BACKGROUND: Knee osteoarthritis (OA) is still challenging to prevent or treat. Enhanced endoplasmic reticulum (ER) stress and increased pyroptosis in chondrocytes may be responsible for cartilage degeneration. This study aims to investigate the effect of ER stress on chondrocyte pyroptosis and the upstream regulatory mechanisms, which have rarely been reported. METHODS: The expression of the histone methyltransferase enhancer of zeste homolog 2 (EZH2), microRNA-142-3p (miR-142-3p), and high mobility group box 1 (HMGB1) and the levels of ER stress, pyroptosis, and metabolic markers in normal and OA chondrocytes were investigated by western blotting, quantitative polymerase chain reaction, immunohistochemistry, fluorescence in situ hybridization, fluorescein amidite-tyrosine-valine-alanine-aspartic acid-fluoromethyl ketone (FAM-YVAD-FMK)/Hoechst 33342/propidium iodide (PI) staining, lactate dehydrogenase (LDH) release assays, and cell viability assessments. The effects of EZH2, miR-142-3p, and HMGB1 on ER stress and pyroptosis and the hierarchical regulatory relationship between them were analyzed by chromatin immunoprecipitation, luciferase reporters, gain/loss-of-function assays, and rescue assays in interleukin (IL)-1β-induced OA chondrocytes. The mechanistic contribution of EZH2, miR-142-3p, and HMGB1 to chondrocyte ER stress and pyroptosis and therapeutic prospects were validated radiologically, histologically, and immunohistochemically in surgically induced OA rats. RESULTS: Increased EZH2 and HMGB1, decreased miR-142-3p, enhanced ER stress, and activated pyroptosis in chondrocytes were associated with OA occurrence and progression. EZH2 and HMGB1 exacerbated and miR-142-3p alleviated ER stress and pyroptosis in OA chondrocytes. EZH2 transcriptionally silenced miR-142-3p via H3K27 trimethylation, and miR-142-3p posttranscriptionally silenced HMGB1 by targeting the 3'-UTR of the HMGB1 gene. Moreover, ER stress mediated the effects of EZH2, miR-142-3p, and HMGB1 on chondrocyte pyroptosis. In vivo experiments mechanistically validated the hierarchical regulatory relationship between EZH2, miR-142-3p, and HMGB1 and their effects on chondrocyte ER stress and pyroptosis. CONCLUSIONS A novel EZH2/miR-142-3p/HMGB1 axis mediates chondrocyte pyroptosis and cartilage degeneration by regulating ER stress in OA, contributing novel mechanistic insights into OA pathogenesis and providing potential targets for future therapeutic research.
Enhancer of Zeste Homolog 2 Protein/genetics* ; Osteoarthritis, Knee/pathology* ; Chondrocytes/metabolism* ; Pyroptosis/physiology* ; HMGB1 Protein/genetics* ; MicroRNAs/metabolism* ; Endoplasmic Reticulum Stress/genetics* ; Humans ; Animals ; Rats ; Male ; Rats, Sprague-Dawley ; Middle Aged

OBJECTIVES: To investigate the effect of nuclear protein (Nupr) 1 on the pathological progression of osteoarthritis and its relationship with ferroptosis of chondrocytes. METHODS: Chondrocytes from mouse knees were divided into small interfering RNA (siRNA) control group, small interfering RNA targeting Nupr1 (siNupr1) group, siRNA control+IL-1β group (siRNA control interference for 24 h followed by 10 ng/mL IL-1β) and siNupr1+IL-1β group (siNupr1 interference for 24 h followed by 10 ng/mL IL-1β). The protein and mRNA expressions of Nupr1 were detected by Western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell proliferation viabilities were measured using the cell counting kit-8 method. The levels of ferrous ions were detected by FerroOrange staining. Lipid peroxidation levels were detected by C11-BODIPY-591 fluorescence imaging. The contents of malondialdehyde (MDA) and glutathione (GSH) were detected by enzyme-linked immunosorbent assay. The protein expressions of acyl-CoA synthetase long-chain family (ACSL) 4, P53, glutathione peroxidase (GPX) 4 and solute carrier family 7 member 11 gene (SLC7A11) were detected by Western blotting. The osteoarthritis model was constructed by destabilization of the medial meniscus (DMM) surgery in 7-week-old male C57BL/6J mice. The mice were randomly divided into four groups with 10 animals in each group: sham surgery (Sham)+adeno-associated virus serotype 5 (AAV5)-short hairpin RNA (shRNA) control group, Sham+AAV5-shRNA control targeting Nupr1 (shNupr1) group, DMM+AAV5-shRNA control group, and DMM+AAV5-shNupr1 group. Hematoxylin and eosin staining and Safranin O-Fast Green staining were used to observe the morphological changes in cartilage tissue. The Osteoarthritis Research Society International (OARSI) osteoarthritis cartilage histopathology assessment system was used to evaluate the degree of cartilage degeneration in mice. The mRNA expressions of matrix metallopeptidase (MMP) 13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 5, cyclooxy-genase (COX) 2, and GPX4 were detected by qRT-PCR. RESULTS: In vitro experiments showed that knocking down Nupr1 alleviated the decrease of chondrocyte proliferation activity induced by IL-1β, reduced iron accumulation in mouse chondrocytes, lowered lipid peroxidation, downregulated ACSL4 and P53 protein expression and upregulated GPX4 and SLC7A11 protein expression (all P<0.01), thereby inhibiting ferroptosis in mouse chondrocytes. Meanwhile, in vivo animal experiments demonstrated that knocking down Nupr1 delayed the degeneration of articular cartilage in osteoarthritis mice, improved the OARSI score, slowed down the degradation of the extracellular matrix in osteoarthritis cartilage, and reduced the expression of the key ferroptosis regulator GPX4 (all P<0.01). CONCLUSIONS Knockdown of Nupr1 can delay the pathological progression of osteoarthritis through inhibiting ferroptosis in mouse chondrocytes.
Animals ; Ferroptosis ; Mice ; Chondrocytes/metabolism* ; Osteoarthritis/pathology* ; RNA, Small Interfering/genetics* ; Basic Helix-Loop-Helix Transcription Factors/genetics* ; Interleukin-1beta/metabolism* ; Phospholipid Hydroperoxide Glutathione Peroxidase/genetics* ; Coenzyme A Ligases/genetics* ; Tumor Suppressor Protein p53/metabolism* ; Mice, Inbred C57BL ; DNA-Binding Proteins ; Neoplasm Proteins ; Amino Acid Transport System y+ ; Nuclear Receptor Subfamily 1, Group D, Member 1

To investigate the mechanism by which Cangxi Tongbi Capsules promote chondrocyte autophagy to inhibit knee osteoarthritis(KOA) progression by regulating the circRNA＿0008365/miR-1271/p38 mitogen-activated protein kinase(MAPK) pathway. The cell and animal models of KOA were established and intervened with Cangxi Tongbi Capsules, si-circRNA＿0008365, si-NC, and Cangxi Tongbi Capsules combined with si-circRNA＿0008365. Flow cytometry and transmission electron microscopy were employed to determine the level of apoptosis and observe autophagosomes, respectively. Western blot was employed to reveal the changes in the protein levels of microtubule-associated protein light chain 3(LC3)Ⅱ/Ⅰ, Beclin-1, selective autophagy junction protein p62/sequestosome 1, collagen Ⅱ, a disintegrin and metalloproteinase with thrombospondin motifs 5(ADAMTS-5), and p38 MAPK. The mRNA levels of circRNA＿0008365, miR-1271, collagen Ⅱ, and ADAMTS-5 were determined by qRT-PCR. Hematoxylin-eosin staining was employed to reveal the pathological changes of the cartilage tissue of the knee, and enzyme-linked immunosorbent assay to measure the levels of interleukin-1β(IL-1β) and tumor necrosis factor-alpha(TNF-α). The chondrocytes treated with IL-1β showed down-regulated expression of circRNA＿0008365, up-regulated expression of miR-1271 and p38 MAPK, lowered autophagy level, increased apoptosis rate, and accelerated catabolism of extracellular matrix. The intervention with Cangxi Tongbi Capsules up-regulated the expression of circRNA＿0008365, down-regulated the expression of miR-1271 and p38 MAPK, increased the autophagy level, decreased the apoptosis rate, and weakened the catabolism of extracellular matrix. However, the effect of Cangxi Tongbi Capsules was suppressed after interfering with circRNA＿0008365. The in vivo experiments showed that Cangxi Tongbi Capsules dose-dependently inhibited the p38 MAPK pathway, enhanced chondrocyte autophagy, and mitigated articular cartilage damage and inflammatory response, thereby inhibiting the progression of KOA in rats. This study indicated that Cangxi Tongbi Capsules promoted chondrocyte autophagy by regulating the circRNA＿0008365/miR-1271/p38 MAPK pathway to inhibit the development of KOA.
Rats ; Animals ; Chondrocytes ; Osteoarthritis, Knee/pathology* ; RNA, Circular/pharmacology* ; p38 Mitogen-Activated Protein Kinases/metabolism* ; MicroRNAs/metabolism* ; Apoptosis ; Autophagy/genetics* ; Collagen/metabolism*

Articular cartilage is a connective tissue consisting of a specialized extracellular matrix (ECM) that dominates the bulk of its wet and dry weight. Type II collagen and aggrecan are the main ECM proteins in cartilage. However, little attention has been paid to less abundant molecular components, especially minor collagens, including type IV, VI, IX, X, XI, XII, XIII, and XIV, etc. Although accounting for only a small fraction of the mature matrix, these minor collagens not only play essential structural roles in the mechanical properties, organization, and shape of articular cartilage, but also fulfil specific biological functions. Genetic studies of these minor collagens have revealed that they are associated with multiple connective tissue diseases, especially degenerative joint disease. The progressive destruction of cartilage involves the degradation of matrix constituents including these minor collagens. The generation and release of fragmented molecules could generate novel biochemical markers with the capacity to monitor disease progression, facilitate drug development and add to the existing toolbox for in vitro studies, preclinical research and clinical trials.
Aggrecans ; chemistry ; genetics ; metabolism ; Animals ; Biomarkers ; metabolism ; Cartilage, Articular ; chemistry ; metabolism ; pathology ; Collagen ; chemistry ; classification ; genetics ; metabolism ; Extracellular Matrix Proteins ; chemistry ; genetics ; metabolism ; Gene Expression ; Humans ; Osteoarthritis ; diagnosis ; genetics ; metabolism ; pathology ; Protein Isoforms ; chemistry ; classification ; genetics ; metabolism

To observe the influence of matrix metalloproteinases-2 (MMP-2), monocyte chemoattractant protein-1 (MCP-1), CD47, L-selectin and advanced oxidation proteinproducts (AOPP) in osteoarthritis and the intervention of curcumin.A total of 20 male C57BL/6 mice (10.05-15.00 g) were randomly divided into control group, OA group, Cur25 group and Cur50 group (intraperitoneal injected 25 μmol/L or 50 μmol/L of curcumin everyday after modeling). After 4 weeks treatment, we observed the morphological changes of the gross specimen by immunohistochemical method, and observed the ultrastructure of cartilage tissue under electron microscope. The expression of MMP-2, MCP-1 and CD47 were detected by western blotting, and L-selectin and AOPP were detected by ELISA and spectrophotometer, respectively.In the cartilage tissue morphology, the chondrocytes of OA group showed obvious change, while Cur25 and Cur50 groups maintained the good cartilage cell membrane intact. Compared with control group, the expressions of MMP-2, MCP-1, L-selectin and AOPP in OA group, Cur25 group and Cur50 group were increased (all<0.05), while CD47 levels were decreased (all<0.05). Compared with OA group, the expressions of MMP-2, MCP-1, L-selectin and AOPP in Cur25 group and Cur50 group were decreased (all<0.05), while CD47 levels were increased (all<0.05), and such changes were more significant in Cur50 group (all<0.05).The MMP-2, MCP-1, CD47, L-selectin and AOPP are closely associated with the pathology course of OA. Curcumin has protection effect on cartilage, which can relieve joint cartilage degeneration, reduce cartilage inflammation and increase the metabolic activity of chondrocytes.
Advanced Oxidation Protein Products ; metabolism ; Animals ; Biomarkers ; CD47 Antigen ; metabolism ; Cartilage ; chemistry ; drug effects ; pathology ; Chemokine CCL2 ; metabolism ; Chondrocytes ; drug effects ; pathology ; Curcumin ; administration & dosage ; pharmacology ; Cytokines ; L-Selectin ; metabolism ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Mice, Inbred C57BL ; Osteoarthritis ; genetics ; pathology ; physiopathology ; Oxidative Stress

To investigate the effect of diosgenin (Dgn) on chondrocytes and its relation to JAK2/STAT3 signaling pathway in mice with osteoarthritis (OA).Fifteen male C57BL/6 mice were randomly divided into three groups:control group, OA group and OA+Dgn group. After 4 weeks of treatment, the histopathological changes of cartilage tissue were observed by toluidine blue staining under light microscopy and the ultrastructure of chondrocytes was observed under electron microscopy. The primarily cultured chondrocytes of OA mice were randomly divided into 4 groups:(1) OA group, (2) Dgn group, (3) Dgn+AG490 group, (4) AG490 group. The expression of p-JAK2, p-STAT3, Bax, succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) were detected by Western blotting, and superoxide dismutase (SOD) was detected using colorimetric method.The morphological observation showed that the chondrocytes of OA group presented considerable pathological changes, while the chondrocytes in OA+Dgn group maintained intact membrane. Electron microscopy observation found obvious injury in cartilage tissues of OA group, while that in OA+Dgn group remained smooth. Compared with OA group, the expressions of p-JAK2 and p-STAT3 in chondrocytes of Dgn group were increased (all<0.05), and the expressions of Bax protein, SDH, COX and SOD were decreased (all<0.05). While compared with Dgn group, the expressions of p-JAK2, p-STAT3, SDH, COX and SOD in chondrocytes of Dgn+AG490 group were decreased (all<0.05), and the expression of Bax protein was increased (<0.05).Diosgenin can inhibit apoptosis and increase mitochondrial oxidative stress capacity of chondrocytes in mice with osteoarthritis, which is closely related to the activation of JAK2/STAT3 signaling pathway.
Animals ; Apoptosis ; drug effects ; Cartilage ; drug effects ; pathology ; Chondrocytes ; chemistry ; drug effects ; pathology ; Diosgenin ; pharmacology ; Electron Transport Complex IV ; metabolism ; Janus Kinase 2 ; drug effects ; Male ; Mice ; Mice, Inbred C57BL ; Mitochondria ; drug effects ; genetics ; Osteoarthritis ; genetics ; physiopathology ; Oxidative Stress ; drug effects ; STAT3 Transcription Factor ; drug effects ; Signal Transduction ; Succinate Dehydrogenase ; metabolism ; Superoxide Dismutase ; metabolism ; Tyrphostins ; pharmacology ; bcl-2-Associated X Protein ; metabolism

This study investigated the effects of SIRT1 gene knock-out on osteoarthritis in mice, and the possible roles of SREBP2 protein and the PI3K/AKT signaling pathway in the effects. Mice were randomly divided into a normal group and a SIRT1 gene knock-out group (6 mice in each group). In these groups, one side of the knee anterior cruciate ligament was traversed, and the ipsilateral medial meniscus was cut to establish an osteoarthritis model of knee joint. The countralateral synovial bursa was cut out, serving as controls. The knee joint specimens were then divided into four groups: SIRT1control group (group A, n=6); SIRT1osteoarthritis group (group B, n=6); SIRT1control group (group C, n=6); SIRT1osteoarthritis group (group D, n=6). HE staining, Masson staining, Safranin O-Fast Green staining and Van Gieson staining were used to observe the morphological changes in the articular cartilage of the knee. Immunohistochemical staining was employed to detect the expression of SIRT1, SREBP2, VEGF, AKT, HMGCR and type II collagen proteins. SA-β-gal staining was utilized to evaluate chondrocyte aging. The results showed clear knee joint cartilage destruction and degeneration in the SIRT1osteoarthritis group. The tidal line was twisted and displaced anteriorly. Type II collagen was destroyed and distributed unevenly. Compared with the SIRT1osteoarthritis group and SIRT1control group, SIRT1 protein expression was not obviously changed in the SIRT1osteoarthritis group (P>0.05), while the expression levels of the SREBP2, VEGF and HMGCR proteins were significantly increased (P<0.05) and the levels of AKT and type II collagen proteins were significantly decreased (P<0.05). SIRT1 gene knock-out may aggravate cartilage degeneration in osteoarthritis by activating the SREBP2 protein-mediated PI3K/AKT signalling pathway, suggesting that SIRT1 gene may play a protective role against osteoarthritis.
Animals ; Cartilage ; pathology ; Chondrocytes ; metabolism ; Collagen Type II ; metabolism ; Disease Models, Animal ; Humans ; Knee Joint ; metabolism ; pathology ; Mice ; Mice, Knockout ; Oncogene Protein v-akt ; genetics ; Osteoarthritis ; genetics ; pathology ; Phosphatidylinositol 3-Kinases ; genetics ; Signal Transduction ; genetics ; Sirtuin 1 ; genetics ; Sterol Regulatory Element Binding Protein 2 ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor A ; biosynthesis

IL-6 is an inflammatory cytokine and its overexpression plays an important role in osteoarthritis (OA) pathogenesis. Expression of IL-6 is regulated post-transcriptionally by MCPIP1. The 3' untranslated region (UTR) of MCPIP1 mRNA harbors a miR-139 'seed sequence', therefore we examined the post-transcriptional regulation of MCPIP1 by miR-139 and its impact on IL-6 expression in OA chondrocytes. Expression of miR-139 was found to be high in the damaged portion of the OA cartilage compared with unaffected cartilage from the same patient and was also induced by IL-1beta in OA chondrocytes. Inhibition of miR-139 decreased the expression of IL-6 mRNA by 38% and of secreted IL-6 protein by 40%. However, overexpression of miR-139 increased the expression of IL-6 mRNA by 36% and of secreted IL-6 protein by 56%. These data correlated with altered expression profile of MCPIP1 in transfected chondrocytes. Studies with a luciferase reporter construct confirmed the interactions of miR-139 with the 'seed sequence' located in the 3' UTR of MCPIP mRNA. Furthermore, miR-139 overexpression increased the catabolic gene expression but expression of anabolic markers remained unchanged. Overexpression of miR-139 also induced apoptosis in OA chondrocytes. Importantly, we also discovered that IL-6 is a potent inducer of miR-139 expression in OA chondrocytes. These findings indicate that miR-139 functions as a post-transcriptional regulator of MCPIP1 expression and enhances IL-6 expression, which further upregulates miR-139 expression in OA chondrocytes. These results support our hypothesis that miR-139-mediated downregulation of MCPIP1 promotes IL-6 expression in OA. Therefore, targeting miR-139 could be therapeutically beneficial in the management of OA.
3' Untranslated Regions ; Aged ; *Apoptosis ; Chondrocytes/*metabolism/pathology ; Down-Regulation ; Female ; Gene Expression Regulation ; Humans ; Interleukin-6/*genetics ; Male ; MicroRNAs/*genetics ; Middle Aged ; Osteoarthritis/*genetics/pathology ; RNA, Messenger/genetics ; Ribonucleases/*genetics ; Transcription Factors/*genetics ; Up-Regulation

Rheumatoid arthritis (RA) and osteoarthritis (OA), two common types of arthritis, affect the joints mainly by targeting the synovium and cartilage. Increasing evidence indicates that a significant network connects synovitis and cartilage destruction during the progression of arthritis. We recently demonstrated that hypoxia-inducible factor (HIF)-2alpha causes RA and OA by regulating the expression of catabolic factors in fibroblast-like synoviocytes (FLS) or chondrocytes. To address the reciprocal influences of HIF-2alpha on FLS and chondrocytes, we applied an in vitro co-culture system using a transwell apparatus. When co-cultured with HIF-2alpha-overexpressing chondrocytes, FLS exhibited increased expression of matrix metalloproteinases and inflammatory mediators, similar to the effects induced by tumor-necrosis factor (TNF)-alpha treatment of FLS. Moreover, chondrocytes co-cultured with HIF-2alpha-overexpressing FLS exhibited upregulation of Mmp3 and Mmp13, which is similar to the effects induced by interleukin (IL)-6 treatment of chondrocytes. We confirmed these differential HIF-2alpha-induced effects via distinct secretory mediators using Il6-knockout cells and a TNF-alpha-blocking antibody. The FLS-co-culture-induced gene expression changes in chondrocytes were significantly abrogated by IL-6 deficiency, whereas TNF-alpha neutralization blocked the alterations in gene expression associated with co-culture of FLS with chondrocytes. Our results further suggested that the observed changes might reflect the HIF-2alpha-induced upregulation of specific receptors for TNF-alpha (in FLS) and IL-6 (in chondrocytes). This study broadens our understanding of the possible regulatory mechanisms underlying the crosstalk between the synovium and cartilage in the presence of HIF-2alpha, and may suggest potential new anti-arthritis therapies.
Animals ; Arthritis/genetics/*immunology/pathology ; Arthritis, Rheumatoid/genetics/immunology/pathology ; Basic Helix-Loop-Helix Transcription Factors/genetics/*immunology ; Cells, Cultured ; Chondrocytes/immunology/metabolism/*pathology ; Coculture Techniques ; Fibroblasts/immunology/metabolism/*pathology ; Gene Expression Regulation ; Interleukin-6/genetics/*immunology ; Male ; Mice ; Mice, Inbred C57BL ; Osteoarthritis/genetics/immunology/pathology ; Synovial Membrane/immunology/metabolism/*pathology ; Tumor Necrosis Factor-alpha/genetics/*immunology ; Up-Regulation

Osteoarthritis is a common cause of functional deterioration in older adults and is an immense burden on the aging population. Altered chondrogenesis is the most important pathophysiological process involved in the development of osteoarthritis. However, the molecular mechanism underlying the regulation of chondrogenesis in patients with osteoarthritis requires further elucidation, particularly with respect to the role of microRNAs. MiR-21 expression in cartilage specimens was examined in 10 patients with knee osteoarthritis and 10 traumatic amputees. The effect of miR-21 on chondrogenesis was also investigated in a chondrocyte cell line. The effect of miR-21 on the expression of growth differentiation factor 5 (GDF-5) was further assessed by luciferase reporter assay and western blot. We found that endogenous miR-21 is upregulated in osteoarthritis patients, and overexpression of miR-21 could attenuate the process of chondrogenesis. Furthermore, we identified GDF-5 as the direct target of miR-21 during the regulation of chondrogenesis. Our data suggest that miR-21 has an important role in the pathogenesis of osteoarthritis and is a potential therapeutic target.
Cartilage/metabolism/pathology ; Case-Control Studies ; Cell Line ; Chondrocytes/*metabolism/pathology ; Growth Differentiation Factor 5/genetics/*metabolism ; Humans ; MicroRNAs/genetics/*metabolism ; Osteoarthritis/*metabolism/pathology ; Up-Regulation