DEZ alleviated the inflammation and oxidative stress, evidenced by decreased levels of IL-6, TNF-α, ROS, MDA, p-NF-κB p65, NF-κB p65 in nucleus, cox-2 and increased levels of NF-κB p65 in cytoplasm, GSH, SOD1 and SOD2. Moreover, DEZ notably inhibited IL-1ß-induced apoptosis of HNPCs. Furthermore, DEZ suppressed the levels of ERS-related proteins. The levels of related proteins in MAPK signaling including p-P38 and p-ERK1/2 were remarkably reduced after DEZ administration. By contrast, PMA crippled the impacts of DEZ on inflammation, oxidative stress and apoptosis of HNPCs induced by IL-1ß. Collectively, DEZ ameliorates IL-1ß-induced HNPCs injury via inhibiting MAPK signaling.

The protective effects of etomidate against interleukin-1ß (IL-1ß)-induced oxidative stress, extracellular matrix alteration and cellular senescence in chondrocytes.

Osteoarthritis (OA) is a common chronic inflammatory disease associated with aging. Etomidate is an intravenous anesthetic with profound antioxidant and anti-inflammatory effects. We speculated that etomidate might exert a beneficial effect on OA. Herein, we explored the effects of etomidate on interleukin-1ß (IL-1ß)- induced chondrocytes. Our results prove that etomidate ameliorated the IL-1ß-induced oxidative stress in C28/12 chondrocytes by decreasing and increasing the reactive oxygen species (ROS) and glutathione peroxidase (GPx) levels, respectively. Etomidate prevented the IL-1ß-induced increase in the expressions of matrix metalloproteinase-3 (MMP-3) and matrix metalloproteinase-13 (MMP-13) in C28/I2 chondrocytes at both mRNA and protein levels. It also caused a significant reduction in the percentage of senescence-associated-ß-galactosidase (SA-ß-Gal)-stained chondrocytes, while inducing elevated telomerase activity in IL-1ß-treated C28/I2 chondrocytes. The expression levels of senescence regulators, plasminogen activator inhibitor-1 (PAI-1) and p16, were also inhibited by etomidate in IL-1ß-treated C28/I2 chondrocytes. In addition, etomidate caused the activation of Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), along with upregulated expression levels of phosphorylated AMPKα and phosphorylated acetyl-Co A carboxylase (ACC). Moreover, blockage of AMPK using compound C abolished the protective effects of etomidate on IL-1ß-challenged C28/I2 chondrocytes. Taken together, these results demonstrate that etomidate protected C28/I2 chondrocytes from IL-1ß-induced oxidative stress, ECM degradation, and cellular senescence via activating AMPK signaling.

High-Molecular-Weight Hyaluronic Acid Inhibits IL-1ß-Induced Synovial Inflammation and Macrophage Polarization through the GRP78-NF-κB Signaling Pathway.

Recent evidence has suggested that synovial inflammation and macrophage polarization were involved in the pathogenesis of osteoarthritis (OA). Additionally, high-molecular-weight hyaluronic acid (HMW-HA) was often used clinically to treat OA. GRP78, an endoplasmic reticulum (ER) stress chaperone, was suggested to contribute to the hyperplasia of synovial cells in OA. However, it was still unclear whether HMW-HA affected macrophage polarization through GRP78. Therefore, we aimed to identify the effect of HMW-HA in primary synovial cells and macrophage polarization and to investigate the role of GRP78 signaling. We used IL-1ß to treat primary synoviocytes to mimic OA, and then treated them with HMW-HA. We also collected conditioned medium (CM) to culture THP-1 macrophages and examine the changes in the phenotype. IL-1ß increased the expression of GRP78, NF-κB (p65 phosphorylation), IL-6, and PGE2 in primary synoviocytes, accompanied by an increased macrophage M1/M2 polarization. GRP78 knockdown significantly reversed the expression of IL-1ß-induced GRP78-related downstream molecules and macrophage polarization. HMW-HA with GRP78 knockdown had additive effects in an IL-1ß culture. Finally, the synovial fluid from OA patients revealed significantly decreased IL-6 and PGE2 levels after the HMW-HA treatment. Our study elucidated a new form of signal transduction for HMW-HA-mediated protection against synovial inflammation and macrophage polarization and highlighted the involvement of the GRP78-NF-κB signaling pathway.

Montelukast attenuates interleukin IL-1ß-induced oxidative stress and apoptosis in chondrocytes by inhibiting CYSLTR1 (Cysteinyl Leukotriene Receptor 1) and activating KLF2 (Kruppel Like Factor 2).

Montelukast is a cysteinyl leukotriene receptor 1 (CysLTR1) antagonist widely used to suppress the inflammatory response in asthma and allergic rhinitis. This study aimed to investigate the potential impacts of montelukast on osteoarthritis (OA) progression. To determine the role of montelukast in OA, the expression of CysLTR1 was first examined by quantitative reverse transcription PCR (RT-qPCR) and western blot in IL-1ß-induced ATDC5 cells treated with or without montelukast. Subsequently, the impacts of montelukast on cell viability and oxidative stress were measured by Cell-Counting-Kit-8 (CCK-8), commercial kits and western blot. Oxidative stress-related protein expressions were determined by western blot analysis in Il-1ß-induced ATDC5 cells. Cell apoptosis and cartilage degradation were examined by TdT-mediated dUTP Nick-End Labeling (TUNEL) assay, western blot and RT-qPCR. KLF2 expression was measured in IL-1ß-induced ATDC5 cells treated with montelukast. After interference with small interfering RNA (siRNA)-KLF2 in ATDC5 cells, the loss-of-function assays were also performed in same ways. CysLTR1 expression was elevated in IL-1ß-induced ATDC5 cells but inhibited significantly by montelukast. Montelukast attenuated the oxidative stress and apoptosis, improved cell viability. Moreover, montelukast enhanced KLF2 expression. After transfected with siRNA-KLF2, montelukast attenuated cell injury, oxidative stress, apoptosis and cartilage degradation in IL-1ß-induced ATDC5 cells by activating KLF2.In summary, this work elaborates the evidence that montelukast could attenuate oxidative stress and apoptosis in IL-1ß-induced chondrocytes by inhibiting CysLTR1 and activating KLF2, which can guide the therapeutic strategies of montelukast for OA development in the future.

Ultra-Low Dose Cytokines in Rheumatoid Arthritis, Three Birds with One Stone as the Rationale of the 2LARTH® Micro-Immunotherapy Treatment.

Tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) are two cytokines involved in the perpetuation of the chronic inflammation state characterizing rheumatoid arthritis (RA). Significant advances in the treatment of this pathology have been made over the past ten years, partially through the development of anti-TNF and anti-IL-1 therapies. However, major side effects still persist and new alternative therapies should be considered. The formulation of the micro-immunotherapy medicine (MIM) 2LARTH® uses ultra-low doses (ULD) of TNF-α, IL-1ß, and IL-2, in association with other immune factors, to gently restore the body's homeostasis. The first part of this review aims at delineating the pivotal roles played by IL-1ß and TNF-α in RA physiopathology, leading to the development of anti-TNF and anti-IL-1 therapeutic agents. In a second part, an emphasis will be made on explaining the rationale of using multiple therapeutic targets, including both IL-1ß and TNF-α in 2LARTH® medicine. Particular attention will be paid to the ULD of those two main pro-inflammatory factors in order to counteract their overexpression through the lens of their molecular implication in RA pathogenesis.

Protective Effects of a Hyaluronan-Binding Peptide (P15-1) on Mesenchymal Stem Cells in an Inflammatory Environment.

Mesenchymal stem cells (MSCs) obtained from various sources, including bone marrow, have been proposed as a therapeutic strategy for the improvement of tissue repair/regeneration, including the repair of cartilage defects or lesions. Often the highly inflammatory environment after injury or during diseases, however, greatly diminishes the therapeutic and reparative effectiveness of MSCs. Therefore, the identification of novel factors that can protect MSCs against an inflammatory environment may enhance the effectiveness of these cells in repairing tissues, such as articular cartilage. In this study, we investigated whether a peptide (P15-1) that binds to hyaluronan (HA), a major component of the extracellular matrix of cartilage, protects bone-marrow-derived MSCs (BMSCs) in an inflammatory environment. The results showed that P15-1 reduced the mRNA levels of catabolic and inflammatory markers in interleukin-1beta (IL-1ß)-treated human BMSCs. In addition, P15-1 enhanced the attachment of BMSCs to HA-coated tissue culture dishes and stimulated the chondrogenic differentiation of the multipotential murine C3H/10T1/2 MSC line in a micromass culture. In conclusion, our findings suggest that P15-1 may increase the capacity of BMSCs to repair cartilage via the protection of these cells in an inflammatory environment and the stimulation of their attachment to an HA-containing matrix and chondrogenic differentiation.

Identification and validation of key long non-coding RNAs in resveratrol protect against IL-1ß-treated chondrocytes via integrated bioinformatic analysis.

BACKGROUND: Long non-coding RNAs (lncRNAs) participate in regulation of gene transcription, but little is known about the correlation among resveratrol and lncRNAs. This study aimed to identify and validate the key lncRNAs in resveratrol protect against IL-1ß-treated chondrocytes. METHODS: In this experiment, high-throughput sequencing technique was performed to identify the differentially expressed lncRNAs, miRNAs, and mRNAs between IL-1ß-treated chondrocytes with or not resveratrol. Moreover, gene ontology and KEGG pathway of the differentially expressed genes were carried out by R software. Then, lncRNA-miRNA-mRNA network was constructed by Cytoscape software. Venn diagram was performed to identify the potentially target miRNAs of LINC00654. Then, real-time polymerase chain reaction (RT-PCR) was performed to validate the most significantly differentially expressed lncRNAs. RESULTS: Totally, 1016 differentially expressed lncRNAs were identified (493 downregulated) between control and resveratrol-treated chondrocytes. Totally, 75 differentially expressed miRNAs were identified (downregulated = 54, upregulated = 21). Totally, 3308 differentially expressed miRNAs were identified (downregulated = 1715, upregulated = 1593). GO (up) were as follows: skin development, response to organophosphorus. GO (down) mainly included visual perception, single fertilization, and sensory perception of smell. KEGG (up) were as follows: TNF signaling pathway and TGF-beta signaling pathway. KEGG (down) were as follows: viral protein interaction with cytokine and cytokine receptor. We identified that LINC00654 and OGFRL1 were upregulated in resveratrol-treated chondrocytes. However, miR-210-5p was downregulated in resveratrol-treated chondrocytes. CONCLUSION: In sum, the present study for the first time detected the differential expressed lncRNAs involved in resveratrol-treated chondrocytes via employing bioinformatic methods.

Susceptibility of cyclin-dependent kinase inhibitor 1-deficient mice to rheumatoid arthritis arising from interleukin-1ß-induced inflammation.

We recently reported that cyclin-dependent kinase inhibitor 1 (p21) deficiency induces osteoarthritis susceptibility. Here, we determined the mechanism underlying the effect of p21 in synovial and cartilage tissues in RA. The knee joints of p21-knockout (p21-/-) (n = 16) and wild type C57BL/6 (p21+/+) mice (n = 16) served as in vivo models of collagen antibody-induced arthritis (CAIA). Arthritis severity was evaluated by immunological and histological analyses. The response of p21 small-interfering RNA (siRNA)-treated human RA FLSs (n = 5 per group) to interleukin (IL)-1ß stimulation was determined in vitro. Arthritis scores were higher in p21-/- mice than in p21+/+ mice. More severe synovitis, earlier loss of Safranin-O staining, and cartilage destruction were observed in p21-/- mice compared to p21+/+ mice. p21-/- mice expressed higher levels of IL-1ß, TNF-α, F4/80, CD86, p-IKKα/ß, and matrix metalloproteinases (MMPs) in cartilage and synovial tissues via IL-1ß-induced NF-kB signaling. IL-1ß stimulation significantly increased IL-6, IL-8, and MMP expression, and enhanced IKKα/ß and IκBα phosphorylation in human FLSs. p21-deficient CAIA mice are susceptible to RA phenotype alterations, including joint cartilage destruction and severe synovitis. Therefore, p21 may have a regulatory role in inflammatory cytokine production including IL-1ß, IL-6, and TNF-α.

Circ-SPG11 knockdown hampers IL-1ß-induced osteoarthritis progression via targeting miR-337-3p/ADAMTS5.

BACKGROUND: Osteoarthritis (OA) is responsible for the impotent disability in old people. Circular RNA (circRNA) has been reported to be related to the development of diseases. The lack of research on the role of circRNA spastic paraplegia 11 (circ-SPG11) results in conducting this study. METHODS: The expression of circ-SPG11, microRNA-337-3p (miR-337-3p), and aggrecanases like a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) mRNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot was used to measure the protein expression of extracellular matrix (ECM) degradation-related markers and ADAMTS5. Ribonuclease R (RNase R) was applied to test the stability of circ-SPG11 in CHON-001 cells. The viability, apoptosis, TNF-α and IL-6 production were determined by cell counting kit-8 (CCK-8) assay, flow cytometry assay, and enzyme-linked immunosorbent assay (ELISA), respectively. Meanwhile, the interaction between miR-337-3p and circ-SPG11 or ADAMTS5 was respectively predicted by Circinteractome or Starbase2.0, which was further verified by dual-luciferase reporter system and RNA binding protein immunoprecipitation (RIP) assay. RESULTS: Circ-SPG11 and ADAMTS5 were upregulated and miR-337-3p was downregulated in OA tissues and OA model cells. Circ-SPG11 knockdown allayed interleukin 1ß (IL-1ß)-induced restraint in viability and promotion in apoptosis, TNF-α, and IL-6 generation and ECM degradation in CHON-001 cells. Anti-miR-337-3p or ADAMTS5 overexpression correspondingly reversed si-circ-SPG11 or miR-337-3p overexpression-mediated facilitation in viability, and inhibition in apoptosis, TNF-α and IL-6 generation and ECM degradation in OA model cells. Moreover, anti-miR-337-3p ameliorated si-circ-SPG11-mediated inhibition in ADAMTS5 mRNA and protein expression in OA model cells. CONCLUSION: Circ-SPG11 facilitated OA development via regulating miR-337-3p/ADAMTS5 axis. This finding might contribute to the improvement of OA therapy.

MiR-24-3p attenuates IL-1ß-induced chondrocyte injury associated with osteoarthritis by targeting BCL2L12.

BACKGROUND: MiR-24-3p has been reported to be involved in an osteoarthritis (OA)-resembling environment. However, the functional role and underlying mechanism of miR-24-3p in chondrocyte injury associated with OA remains unknown. METHODS: The expression of miR-24-3p was determined using reverse transcription quantitative PCR analysis in OA cases and control patients, as well as IL-1ß-stimulated chondrocyte cell line CHON-001. The cell viability was analyzed by CCK-8 assay. Apoptosis status was assessed by caspase-3 activity detection. The pro-inflammatory cytokines (TNF-α and IL-18) were determined using ELISA assay. The association between miR-24-3p and B cell leukemia 2-like 12 (BCL2L12) was confirmed by luciferase reporter assay. RESULTS: We first observed that miR-24-3p expression level was lower in the OA cases than in the control patients and IL-1ß decreased the expression of miR-24-3p in the chondrocyte CHON-001. Functionally, overexpression of miR-24-3p significantly attenuated IL-1ß-induced chondrocyte injury, as reflected by increased cell viability, decreased caspase-3 activity, and pro-inflammatory cytokines (TNF-α and IL-18). Western blot analysis showed that overexpression of miR-24-3p weakened IL-1ß-induced cartilage degradation, as reflected by reduction of MMP13 (Matrix Metalloproteinase-13) and ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs-5) protein expression, as well as markedly elevation of COL2A1 (collagen type II). Importantly, BCL2L12 was demonstrated to be a target of miR-24-3p. BCL2L12 knockdown imitated, while overexpression significantly abrogated the protective effects of miR-24-3p against IL-1ß-induced chondrocyte injury. CONCLUSIONS: In conclusion, our work provides important insight into targeting miR-24-3p/BCL2L12 axis in OA therapy.

Investigation of synovial fluid lubricants and inflammatory cytokines in the horse: a comparison of recombinant equine interleukin 1 beta-induced synovitis and joint lavage models.

BACKGROUND: Lameness is a debilitating condition in equine athletes that leads to more performance limitation and loss of use than any other medical condition. There are a limited number of non-terminal experimental models that can be used to study early inflammatory and synovial fluid biophysical changes that occur in the equine joint. Here, we compare the well-established carpal IL-1ß-induced synovitis model to a tarsal intra-articular lavage model, focusing on serial changes in synovial fluid inflammatory cytokines/chemokines and the synovial fluid lubricating molecules lubricin/proteoglycan 4 and hyaluronic acid. The objectives of this study were to evaluate clinical signs; synovial membrane and synovial fluid inflammation; and synovial fluid lubricants and biophysical properties in response to carpal IL-1ß synovitis and tarsal intra-articular lavage. RESULTS: Hyaluronic acid (HA) concentrations, especially high molecular weight HA, and synovial fluid viscosity decreased after both synovitis and lavage interventions. Synovial fluid lubricin concentrations increased 17-20-fold for both synovitis and lavage models, with similar changes in both affected and contralateral joints, suggesting that repeated arthrocentesis alone resulted in elevated synovial fluid lubricin concentrations. Synovitis resulted in a more severe inflammatory response based on clinical signs (temperature, heart rate, respiratory rate, lameness and joint effusion) and clinicopathological and biochemical parameters (white blood cell count, total protein, prostaglandin E2, sulfated glycosaminoglycans, tumor necrosis factor-α and CC chemokine ligands - 2, - 3, - 5 and - 11) as compared to lavage. CONCLUSIONS: Synovial fluid lubricin increased in response to IL-1ß synovitis and joint lavage but also as a result of repeated arthrocentesis. Frequent repeated arthrocentesis is associated with inflammatory changes, including increased sulfated glycosaminoglycan concentrations and decreased hyaluronic acid concentrations. Synovitis results in more significant inflammatory changes than joint lavage. Our data suggests that synovial fluid lubricin, TNF-α, CCL2, CCL3, CCL5, CCL11 and sGAG may be useful biomarkers for synovitis and post-lavage joint inflammation. Caution should be exercised when performing repeated arthrocentesis clinically or in experimental studies due to the inflammatory response and loss of HA and synovial fluid viscosity.

Proinflammatory Cytokine Interleukin 1ß Disrupts ß-cell Circadian Clock Function and Regulation of Insulin Secretion.

Intrinsic ß-cell circadian clocks are important regulators of insulin secretion and overall glucose homeostasis. Whether the circadian clock in ß-cells is perturbed following exposure to prodiabetogenic stressors such as proinflammatory cytokines, and whether these perturbations are featured during the development of diabetes, remains unknown. To address this, we examined the effects of cytokine-mediated inflammation common to the pathophysiology of diabetes, on the physiological and molecular regulation of the ß-cell circadian clock. Specifically, we provide evidence that the key diabetogenic cytokine IL-1ß disrupts functionality of the ß-cell circadian clock and impairs circadian regulation of glucose-stimulated insulin secretion. The deleterious effects of IL-1ß on the circadian clock were attributed to impaired expression of key circadian transcription factor Bmal1, and its regulator, the NAD-dependent deacetylase, Sirtuin 1 (SIRT1). Moreover, we also identified that Type 2 diabetes in humans is associated with reduced immunoreactivity of ß-cell BMAL1 and SIRT1, suggestive of a potential causative link between islet inflammation, circadian clock disruption, and ß-cell failure. These data suggest that the circadian clock in ß-cells is perturbed following exposure to proinflammatory stressors and highlights the potential for therapeutic targeting of the circadian system for treatment for ß-cell failure in diabetes.

Interleukin-1ß-induced pancreatitis promotes pancreatic ductal adenocarcinoma via B lymphocyte-mediated immune suppression.

OBJECTIVE: Long-standing chronic pancreatitis is an established risk factor for pancreatic ductal adenocarcinoma (PDAC). Interleukin-1ß (IL-1ß) has been associated in PDAC with shorter survival. We employed murine models to investigate the mechanisms by which IL-1ß and chronic pancreatitis might contribute to PDAC progression. DESIGN: We crossed LSL-Kras+/G12D;Pdx1-Cre (KC) mice with transgenic mice overexpressing IL-1ß to generate KC-IL1ß mice, and followed them longitudinally. We used pancreatic 3D in vitro culture to assess acinar-to-ductal metaplasia formation. Immune cells were analysed by flow cytometry and immunohistochemical staining. B lymphocytes were adoptively transferred or depleted in Kras-mutant mice. B-cell infiltration was analysed in human PDAC samples. RESULTS: KC-IL1ß mice developed PDAC with liver metastases. IL-1ß treatment increased Kras+/G12D pancreatic spheroid formation. CXCL13 expression and B lymphocyte infiltration were increased in KC-IL1ß pancreata. Adoptive transfer of B lymphocytes from KC-IL1ß mice promoted tumour formation, while depletion of B cells prevented tumour progression in KC-IL1ß mice. B cells isolated from KC-IL1ß mice had much higher expression of PD-L1, more regulatory B cells, impaired CD8+ T cell activity and promoted tumorigenesis. IL-35 was increased in the KC-IL1ß pancreata, and depletion of IL-35 decreased the number of PD-L1+ B cells. Finally, in human PDAC samples, patients with PDAC with higher B-cell infiltration within tumours showed significantly shorter survival. CONCLUSION: We show here that IL-1ß promotes tumorigenesis in part by inducing an expansion of immune-suppressive B cells. These findings point to the growing significance of B suppressor cells in pancreatic tumorigenesis.

Maslinic acid prevents IL-1ß-induced inflammatory response in osteoarthritis via PI3K/AKT/NF-κB pathways.

Osteoarthritis (OA) is a degenerative joint disease characterized by destruction of articular cartilage. The inflammatory response is the most important factor affecting the disease process. As interleukin-1ß (IL-1ß) stimulates several key mediators in the inflammatory response, it plays a major role in the pathogenesis of OA. Maslinic acid (MA) is a natural compound distributed in olive fruit. Previous studies have found that maslinic acid has an inhibitory effect on inflammation, but its specific role in the progression of OA disease has not been studied so far. In this study, we aim to assess the protective effect of MA on OA progression by in vitro and in vivo experiments. Our results indicate that, in IL-1ß-induced inflammatory response, MA is effective in attenuating some major inflammatory mediators such as nitric oxide (NO) and prostaglandin E2, and inhibits the expression of IL-6, inducible nitric oxide synthase, cyclooxygenase-2, and tumor necrosis factor-α (TNF-α) in a concentration-dependent manner. Also, MA downregulated the expression levels of thrombospondin motif 5 (ADAMTS5) and matrix metalloproteinase 13 in chondrocytes, resulting in reduced degradation of its extracellular matrix. Mechanistically, MA exhibits an anti-inflammatory effect by inactivating the PI3K/AKT/NF-κB pathway. In vivo, the protective effect of MA on OA development can be detected in a surgically induced mouse OA model. In summary, these findings suggest that MA can be used as a safe and effective potential OA therapeutic strategy.

Resveratrol alleviates the interleukin-1ß-induced chondrocytes injury through the NF-κB signaling pathway.

BACKGROUND: Osteoarthritis (OA) is a regular age-related disease that affects millions of people. Resveratrol (RSV) is a flavonoid with a stilbene structure with different pharmacological effects. The purpose of the experiment was to evaluate the protective role of RSV against the human OA chondrocyte injury induced by interleukin-1ß (IL-1ß). METHODS: Chondrocytes were isolated from OA patients and identified by type II collagen, safranin O staining, and toluidine blue staining. Differentially expressed genes in chondrocytes treated RSV were identified by RNA sequencing. Kyoto encyclopedia of genes and genomes (KEGG) pathway as well as gene ontology (GO) were further conducted through Metascape online tool. A cell counting kit-8 (CCK-8) assay was applied to discover the viability of chondrocytes (6, 12, 24, and 48 µM). Many genes associated with inflammation and matrix degradation are evaluated by real-time PCR (RT-PCR) as well as western blot (WB). The mechanism of RSV for protecting IL-1ß induced chondrocytes injury was further measured through immunofluorescence and WB assays. RESULTS: A total of 845 differentially expressed genes (upregulated = 499, downregulated = 346) were found. These differentially expressed genes mainly enriched into negative regulation of catabolic process, autophagy, and cellular catabolic process, intrinsic apoptotic, apoptotic, and regulation of apoptotic signaling pathway, cellular response to abiotic stimulus, external stimuli, stress, and radiation. These differentially expressed genes were obviously enriched in NF-kB signaling pathway. RSV at the concentration of 48 µM markedly weakened the viability of the cells after 24 h of treatment (87% vs 100%, P < 0.05). No obvious difference was observed between the 6, 12, and 24 µM groups (106% vs 100%, 104% vs 100%, 103% vs 100%, P > 0.05). RSV (24 µM) also markedly depressed the levels of PGE2 and NO induced by IL-1ß by 25% and 29% respectively (P < 0.05). Our experiment pointed out that RSV could dramatically inhibit the inflammatory response induced by IL-1ß, including the MMP-13, MMP-3, and MMP-1 in human OA chondrocytes by 50%, 35%, and 33% respectively. On the other hand, RSV inhibited cyclooxygenase-2 (COX-2), matrix metalloproteinase-1 (MMP-1), MMP-3, MMP-13, and inducible nitric oxide synthase (iNOs) expression (P < 0.05), while increased collagen-II and aggrecan levels (P < 0.05). From a mechanistic perspective, RSV inhibited the degradation of IκB-α as well as the activation of nuclear factor-kappa B (NF-κB) induced by IL-1ß. CONCLUSION: In summary, RSV regulates the signaling pathway of NF-κB, thus inhibiting inflammation and matrix degradation in chondrocytes. More studies should be focused on the treatment efficacy of RSV for OA in vivo.

Macroscale mesenchymal condensation to study cytokine-driven cellular and matrix-related changes during cartilage degradation.

Understanding the pathophysiological processes of cartilage degradation requires adequate model systems to develop therapeutic strategies towards osteoarthritis (OA). Although different in vitro or in vivo models have been described, further comprehensive approaches are needed to study specific disease aspects. This study aimed to combine in vitro and in silico modeling based on a tissue-engineering approach using mesenchymal condensation to mimic cytokine-induced cellular and matrix-related changes during cartilage degradation. Thus, scaffold-free cartilage-like constructs (SFCCs) were produced based on self-organization of mesenchymal stromal cells (mesenchymal condensation) and (i) characterized regarding their cellular and matrix composition or secondly (ii) treated with interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNFα) for 3 weeks to simulate OA-related matrix degradation. In addition, an existing mathematical model based on partial differential equations was optimized and transferred to the underlying settings to simulate the distribution of IL-1ß, type II collagen degradation and cell number reduction. By combining in vitro and in silico methods, we aimed to develop a valid, efficient alternative approach to examine and predict disease progression and effects of new therapeutics.