u-0126 and Osteoarthritis

Osteoarthritis (OA) is a chronic, age‑related osteoarthropathy that causes a considerable decline in quality of life, as well as economic losses due to its high incidence and poor prognosis. Mitogen‑activated protein kinases (MAPKs) regulate multiple cellular processes, including proliferation, differentiation and apoptosis, in certain diseases, such as cancer, diabetes and Alzheimer's disease. The present study aimed to investigate the regulatory role of the MAPK signaling pathway in early‑stage OA. A rabbit model of early‑stage OA was induced by treatment with the enzyme papain. U0126 [an extracellular signal‑regulated kinase (ERK) inhibitor], SP600125 [a Jun NH2‑terminal kinase (JNK) inhibitor] and SB203580 (a p38 inhibitor) were administered to the rabbits via intra‑articular injection. The severity of OA was assessed by histological examination using H&E, toluidine blue and safranin‑O/fast green staining, as well by analyzing the glycosaminoglycan (GAG) content and determining the OA Research Society International (OARSI) score. Western blotting was used to detect the protein expression levels of matrix metalloproteinase‑3 (MMP3), ERK, phosphorylated (p)‑ERK, p38, p‑p38, JNK, p‑JNK, Beclin1, UNC‑51‑like kinase 1 (ULK1) and microtubule‑associated protein 1 light chain 3 (LC3)II/I. U0126, SP600125 or SB203580 treatment significantly decreased the OARSI scores and significantly increased the GAG levels in the cartilaginous tissues of OA model rabbits. These results indicated that the MAPK inhibitors reduced the severity of OA‑induced injury at the early stage. Western blotting results demonstrated that MAPK inhibition significantly decreased the protein expression levels of MMP3 in OA cartilage. The protective effect of MAPK inhibitors in OA was mediated via the activation of autophagy, as demonstrated by the increased protein expression levels of LC3II/I, ULK1 and Beclin1. Overall, the data indicated that MAPK inhibitors may exert a protective effect against OA by restoring compromised autophagy. Furthermore, the present study suggested that MAPK inhibitors may represent a potential pharmacological strategy for treating OA in the future.

Topics: Animals; Anthracenes; Autophagy; Butadienes; Disease Models, Animal; Imidazoles; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Nitriles; Osteoarthritis; Protein Kinase Inhibitors; Pyridines; Rabbits; Severity of Illness Index

Osteoarthritis (OA) is a degenerative disease characterized by progressive articular cartilage destruction and joint marginal osteophyte formation with different degrees of synovitis. Docosahexaenoic acid (DHA) is an unsaturated fatty acid with anti-inflammatory, antioxidant, and antiapoptotic functions. In this study, the human chondrosarcoma cell line SW1353 was cultured in vitro, and an OA cell model was constructed with inflammatory factor IL-1β stimulation. After cells were treated with DHA, cell apoptosis was measured. Western blot assay was used to detect protein expression of apoptosis-related factors (Bax, Bcl-2, and cleaved caspase-3) and mitogen-activated protein kinase (MAPK) signaling pathway family members, including extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK), and p38 MAPK. Our results show that IL-1β promotes the apoptosis of SW1353 cells, increases the expression of Bax and cleaved caspase-3, and activates the MAPK signaling pathway. In contrast, DHA inhibits the expression of IL-1β, inhibits IL-1β-induced cell apoptosis, and has a certain inhibitory effect on the activation of the MAPK signaling pathway. When the MAPK signaling pathway is inhibited by its inhibitors, the effects of DHA on SW1353 cells are weakened. Thus, DHA enhances the apoptosis of SW1353 cells through the MAPK signaling pathway.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Bone Neoplasms; Butadienes; Cell Line, Tumor; Chondrosarcoma; Docosahexaenoic Acids; Drug Evaluation, Preclinical; Enzyme Activation; Enzyme Induction; Gene Expression Regulation, Neoplastic; Humans; Interleukin-1beta; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Nitriles; Osteoarthritis; Protein Kinase Inhibitors

We hypothesised that a potentially disease-modifying osteoarthritis (OA) drug such as hyaluronic acid (HA) given in combination with anti-inflammatory signalling agents such as mitogen-activated protein kinase kinase-extracellular signal-regulated kinase (MEK-ERK) signalling inhibitor (U0126) could result in additive or synergistic effects on preventing the degeneration of articular cartilage. Chondrocyte differentiation and hypertrophy were evaluated using human OA primary cells treated with either HA or U0126, or the combination of HA + U0126. Cartilage degeneration in menisectomy (MSX) induced rat OA model was investigated by intra-articular delivery of either HA or U0126, or the combination of HA + U0126. Histology, immunostaining, RT-qPCR, Western blotting and zymography were performed to assess the expression of cartilage matrix proteins and hypertrophic markers. Phosphorylated ERK (pERK)1/2-positive chondrocytes were significantly higher in OA samples compared with those in healthy control suggesting the pathological role of that pathway in OA. It was noted that HA + U0126 significantly reduced the levels of pERK, chondrocyte hypertrophic markers (COL10 and RUNX2) and degenerative markers (ADAMTs5 and MMP-13), however, increased the levels of chondrogenic markers (COL2) compared to untreated or the application of HA or U0126 alone. In agreement with the results in vitro, intra-articular delivery of HA + U0126 showed significant therapeutic improvement of cartilage in rat MSX OA model compared with untreated or the application of HA or U0126 alone. Our study suggests that the combination of HA and MEK-ERK inhibition has a synergistic effect on preventing cartilage degeneration.

Topics: Animals; Anti-Inflammatory Agents; Butadienes; Cartilage, Articular; Cells, Cultured; Chondrocytes; Drug Synergism; Humans; Hyaluronic Acid; Male; MAP Kinase Signaling System; Nitriles; Osteoarthritis; Phosphorylation; Protein Kinase Inhibitors; Rats; Signal Transduction

To investigate apoptosis of osteoarthritic (OA) chondrocytes stimulated with different inhibitors targeting tumor necrosis factor-alpha (TNFα) pathway, we isolated first passage chondrocytes from OA patients and then treated them with the inhibitors in combination with TNFα, and then collected the stimulated chondrocytes for Western blotting. Chondrocytes from OA patients expressed cleaved caspase-3 and PARP, suggesting apoptotic background. We here, validated that 10 ng/ml of TNFα couldn't induce more chondrocytes apoptosis. PI3K inhibitor LY294002 or NF-κB inhibitor CAPE, but not mTOR inhibitor rapamycin and MEK1/2 inhibitor U0126 in combination with TNFα could facilitate apoptosis. CAPE-induced more apoptosis could be explained by c-FLIP downregulation more than cIAP1 upregulation. And, we showed the first time that PI3K-NF-κB pathway, but not mTOR pathway could prevent chondrocytes apoptosis induced by a pro-apoptotic factor TNFα and call for attention while trying to inhibit NF-κB as a therapeutic target.

Topics: Aged; Apoptosis; Butadienes; Caffeic Acids; Caspase 3; Cells, Cultured; Chondrocytes; Chromones; Female; Humans; Middle Aged; Morpholines; NF-kappa B; Nitriles; Osteoarthritis; Phenylethyl Alcohol; Phosphoinositide-3 Kinase Inhibitors; Poly(ADP-ribose) Polymerases; Sirolimus; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha

To examine the relative importance of tumour necrosis factor-receptor 1 (TNF-R1) and TNF-R2 and their signalling pathways for pro-inflammatory and pro-destructive features of early-passage synovial fibroblasts (SFB) from rheumatoid arthritis (RA) and osteoarthritis (OA).. Cells were stimulated with tumour necrosis factor (TNF)alpha or agonistic anti-TNF-R1/TNF-R2 monoclonal antibodies. Phosphorylation of p38, ERK and JNK kinases was assessed by western blot; proliferation by bromodesoxyuridine incorporation; interleukin (IL)6, IL8, prostaglandin E(2) (PGE(2)) and matrix metalloproteinase (MMP)-1 secretion by ELISA; and MMP-3 secretion by western blot. Functional assays were performed with or without inhibition of p38 (SB203580), ERK (U0126) or JNK (SP600125).. In RA- and OA-SFB, TNFalpha-induced phosphorylation of p38, ERK or JNK was exclusively mediated by TNF-R1. Reduction of proliferation and induction of IL6, IL8 and MMP-1 were solely mediated by TNF-R1, whereas PGE(2) and MMP-3 secretion was mediated by both TNF-Rs. In general, inhibition of ERK or JNK did not significantly alter the TNFalpha influence on these effector molecules. In contrast, inhibition of p38 reversed TNFalpha effects on proliferation and IL6/PGE(2) secretion (but not on IL8 and MMP-3 secretion). The above effects were comparable in RA- and OA-SFB, except that TNFalpha-induced MMP-1 secretion was reversed by p38 inhibition only in OA-SFB.. In early-passage RA/OA-SFB, activation of MAPK cascades and pro-inflammatory/pro-destructive features by TNFalpha is predominantly mediated by TNF-R1 and, for proliferation and IL6/PGE(2) secretion, exclusively regulated by p38. Strikingly, RA-SFB are insensitive to p38 inhibition of MMP-1 secretion. This indicates a resistance of RA-SFB to the inhibition of pro-destructive functions and suggests underlying structural/functional alterations of the p38 pathway, which may contribute to the pathogenesis or therapeutic sensitivity of RA, or both.

Topics: Anthracenes; Arthritis, Rheumatoid; Blotting, Western; Butadienes; Case-Control Studies; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Humans; Imidazoles; MAP Kinase Kinase 4; Matrix Metalloproteinase 1; Matrix Metalloproteinase Inhibitors; Nitriles; Osteoarthritis; p38 Mitogen-Activated Protein Kinases; Pyridines; Receptors, Tumor Necrosis Factor, Type I; Receptors, Tumor Necrosis Factor, Type II; Signal Transduction; Statistics, Nonparametric; Synovial Membrane; Tumor Necrosis Factor-alpha