Regulation of circADAMTS6-miR-324-5p-PIK3R3 ceRNA pathway may be a novel mechanism of IL-1ß-induced osteoarthritic chondrocytes.

INTRODUCTION: A disintegrin and metallopeptidase with thrombospondin type 1 motif 6 (ADAMTS6)-derived circular RNA (circADAMTS6; hsa_circ_0008667) is a novel regulator in interleukin (IL)-1ß-induced apoptosis of human chondrocytes (HCs). Here, we planned to probe into its role and mechanism underlying HCs injury in osteoarthritis. MATERIALS AND METHODS: Real time-quantitative PCR and immunoblotting estimated the abundance of RNA and protein, respectively. Cell proliferation and apoptosis were measured by WST-8, EdU, fluorescein isothiocyanate, and caspase3/7 activity assays. Levels of inflammatory cytokines (IL-6 and tumor necrosis factor-α), apoptosis-related proteins (Bcl-2 and Bcl-2-associated X protein), extracellular matrix (ECM)-related proteins (matrix metalloproteinase-13 and collagen type II alpha-1), and PI3K/AKT/mTOR signaling pathway-related proteins (AKT, mTOR, phosphorylated-AKT, and phosphorylated-mTOR) were evaluated by enzyme-linked immunosorbent assays and immunoblotting. Target relationship was confirmed by dual-luciferase reporter, Argonaute-2 immunoprecipitation and RNA pull-down assays. RESULTS: Abundances of circADAMTS6 and phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) were underexpressed, and microRNA (miR)-324-5p was elevated in human osteoarthritic tissues and IL-1ß-induced HCs. Overexpressing circADAMTS6 and inhibiting miR-324-5p enhanced proliferation and ECM synthesis, but suppressed apoptosis and inflammatory response in IL-1ß-challenged HCs. Besides, silencing circADAMTS6 caused similar effects of IL-1ß stress on HCs. Mechanically, there was a direct interaction between miR-324-5p and circADAMTS6 or PIK3R3, and IL-1ß-induced activation of PI3K/AKT/mTOR signaling pathway was suppressed by circADAMTS6 overexpression and miR-324-5p silencing. Furthermore, counteractive effects of miR-324-5p upregulation on circADAMTS6 overexpression and PIK3R3 knockdown on miR-324-5p silencing were observed. CONCLUSION: CircADAMTS6-miR-324-5p-PIK3R3 axis might participate in IL-1ß-induced HCs dysfunction via competing endogenous RNA mechanism and the PI3K/AKT/mTOR signaling pathway.

Cyanidin attenuates the apoptosis of rat nucleus pulposus cells and the degeneration of intervertebral disc via the JAK2/STAT3 signal pathway in vitro and in vivo.

CONTEXT: Cyanidin has been shown to have therapeutic potential in osteoarthritis. However, it is unclear whether cyanidin prevents the progression of intervertebral disc degeneration (IVDD). OBJECTIVE: This study evaluates the effects of cyanidin on IVDD in vitro and in vivo. MATERIALS AND METHODS: Nucleus pulposus cells (NPCs) isolated from lumbar IVD of 4-week-old male Sprague-Dawley (SD) rats were exposed to 20 ng/mL IL-1ß, and then treated with different doses (0-120 µM) of cyanidin for 24 h. SD rats were classified into three groups (n = 8) and treated as follows: control (normal saline), IVDD (vehicle), IVDD + cyanidin (50 mg/kg). Cyanidin was administered intraperitoneally for 8 weeks. RESULTS: The IC50 of cyanidin for NPCs was 94.78 µM, and cyanidin had no toxicity at concentrations up to 500 mg/kg in SD rats. Cyanidin inhibited the apoptosis of NPCs induced by IL-1ß (12.73 ± 0.61% vs. 18.54 ± 0.60%), promoted collagen II (0.82-fold) and aggrecan (0.81-fold) expression, while reducing MMP-13 (1.02-fold) and ADAMTS-5 (1.40-fold) expression. Cyanidin increased the formation of autophagosomes in IL-1ß-induced NPCs, and promoted LC3II/LC3I (0.83-fold) and beclin-1 (0.85-fold) expression, which could be reversed by chloroquine. Cyanidin inhibited the phosphorylation of JAK2 (0.47-fold) and STAT3 (0.53-fold) in IL-1ß-induced NPCs. The effects of cyanidin could be enhanced by AG490. Furthermore, cyanidin mitigated disc degeneration in IVDD rats in vivo. DISCUSSION AND CONCLUSIONS: Cyanidin improved the function of NPCs in IVDD by regulating the JAK2/STAT3 pathway, which may provide a novel alternative strategy for IVDD. The mechanism of cyanidin improving IVDD still needs further work for in-depth investigation.

Intrathecal interleukin-1ß decreases sigma-1 receptor expression in spinal astrocytes in a murine model of neuropathic pain.

The sigma-1 receptor (Sig-1R) plays an important role in spinal pain transmission by increasing phosphorylation of the N-methyl-D-aspartate (NMDA) receptor GluN1 subunit (pGluN1). As a result Sig-1R has been suggested as a novel therapeutic target for prevention of chronic pain. Here we investigated whether interleukin-1ß (IL-1ß) modulates the expression of the Sig-1R in spinal astrocytes during the early phase of nerve injury, and whether this modulation affects spinal pGluN1 expression and the development of neuropathic pain following chronic constriction injury (CCI) of the sciatic nerve. Repeated intrathecal (i.t.) administration of IL-1ß from days 0-3 post-surgery significantly reduced the increased pGluN1 expression at the Ser896 and Ser897 sites in the ipsilateral spinal cord, as well as, the development of mechanical allodynia and thermal hyperalgesia in the ipsilateral hind paw of CCI mice, which were restored by co-administration of IL-1 receptor antagonist with IL-1ß. Sciatic nerve injury increased the expression of Sig-1R in astrocytes of the ipsilateral spinal cord, and this increase was suppressed by i.t. administration of IL-1ß. Agonistic stimulation of the Sig-1R with PRE084 restored pGluN1 expression and the development of mechanical allodynia that were originally suppressed by IL-1ß in CCI mice. Collectively these results demonstrate that IL-1ß administration during the induction phase of neuropathic pain produces an analgesic effect on neuropathic pain development by controlling the expression of Sig-1R in spinal astrocytes.

Inhibition of neutral sphingomyelinase 2 reduces extracellular vesicle release from neurons, oligodendrocytes, and activated microglial cells following acute brain injury.

Extracellular Vesicles (EVs) are implicated in the spread of pathogenic proteinsin a growing number of neurological diseases. Given this, there is rising interest in developing inhibitors of Neutral Sphingomyelinase 2 (nSMase2), an enzyme critical in EV biogenesis. Our group recently discovered phenyl(R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)carbamate (PDDC), the first potent, selective, orally-available, and brain-penetrable nSMase2 inhibitor, capable of dose-dependently reducing EVs release in vitro and in vivo. Herein, using multiplexed Surface Plasmon Resonance imaging (SPRi), we evaluated which brain cell-derived EVs were affected by PDDC following acute brain injury. Mice were fed PDDC-containing chow at doses which gave steady PDDC brain exposures exceeding its nSMase2 IC50. Mice were then administered an intra-striatal IL-1ß injection and two hours later plasma and brain were collected. IL-1ß injection significantly increased striatal nSMase2 activity which was completely normalized by PDDC. Using SPRi, we found that IL-1ß-induced injury selectively increased plasma levels of CD171 + and PLP1 + EVs; this EV increase was normalized by PDDC. In contrast, GLAST1 + EVs were unchanged by IL-1ß or PDDC. IL-1ß injection selectively increased EVs released from activated versus non-activated microglia, indicated by the CD11b+/IB4 + ratio. The increase in EVs from CD11b + microglia was dramatically attenuated with PDDC. Taken together, our data demonstrate that following acute injury, brain nSMase2 activity is elevated. EVs released from neurons, oligodendrocytes, and activated microglial are increased in plasma and inhibition of nSMase2 with PDDC reduced these IL-1ß-induced changes implicating nSMase2 inhibition as a therapeutic target for acute brain injury.

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.

The pro-inflammatory and anti-inflammatory role of hyaluronic acid in endometriosis.

OBJECTIVE: Endometriosis is a bothersome disease affected women worldwide, the mechanism of disease development is still under investigation. Several inflammatory responses after clinical hyaluronic acid (HA) use were reported. Cyclooxygenase (COX)-2 mediated inflammation pathway is involved in the pathogenesis of endometriosis. Thus, we tried to investigate the inflammatory role of hyaluronic acid in endometriosis. MATERIALS AND METHODS: Peritoneal fluid was collected in endometriosis and disease-free patients for the measurement of HA. Endometriotic stromal cells were treated with IL-1ß and HA and expression of COX-2 was evaluated. Mice model of endometriosis was established and treated with fluid or gel form of HA. Endometriotic lesion size and weight were recorded and level of COX-2 was evaluated by immunohistochemistry staining. RESULTS: The level of HA in the peritoneal fluid had no statistically significant difference between normal, early and advanced stage endometriosis patients. The overexpression of COX-2 protein was detected when treating endometriotic stromal cell with HA in the presence of IL-1ß (P < 0.001). The endometriotic lesion size was reduced in mice model when treated with higher concentration gel form HA. It further showed less proportion of strong COX-2 expression compare of gel form HA to fluid form treatment in COX-2 expression score of endometriosis lesion. CONCLUSION: Both proinflammatory evidence, elevated COX-2 expression, and anti-inflammatory result, reduced endometriosis lesion size and COX-2 expression score, were noted in our study after treating HA in in vivo and in vitro models. We hypothesized it is possible that HA may induce an acute proinflammatory response followed by chronic anti-inflammatory reaction in the formation of endometriosis.

Chondroprotective and antiarthritic effects of galangin in osteoarthritis: An in vitro and in vivo study.

Osteoarthritis (OA) is a common degenerative joint disease blamed for pain and disability in the elderly. Galangin (GAL) is a natural flavonoid that exhibits anti-inflammatory properties in various inflammation diseases. However, the role of GAL in OA remains unclear. In this study, we investigate the role of GAL in the progress and development of OA in vitro and vivo. The results showed that IL-1ß exposure resulted in increased expression of iNOS, COX-2, MMP1, MMP3, MMP13 and ADAMTS5 in rat chondrocytes. However, co-treatment with GAL significantly decreased theses inflammatory cytokines and catabolic factors expression. In addition, GAL reduced IL-1ß-induced degradation of collagen II and aggrecan in chondrocytes. Furthermore, GAL significantly suppressed IL-1ß-induced Akt phosphorylation and NF-κB activation in rat chondrocytes. In vivo, intra-articular injection of GAL could also reduce the cartilage degradation in the ACLT rat model. This study reveals galangin may act as a promising novel agent in the treatment of OA.

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.

LncRNA RMRP knockdown promotes proliferation and inhibits apoptosis in osteoarthritis chondrocytes by miR-206/CDK9 axis.

The etiology of osteoarthritis (OA) has been discussed widely, but the molecular mechanisms beneath OA aggravation have not yet been investigated in detail. This study focused on the role of lncRNA RMRP (RMRP) on OA progression. We found that the expression of RMRP was significantly increased in cartilage tissues of patients with OA. CCK-8 and colony formation assays showed that RMRP knockdown promoted proliferation of chondrocytes treated with IL-1ß. Flow cytometry and caspase-3 activity analysis indicated that RMRP silence inhibited apoptosis of chondrocytes treated with IL-1ß. Moreover, luciferase reporter, RNA pull-down and RIP assays showed that RMRP competing with miR-206. Additionally, CDK9 acted as a direct target of miR-206. Moreover, rescue assays indicated that miR-206 inhibitor or pcDNA-CDK9 reversed the effects of RMRP suppression on the proliferation and apoptosis of chondrocytes. Taken together, our results indicated that RMRP knockdown could promote proliferation and inhibit apoptosis in OA chondrocytes via the miR-206/CDK9 axis.

Maternal administration of probiotics promotes gut development in mouse offsprings.

Necrotizing enterocolitis is the most common gastrointestinal disorder in premature neonates. This disease is characterized by massive epithelial necrosis, gut barrier dysfunction and improper mucosal defense development. Studies have shown that probiotic administration can decrease NEC incidence and mortality. The proposed mechanisms of probiotics for the prevention of NEC are: promotion of intestinal development; improved barrier function through decreased apoptosis and improved mucin production; decreased expression of proinflammatory cytokines IL6, IL8, and TNFα, and modulation of microbiota dysbiosis in preterm infants. However, reported sepsis in the immunocompromised preterm host has deterred routine prophylactic administration of probiotics in the neonatal intensive care unit. We hypothesize that maternal administration of probiotics to pregnant mouse dams can recapitulate the beneficial effects observed in neonates fed with probiotics directly. We exposed pregnant mice to the probiotics and monitored the changes in the developing intestines of the offspring. Pregnant mice were fed daily with the probiotics Lactobacillus acidophilus and Bifidobacterium infantis (LB) from embryonic day15 to 2-week-old postnatally. Intraperitoneal administration of IL-1ß in the pups was used to model proinflammatory insults. Sera were collected at 2 weeks of age and evaluated for inflammatory cytokines by enzyme-linked-immunosorbent-assay and gut permeability by Fluorescein isothiocyanate-dextran tracer assay. Ileal tissues were collected for the evaluation of apoptosis and proliferation of the intestinal epithelium; as well as mucin and tight junction integrity at mucosal surface by immunofluorescent staining. We find that maternal LB exposure facilitated intestinal epithelial cell differentiation, prevented loss of mucin and preserved the intestinal integrity and barrier function and decreased serum levels of IL-1ß, TNF-α and IL-6 in the preweaned offsprings. in LB exposed pups. We demonstrate that maternal probiotic supplementation promotes gut maturation in developing offspring. This is potentially a safe alternative therapy to induce intestinal maturation and prevent prematurity-associated neonatal disorders.

Barrier Protection and Recovery Effects of Gut Commensal Bacteria on Differentiated Intestinal Epithelial Cells In Vitro.

Alterations in the gut microbiota composition play a crucial role in the pathogenesis of inflammatory bowel disease (IBD) as specific commensal bacterial species are underrepresented in the microbiota of IBD patients. In this study, we examined the therapeutic potential of three commensal bacterial species, Faecalibacterium prausnitzii (F. prausnitzii), Roseburia intestinalis (R. intestinalis) and Bacteroides faecis (B. faecis) in an in vitro model of intestinal inflammation, by using differentiated Caco-2 and HT29-MTX cells, stimulated with a pro-inflammatory cocktail consisting of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNFα), interferon-γ (IFNγ), and lipopolysaccharide (LPS). Results obtained in this work demonstrated that all three bacterial species are able to recover the impairment of the epithelial barrier function induced by the inflammatory stimulus, as determined by an amelioration of the transepithelial electrical resistance (TEER) and the paracellular permeability of the cell monolayer. Moreover, inflammatory stimulus increased claudin-2 expression and decreased occludin expression were improved in the cells treated with commensal bacteria. Furthermore, the commensals were able to counteract the increased release of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) induced by the inflammatory stimulus. These findings indicated that F. prausnitzii, R. intestinalis and B. faecis improve the epithelial barrier integrity and limit inflammatory responses.

IL-22 promotes tumor growth of breast cancer cells in mice.

Increased interleukin-22 (IL-22) level was reported to associate with progression of breast cancer. Regulation of IL-22 in breast cancer still needs to be elucidated. We assessed the effect of giving IL-22 in tumor growth of mice inoculated with 4T1, MCF7 and MDA-MB-231 breast cancer cells. IL-22-producing cells were analyzed in tumor tissues. We also analyzed the impact of giving IL-1ß and IL-23 on IL-22 levels in tumor tissues. Giving exogenous IL-22 increased tumor size and intra-tumor Ki-67-positive cells in vivo. IL-22 increased phosphorylated STAT3 level and proliferation of breast cancer cells in vitro, an effect blocked by a STAT3-inhibitor stattic. Endogenous IL-22 mRNA level was up-regulated in tumor tissue, compared with normal mammary tissue. Innate lymphoid cell group 3 (ILC3) is a major producer of IL-22 in 4T1 tumor. Giving IL-1ß and/or IL-23 increased cell proliferation in 4T1 tumor, which was reversed by concurrent use of an IL-22 neutralization antibody. IL-1ß and IL-23 increased levels of IL-22 mRNA and IL-22-producing ILC3 in 4T1 tumor. Our findings suggest a mechanism for how IL-22 regulates tumor growth in breast cancer, and indicate blocking IL-22 function might reduce IL-1ß- and IL-23-induced tumor progression of breast cancer.

Effects of adipokine administration to the hypothalamic preoptic area on body temperature in rats.

The effects of adipokine administration to the hypothalamic preoptic area (POA), which is one of the body temperature (BT) regulation centers in the central nervous system, on BT were investigated in male Wistar rats. BT was measured in conscious rats using telemetry. Insulin-like growth factor-1 (IGF-1), interleukin-1ß (IL-1ß), monocyte chemoattractant protein-1 and lipocalin-2 produced hyperthermia, and the effects induced by IL-1ß (25 ng) and IGF-1 (5 µg) were sustainable and remarkable. IL-6 did not show any significant effect. The IGF-1-induced effect was inhibited by pretreatment with IGF binding protein 3 (IGFBP3) or NVP-AEW541 (NVP, a selective inhibitor of type 1 IGF receptor tyrosine kinase, IGF1R TK). NVP-induced inhibition was observed only in the early phase of IGF-1-induced hyperthermia. In addition, IGF-1 increased the IL-1ß concentration in the microdialysate of POA perfusion, but did not increase the IL-1ß concentration in the plasma or the PGE2 concentration in the microdialysate. These findings suggested that IGF-1 produced hyperthermia, which was mediated, at least a part, through an increased IL-1ß concentration after activation of IGF1R TK in the POA, and the IGF-IGFBP system possibly participates in BT homeostasis in the POA.

Acute Inflammation Alters Brain Energy Metabolism in Mice and Humans: Role in Suppressed Spontaneous Activity, Impaired Cognition, and Delirium.

Systemic infection triggers a spectrum of metabolic and behavioral changes, collectively termed sickness behavior, which while adaptive, can affect mood and cognition. In vulnerable individuals, acute illness can also produce profound, maladaptive, cognitive dysfunction including delirium, but our understanding of delirium pathophysiology remains limited. Here, we used bacterial lipopolysaccharide (LPS) in female C57BL/6J mice and acute hip fracture in humans to address whether disrupted energy metabolism contributes to inflammation-induced behavioral and cognitive changes. LPS (250 µg/kg) induced hypoglycemia, which was mimicked by interleukin (IL)-1ß (25 µg/kg) but not prevented in IL-1RI-/- mice, nor by IL-1 receptor antagonist (IL-1RA; 10 mg/kg). LPS suppression of locomotor activity correlated with blood glucose concentrations, was mitigated by exogenous glucose (2 g/kg), and was exacerbated by 2-deoxyglucose (2-DG) glycolytic inhibition, despite preventing IL-1ß synthesis. Using the ME7 model of chronic neurodegeneration in female mice, to examine vulnerability of the diseased brain to acute stressors, we showed that LPS (100 µg/kg) produced acute cognitive dysfunction, selectively in those animals. These acute cognitive impairments were mimicked by insulin (11.5 IU/kg) and mitigated by glucose, demonstrating that acutely reduced glucose metabolism impairs cognition selectively in the vulnerable brain. To test whether these acute changes might predict altered carbohydrate metabolism during delirium, we assessed glycolytic metabolite levels in CSF in humans during inflammatory trauma-induced delirium. Hip fracture patients showed elevated CSF lactate and pyruvate during delirium, consistent with acutely altered brain energy metabolism. Collectively, the data suggest that disruption of energy metabolism drives behavioral and cognitive consequences of acute systemic inflammation.SIGNIFICANCE STATEMENT Acute systemic inflammation alters behavior and produces disproportionate effects, such as delirium, in vulnerable individuals. Delirium has serious short and long-term sequelae but mechanisms remain unclear. Here, we show that both LPS and interleukin (IL)-1ß trigger hypoglycemia, reduce CSF glucose, and suppress spontaneous activity. Exogenous glucose mitigates these outcomes. Equivalent hypoglycemia, induced by lipopolysaccharide (LPS) or insulin, was sufficient to trigger cognitive impairment selectively in animals with existing neurodegeneration and glucose also mitigated those impairments. Patient CSF from inflammatory trauma-induced delirium also shows altered brain carbohydrate metabolism. The data suggest that the degenerating brain is exquisitely sensitive to acute behavioral and cognitive consequences of disrupted energy metabolism. Thus "bioenergetic stress" drives systemic inflammation-induced dysfunction. Elucidating this may offer routes to mitigating delirium.

PHF23 negatively regulates the autophagy of chondrocytes in osteoarthritis.

AIM: Osteoarthritis (OA) is the main cause of disability and joint replacement surgery in the elderly. As a crucial cell survival mechanism, autophagy has been reported to decrease in OA. PHF23 is a new autophagy inhibitor which was first reported by us previously. This study aimed to explore the anti-autophagic mechanism of PHF23 to make it a possible therapeutic target of OA. MAIN METHOD: Lentiviral vectors specific to PHF23 were used on chondrocytes (C28/I2) to establish PHF23 overexpressed or knockdown stable cell strains. Interleukin (IL)-1ß (10 ng/mL) and chloroquine (CQ, 25 uM) were used as an inducer of OA and inhibitor of lysosome, respectively. Autophagy was evaluated by autophagosome formation using transmission electron microscopy (TEM) and western blot analysis of P62 and LC3B on different groups of cells. Effects of PHF23 on OA were evaluated by collagen II immunofluorescent staining and western blot analysis of OA-associated proteins MMP13 and ADAMTS5. Effects of PHF23 on AMPK and mTOR/S6K pathways and mitophagy were determined by western blot analysis. KEY FINDINGS: Knockdown of PHF23 enhanced IL-1ß-induced autophagy, while overexpression of PHF23 exerted the opposite effect. Knockdown of PHF23 protected chondrocytes against IL-1ß-induced OA by decreasing the levels of OA-associated proteins and increasing expression of Collagen II. Knockdown of PHF23 also increased mitophagy level and altered the phosphorylation levels of AMPK, mTOR, and S6K. SIGNIFICANCE: PHF23 downregulates autophagy, mitophagy in IL-1ß-induced OA-like chondrocytes and alters the activities of AMPK and mTOR/S6K, which suggests that PHF23 may be a possible therapeutic target for OA.

Long-chain non-coding RNA HOTAIR promotes the progression of osteoarthritis via sponging miR-20b/PTEN axis.

AIMS: Cumulative evidence suggests that long-chain non-coding RNA (lncRNA) is involved in the pathogenesis of osteoarthritis (OA). The present study aimed to explore the regulatory role and related mechanisms of HOX transcript antisense intergenic RNA (HOTAIR) in OA. MATERIAL AND METHODS: The OA mouse model was constructed by the medial meniscus (DMM) method, and Interleukin (IL)-1ß-induced chondrocytes were used to simulate OA in vitro. KEY FINDINGS: Results found that HOTAIR was significantly up-regulated in articular cartilage tissues of OA mice and IL-1ß-induced chondrocytes, accompanied by down-regulation of miR-20b and increased expression of the phosphatase and tensin homolog (PTEN). HOTAIR silencing improved cartilage tissue damage in OA mice, and promoted the expression of collagen II and aggrecan in cartilage tissue, while inhibited the expression of matrix metalloproteinases (MMP)-13 and ADAMTS-5. Overexpression of HOTAIR inhibited the proliferation of IL-1ß-induced chondrocytes and promoted apoptosis and extracellular matrix (ECM) degradation, whereas the effect of HOTAIR knockdown was reversed. Bioinformatics software and luciferase reporter experiments confirmed that HOTAIR could negatively regulate miR-20b, and PTEN was a target gene of miR-20b. An increase in PTEN expression induced by HOTAIR overexpression could be reversed by the introduction of miR-20b mimic. HOTAIR overexpression significantly reversed miR-20 mimic-mediated inhibition of apoptosis and ECM degradation in IL-1ß-induced chondrocytes, whereas the introduction of si-HOTAIR eliminated anti-miR-20b-mediated apoptosis and ECM degradation. SIGNIFICANCE: HOTAIR can participate in OA by promoting chondrocyte apoptosis and ECM degradation, which may be related to its targeted regulation of miR-20b/PTEN axis.

Anti-Inflammatory Effect of Carprofen Is Enhanced by Avocado/Soybean Unsaponifiables, Glucosamine and Chondroitin Sulfate Combination in Chondrocyte Microcarrier Spinner Culture.

OBJECTIVE: Osteoarthritis is a painful, chronic joint disease affecting man and animals with no known curative therapies. Palliative nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used but they cause adverse side effects prompting the search for safer alternatives. To address this need, we evaluated the anti-inflammatory activity of avocado/soybean unsaponifiables (ASU), glucosamine (GLU), and chondroitin sulfate (CS) with or without the NSAID carprofen. DESIGN: Canine chondrocytes were propagated in microcarrier spinner culture and incubated with (1) control medium, (2) ASU (8.3 µg/mL) + GLU (11 µg/mL) + CS (20 µg/mL) combination for 24 hours; and/or carprofen (40 ng/mL). Cultures were next incubated with control medium alone or IL-1ß (10 ng/mL) for another 24 hours. Production of PGE2, IL-6, IL-8, and MCP-1 (also known as CCL-2) were measured by ELISA. RESULTS: Chondrocytes proliferated in microcarrier spinner culture and produced type II collagen and aggrecan. Stimulation with IL-1ß induced significant increases in PGE2, IL-6, IL-8, and MCP-1 production. The increases in production were suppressed by carprofen as well as [ASU+GLU+CS]. The combination of carprofen and [ASU+GLU+CS] reduced PGE2 production significantly more than either preparation alone. The inhibitory effect of carprofen on IL-6, IL-8, and MCP-1 production was significantly less than that of [ASU+GLU+CS], whereas the combination did not reduce the production of these molecules significantly more than [ASU+GLU+CS] alone. CONCLUSIONS: The potentiating effect of [ASU+GLU+CS] on low-dose carprofen was identified in chondrocyte microcarrier spinner cultures. Our results suggest that the combination of low-dose NSAIDs like carprofen with [ASU+GLU+CS] could offer a safe, effective management for joint pain.

Role of astrocytic GABAergic system on inflammatory cytokine-induced anxiety-like behavior.

Recent studies have shown that not only neurons but astrocytes contain a considerable amount of γ-aminobutyric acid (GABA), which can be released and activate the receptors responsive to GABA. The purpose of this study is to test whether gliotransmitters from astrocytes may play a role in etiology of anxiety symptoms. Intracerebroventricular (i.c.v.) infusion of interleukin-1ß (IL-1ß), one of potent inflammatory cytokines, induced anxiety-like behaviors and activated the glial fibrillary acidic protein (GFAP) in the paraventricular nucleus (PVN) of the hypothalamus. Pretreatment with astrocytes toxin, l-α-aminoadipate (L-AAA) reduced anxiety-like behaviors and the GFAP expression in the PVN. Intraparaventricular nucleus (iPVN) infusion of IL-1ß produced markedly anxiety-like behaviors and increased release of GABA from astrocytes. However, treatment of glial cell inhibitor, L-AAA or blocker of Bestrophin-1 (Best1), 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) markedly inactivated astrocytes and also reduced the anxiety-like behaviors. Treatment of L-AAA or NPPB decreased IL-1ß-induced gliotransmitter GABA release measured by in vivo microdialysis. These results suggest that selective inhibition of astrocytes or astocytic GABA release in the PVN may serve as an effective therapeutic strategy for treating anxiety and affective disorders.

Protein kinase C-ε contributes to a chronic inhibitory effect of IL-1ß on voltage-gated sodium channels in mice with febrile seizure.

This study aimed to understand the role of Interleukin-1ß in mouse febrile seizures. To investigate the chronic effects of raised Interleukin-1ß on seizures, the sodium currents of hippocampal neurons were recorded by whole-cell voltage clamp. Interleukin-1ß inhibited sodium currents in mouse hippocampal neurons and verified that protein kinase C epsilon contributed to the effect of Interleukin-1ß exposure. The inhibitory effect was also identified in neurons from a protein kinase C epsilon null mutant mouse. Action potentials were recorded using a ramp depolarizing current. Peak spike depolarization was significantly reduced by Interleukin-1ß treatment, and was abolished following the administration of a protein kinase C epsilon inhibitor, εV1-2. However, neither Interleukin-1ß nor εV1-2 had any significant effect on spike threshold. Interleukin-1ß reduced the amplitude of action potentials due to its inhibitory effect on sodium channels. This is hypothesised to decrease the release of presynaptic transmitters of neuroexcitability, thus exerting a neuroprotective role in excitotoxicity. To ascertain the role of protein kinase C epsilon on febrile seizures in vivo, a heated water-bath model was used to identify susceptible mice. It was found that protein kinase C epsilon reduced susceptibility to, and frequency of, febrile seizure onset. This may be related to the neuroprotective effect of Interleukin-1ß on hippocampal neurons.

Exposure to systemic and intrauterine inflammation leads to decreased pup survival via different placental mechanisms.

PROBLEM: Exposure to systemic maternal inflammation (i.e., maternal sepsis, influenza, human immunodeficiency virus, or pyelonephritis) and intrauterine (IU) inflammation (i.e., chorioamnionitis or preterm labor) have been associated with adverse perinatal sequelae. Whether systemic and localized inflammation leading to adverse outcomes have similar placental mechanisms remain unclear. METHOD OF STUDY: We conducted a study by murine modeling systemic and localized IU inflammation with injections of either intraperitoneal (IP) or IU interleukin-1ß (IL-1ß) and compared fetoplacental hemodynamic changes, cytokine/chemokine expression, and fetal loss. RESULTS: IU IL-1ß exposure reduced offspring survival by 31.1% and IP IL-1ß exposure by 34.5% when compared with control pups. Despite this similar outcome in offspring survival, Doppler analysis revealed a stark difference: IU group displayed worsened fetoplacental hemodynamic changes while no differences were found between IP and control groups. While both IU and IP groups had increases in pro-inflammatory cytokines and chemokines, specific gene expression trends differed between the two groups, once again highlighting their mechanistic differences. CONCLUSION: While both IP and IU IL-1ß exposure similarly affected pup survival, only IU inflammation resulted in fetoplacental hemodynamic changes. We speculate that exposure to maternal systemic and IU inflammation plays a key role in fetal injury by utilizing different placental inflammatory pathways and mechanisms.