Role of Galectin-3 in intervertebral disc degeneration: an experimental study.

BACKGROUND: This study investigated the role of Galectin-3 in the degeneration of intervertebral disc cartilage. METHODS: The patients who underwent lumbar spine surgery due to degenerative disc disease were recruited and divided into Modic I, Modic II, and Modic III; groups. HE staining was used to detect the pathological changes in endplates. The changes of Galectin-3, MMP3, Aggrecan, CCL3, and Col II were detected by immunohistochemistry, RT-PCR, and Western blot. MTT and flow cytometry were used to detect cartilage endplate cell proliferation, cell cycle, and apoptosis. RESULTS: With the progression of degeneration (from Modic I to III), the chondrocytes and density of the cartilage endplate of the intervertebral disc decreased, and the collagen arrangement of the cartilage endplate of the intervertebral disc was broken and calcified. Meanwhile, the expressions of Aggrecan, Col II, Galectin-3, Aggrecan, and CCL3 gradually decreased. After treatment with Galectin-3 inhibitor GB1107, the proliferation of rat cartilage end plate cells was significantly reduced (P < 0.05). GB1107 (25 µmol/L) also significantly promoted the apoptosis of cartilage endplate cells (P < 0.05). Moreover, the percentage of cartilage endplate cells in the G1 phase was significantly higher, while that in the G2 and S phases was significantly lower (P < 0.05). Additionally, the mRNA and protein expression levels of MMP3, CCL3, and Aggrecan in rat cartilage end plate cells were lower than those in the control group. CONCLUSIONS: Galectin-3 decreases with the progression of the cartilage endplate degeneration of the intervertebral disc. Galectin-3 may affect intervertebral disc degeneration by regulating the degradation of the extracellular matrix.

Whole-Transcriptome Sequencing of Knee Joint Cartilage from Kashin-Beck Disease and Osteoarthritis Patients.

The aim of this study was to provide a comprehensive understanding of similarities and differences in mRNAs, lncRNAs, and circRNAs within cartilage for Kashin-Beck disease (KBD) compared to osteoarthritis (OA). We conducted a comparison of the expression profiles of mRNAs, lncRNAs, and circRNAs via whole-transcriptome sequencing in eight KBD and ten OA individuals. To facilitate functional annotation-enriched analysis for differentially expressed (DE) genes, DE lncRNAs, and DE circRNAs, we employed bioinformatic analysis utilizing Gene Ontology (GO) and KEGG. Additionally, using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), we validated the expression levels of four cartilage-related genes in chondrocytes. We identified a total of 43 DE mRNAs, 1451 DE lncRNAs, and 305 DE circRNAs in KBD cartilage tissue compared to OA (q value < 0.05; |log2FC| > 1). We also performed competing endogenous RNA network analysis, which identified a total of 65 lncRNA-mRNA interactions and 4714 miRNA-circRNA interactions. In particular, we observed that circRNA12218 had binding sites for three miRNAs targeting ACAN, while circRNA12487 had binding sites for seven miRNAs targeting COL2A1. Our results add a novel set of genes and non-coding RNAs that could potentially serve as candidate diagnostic biomarkers or therapeutic targets for KBD patients.

Development of alginate-collagen interpenetrating network for osteoarthritic cartilage by in situ softening.

This study presents an alginate-collagen interpenetrating network (IPN) matrix of incorporating collagen fibrils into an alginate hydrogel by physical mixing and controlled gelation. The resulting matrix closely mimics the physiological and pathological stiffness range of the chondrocyte pericellular matrix (PCM). Chondrocytes were cultured within three-dimensional (3D) alginate-collagen IPN matrices with varying stiffness, namely Firm, Medium, and Soft. Alginate lyase was introduced to study the effects of the changes in stiffness of the Firm on chondrocyte response by in situ softening. The developed alginate-collagen IPN matrix displayed good cell-biocompatibility. Compared with stiffer tissue culture plastic (TCP), chondrocytes grown within Firm displayed a stabilized differentiated phenotype characterized by higher expression levels of aggrecan, collagen II, and SOX-9. Moreover, the developed alginate-collagen IPN matrix exhibited a gradually increased percentage of propidium iodide (PI)-positive dead cells with decreasing stiffness. Softer matrices directed cells towards higher proliferation rates and spherical morphologies while stimulating chondrocyte cluster formation. Furthermore, reducing Firm stiffness by in situ softening decreased aggrecan expression, contributing to matrix degradation similar to that seen in osteoarthritis (OA). Hence, the 3D alginate-collagen IPN constructs hold significant potential for in vitro replicating PCM stiffness changes observed in OA cartilage.

Ginsenoside Rg1 relieves rat intervertebral disc degeneration and inhibits IL-1ß-induced nucleus pulposus cell apoptosis and inflammation via NF-κB signaling pathway.

The study aimed to investigate the effect of ginsenoside Rg1 on intervertebral disc degeneration (IVDD) in rats and IL-1ß-induced nucleus pulposus (NP) cells, and explore its underlying mechanism. Forty IVDD rat models were divided into the IVDD group, low-dose (L-Rg1) group (intraperitoneal injection of 20 mg/kg/d ginsenoside Rg1), medium-dose (M-Rg1) group (intraperitoneal injection of 40 mg/kg/d ginsenoside Rg1), and high-dose (H-Rg1) group (intraperitoneal injection of 80 mg/kg/d ginsenoside Rg1). The pathological change was observed by HE and safranin O-fast green staining. The expression of IL-1ß, IL-6, TNF-α, MMP3, aggrecan, and collagen II was detected. The expression of NF-κB p65 in IVD tissues was detected. Rat NP cells were induced by IL-1ß to simulate IVDD environment and divided into the control group, IL-1ß group, and 20, 50, and 100 µmol/L Rg1 groups. The cell proliferation activity, the apoptosis, and the expression of IL-6, TNF-α, MMP3, aggrecan, collagen II, and NF-κB pathway-related protein were detected. In IVDD rats, ginsenoside Rg1 improved the pathology of IVD tissues; suppressed the expression of IL-1ß, IL-6, TNF-α, aggrecan, and collagen II; and inhibited the expression of p-p65/p65 and nuclear translocation of p65, to alleviate the IVDD progression. In the IL-1ß-induced NP cells, ginsenoside Rg1 also improved the cell proliferation and inhibited the apoptosis and the expression of IL-6, TNF-α, aggrecan, collagen II, p-p65/p65, and IκK in a dose-dependent manner. Ginsenoside Rg1 alleviated IVDD in rats and inhibited apoptosis, inflammatory response, and ECM degradation in IL-1ß-induced NP cells. And Rg1 may exert its effect via inhibiting the activation of NF-κB signaling pathway.

microRNA-365 attenuated intervertebral disc degeneration through modulating nucleus pulposus cell apoptosis and extracellular matrix degradation by targeting EFNA3.

This present study is aimed to investigate the role of microRNA-365 (miR-365) in the development of intervertebral disc degeneration (IDD). Nucleus pulposus (NP) cells were transfected by miR-365 mimic and miR-365 inhibitor, respectively. Concomitantly, the transfection efficiency and the expression level of miRNA were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Meanwhile, NP cells apoptosis was measured through propidium iodide (PI)-AnnexinV-fluorescein isothiocyanate (FITC) apoptosis detection kit. Subsequently, immunofluorescence (IF) staining was performed to assess the expression of collagen II, aggrecan and matrix metalloproteinase 13 (MMP-13). In addition, bioinformatic prediction and Luciferase reporter assay were used to reveal the target gene of miR-365. Finally, we isolated the primary NP cells from rats and injected NP-miR-365 in rat IDD models. The results showed that overexpression of miR-365 could effectively inhibit NP cells apoptosis and MMP-13 expression and upregulate the expression of collagen II and aggrecan. Conversely, suppression of miR-365 enhanced NP cell apoptosis and elevated MMP-13 expression, but decreased the expression of collagen II and aggrecan. Moreover, the further data demonstrated that miR-365 mediated NP cell degradation through targeting ephrin-A3 (EFNA3). In addition, the cells apoptosis and catabolic markers were increased in NP cells when EFNA3 upregulated. More importantly, the vivo data supported that miR-365-NP cells injection ameliorated IDD in rats models. miR-365 could alleviate the development of IDD by regulating NP cell apoptosis and ECM degradation, which is likely mediated by targeting EFNA3. Therefore, miR-365 may be a promising therapeutic avenue for treatment IDD through EFNA3.

Cabozantinib prevents AGEs-induced degradation of type 2 collagen and aggrecan in human chondrocytes.

Osteoarthritis (OA) is a joint degenerative disease commonly observed in the old population, lacks effective therapeutic methods, and markedly impacts the normal lives of patients. Degradation of extracellular matrix (ECM) is reported to participate in OA development, which is a potential target for treating OA. Cabozantinib is an inhibitor of tyrosine kinases and is recently claimed with suppressive properties against inflammation. Herein, the protective function of Cabozantinib on advanced glycation end products (AGEs)-induced damages to chondrocytes was tested. SW1353 chondrocytes were stimulated with 100 µg/ml AGEs with or without 10 and 20 µM Cabozantinib for 24 h. Signally increased reactive oxygen species (ROS) levels, declined reduced glutathione (GSH) levels, and elevated release of inflammatory cytokines were observed in AGEs-stimulated SW1353 chondrocytes, which were markedly reversed by Cabozantinib. Moreover, the notably reduced type II collagen and aggrecan levels, and increased matrix metalloproteinase-13 (MMP-13) and A Disintegrin and Metalloproteinase with Thrombospondin Motifs-5 (ADAMTS-5) levels in AGEs-stimulated SW1353 chondrocytes were largely rescued by Cabozantinib. The downregulated Sry-type high-mobility-group box 9 (SOX-9) observed in AGEs-stimulated SW1353 chondrocytes was abolished by Cabozantinib. Furthermore, the impact of Cabozantinib on type II collagen and aggrecan levels in AGEs-treated SW1353 chondrocytes was abrogated by silencing SOX-9. Collectively, Cabozantinib prevented AGEs-induced degradation of type 2 collagen and aggrecan in human chondrocytes by mediating SOX-9.

Positive Regulation of S-Adenosylmethionine on Chondrocytic Differentiation via Stimulation of Polyamine Production and the Gene Expression of Chondrogenic Differentiation Factors.

S-adenosylmethionine (SAM) is considered to be a useful therapeutic agent for degenerative cartilage diseases, although its mechanism is not clear. We previously found that polyamines stimulate the expression of differentiated phenotype of chondrocytes. We also found that the cellular communication network factor 2 (CCN2) played a huge role in the proliferation and differentiation of chondrocytes. Therefore, we hypothesized that polyamines and CCN2 could be involved in the chondroprotective action of SAM. In this study, we initially found that exogenous SAM enhanced proteoglycan production but not cell proliferation in human chondrocyte-like cell line-2/8 (HCS-2/8) cells. Moreover, SAM enhanced gene expression of cartilage-specific matrix (aggrecan and type II collagen), Sry-Box transcription factor 9 (SOX9), CCN2, and chondroitin sulfate biosynthetic enzymes. The blockade of the methionine adenosyltransferase 2A (MAT2A) enzyme catalyzing intracellular SAM biosynthesis restrained the effect of SAM on chondrocytes. The polyamine level in chondrocytes was higher in SAM-treated culture than control culture. Additionally, Alcian blue staining and RT-qPCR indicated that the effects of SAM on the production and gene expression of aggrecan were reduced by the inhibition of polyamine synthesis. These results suggest that the stimulation of polyamine synthesis and gene expression of chondrogenic differentiation factors, such as CCN2, account for the mechanism underlying the action of SAM on chondrocytes.

Immunohistochemical Analysis of Knee Chondral Defect Repair after Autologous Particulated Cartilage and Platelet-Rich Plasma Treatment in Sheep.

This study performs an analysis that will enable the evaluation of the quality, durability, and structure of repaired cartilaginous extracellular matrix tissue using an autologous-based particulated autograft cartilage and platelet-rich plasma treatment (PACI + PRP). A single-blind controlled experiment was conducted on 28 sheep to evaluate the efficacy of the PACI + PRP treatment for cartilage defects. Full-thickness 8 mm diameter defects were created in the weight-bearing area of both knees. The right knees received PACI + PRP. The left knees were treated with Ringer's lactate solution (RLS) or hyaluronic acid (HA) injections. Sheep were euthanized at 9- or 18-months post-surgery. An extensive immunohistochemical analysis was performed to assess collagen types (I, II, III, V, VI, IX, X, XI) and aggrecan positivity. A semiquantitative scoring system provided a detailed evaluation of immunostaining. Collagens and aggrecan scores in the PACI + PRP groups were similar to healthy cartilage. Significant differences were found in collagens associated with matrix maturity (II and V), degradation (IX), structure and mechanics (VI), and hypertrophy (X) between healthy cartilage and RLS- or HA-repaired cartilage. The PACI + PRP treatment advanced the repair cartilage process in chondral defects with mature hyaline cartilage and enhanced the structural and mechanical qualities with better consistent cartilage, less susceptible to degradation and without hypertrophic formation over time.

[Preliminary study of TRPV4 affects chondrocyte degeneration].

OBJECTIVE: To explore and verify that transient receptor potential vanilloid 4(TRPV4) affects chondrocyte degeneration. METHODS: Neonatal SD rats were selected, primary chondrocytes were extracted, and identified by toluidine blue staining and alcian blue staining;an in vitro chondrocyte inflammation model was constructed by IL-1ß, and TRPV4 inhibitor was used to treat chondrocytes under inflammatory conditions, and the chondrocytes were treated by RT-PCR method was used to detect matrix metallopeptidase 13(MMP-13), a disintegrin and metalloproteinase with thrombospondin 5, (ADAMTS-5)、nitric oxide synthase 2(NOS2)、Collagen, type II alpha 1(Col2α1)and aggrecan (Acan) mRNA in chondrocytes; primary chondrocytes were treated with different concentrations of TRPV4 overexpression plasmid, and the optimal overexpression dose was screened. The mRNA expressions of TRPV4, MMP-13, ADAMTS-5, NOS2, Col2α1 and Acan in chondrocytes under the optimal TRPV4 overexpression dose were detected. RESULTS: Toluidine blue staining and Alcian blue staining identified the extracted cells as primary chondrocytes;RT-PCR showed that TRPV4, MMP-13, ADAMTS-5, NOS2 mRNA in chondrocytes treated with TRPV4 inhibitor under inflammatory conditions. The expression of Col2α1 mRNA was significantly decreased (P<0.05), and the expression of Col2α1 mRNA was increased (P<0.05). Although there was no significant difference in the expression of Acan mRNA, the overall trend was also increasing. The expression of Col2α1 and Acan mRNA in chondrocytes was significantly decreased (P<0.05), and the expression of NOS2 mRNA was increased(P<0.05), but there was no significant difference in MMP-13 and ADAMTS-5 (P>0.05). CONCLUSION: Inhibiting the expression of TRPV4 can down-regulate the expression of genes related to chondrocyte degeneration.

Umbilical cord mesenchymal stem cells relieve osteoarthritis in rats through immunoregulation and inhibition of chondrocyte apoptosis.

This study aims to investigate the effectiveness of umbilical cord mesenchymal stem cells (UCMSCs) in treating osteoarthritis (OA). Sprague-Dawley rats were used in in vivo experiments and divided into four groups: normal, OA model, saline, and UCMSC-treated groups (n = 6). An OA model was established by injecting iodoacetic acid into the joint cavity. The results indicate that UCMSC transplantation significantly reduced joint surface and articular cartilage damage, and the levels of IL-1ß, TNF-α, and MMP13 in the joint fluid were significantly reduced after UCMSC treatment. In vitro experiments showed that co-culturing UCMSCs and chondrocytes promoted the expression of aggrecan, COL2, SOX9, and BCL-2; downregulated the expression of BAX and BAD in chondrocytes; and promoted the expression of IL-10 and TGF-ß1 in UCMSCs. Additionally, the supernatant of UCMSCs inhibited the expression of IL-1ß and TNF-α in the articular cavity and promoted the expression of COL2 and aggrecan in vivo. These effects were impaired when IL-10 and TGF-ß1 were removed. Collectively, UCMSC transplantation appears to improve joint pathology, reduce inflammatory factors, and decrease chondrocyte apoptosis, likely through the involvement of IL-10 and TGF-ß1, thus providing a potential therapeutic option for patients with OA.

GuiLu-ErXian Glue extract promotes mesenchymal stem cells (MSC)-Induced chondrogenesis via exosomes release and delays aging in the MSC senescence process.

ETHNOPHARMACOLOGICAL RELEVANCE: The treatment of osteoarthritis (OA) patients is a challenging problem. Mesenchymal stem cells (MSCs) are multipotent cells and play key roles in regenerative medicine for cartilage degeneration. GuiLu-ErXian Glue (GLEXG) is an herbal remedy widely used in traditional Chinese medicine to treat joint pain and disability in elderly OA patients. However, the mechanisms of how GLEXG affects MSCs-induced chondrogensis remains to be elucidated. AIM OF THE STUDY: The aim of this study was to investigate the effects of GLEXG on MSC-derived chondrogenesis, both in vitro and in vivo and its potential mechanisms. METHODS: Using human MSC (hMSCs) as in vitro model, the effects of HPLC-profiled GLEXG water extract on chondrogenic differentiation were investigated by 3D spheroid cultures under chondrogenesis-inducing medium (CIM) condition. The chondrogenesis process was evaluated by measuring the sphere sizes, chondrogenesis-related genes expression by reverse transcription real-time PCR that targeted type II/X collagens, SOX9, aggrecan, and protein expression by immunostaining. Anti-TGF-ß1 neutralization antibody was used for mechanistic study. Mono-iodoacetate (MIA) induced OA joint was used to evaluate the effects of GLEXG on in vivo model. MSCs-derived exosomes were purified for proteomics study and senescence process was evaluated by cumulative population doublings and senescence-associated ß-Galactosidase staining. RESULTS: The results showed that GLEXG enhanced hMSCs chondrogenesis and upregulated RNA expression of type II/X collagen, SOX9 and aggrecan at 0.1 µg/mL, 0.3 µg/mL in vitro. In vivo, GLEXG at the dose of 0.3 µg intraarticular (i.a.) injection rescued the MIA-induced cartilage defect. Proteomics and ingenuity pathway analysis obtained from MSCs-released exosomes suggested that senescence pathway was less activated in GLEXG group than in vehicle group. Besides, GLEXG was able to increase cumulative population doubling and delayed hMSCs senescence process after four passages in cultures. CONCLUSION: we conclude that GLEXG promotes in vitro MSC-induced chondrogenesis possibly via exosomes release and delays aging in the MSC senescence process and that treatment with GLEXG (0.3 µg, i.a.) rescued cartilage defects in rat OA knee model.

Synergetic enrichment of aggrecan in nucleus pulposus cells by scAAV6-shRNA-mediated knockdown of aggrecanase-1 and aggrecanase-2.

Degenerative disk disease (DDD) that aggravates structural deterioration of intervertebral disks (IVDs) can be accompanied by painful inflammation and immunopathological progressions. Current surgical or pharmacological therapies cannot repair the structure and function of IVDs. Nucleus pulposus (NP) cells are crucial for the preservation or restoration of IVDs by balancing the anabolic and catabolic factors affecting the extracellular matrix. Imbalanced anabolic and catabolic factors cause increased degradation of aggrecan. Aggrecanases A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS)4 and ADAMTS5 are the main degrading enzymes of aggrecan. Previously, we characterized adeno-associated virus (AAV6) as the most suitable serotype with marked NP cellular tropism and demonstrated that ADAMTS4 could be silenced by self-complementary adeno-associated virus grade 6 small helix ribonucleic acid (scAAV6-shRNA) in NP cells of degeneration grade III, which resulted in enrichment of aggrecan. Nonetheless, neither scAAV6-shRNA-mediated inhibition of ADAMTS5 nor joint inhibitions of ADAMTS4 and ADAMTS5 have been investigated, although both enzymes are regulated by analogous proinflammatory cytokines and have the same cleavage sites in aggrecan. Therefore, we attempted scAAV6-shRNA-mediated inhibitions of both enzymes in NP cells of degeneration grade IV to increase efficacies in treatments of DDD. The degeneration grade of IVDs in patients was determined by magnetic resonance imaging (MRI) before surgical operations. After isolation and culturing of NP cells, cells were transduced with scAAV6-shRNAs targeting ADAMTS4 or ADAMTS5. Transduced cells were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR), fluorescence microscopy, flow cytometry-assisted cell sorting (FACS), MTT assay (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay), immunoblotting, and enzyme-linked immunosorbent assay (ELISA). Joint transduction of NP cells exhibited high transduction efficacies (98.1%), high transduction units (TU) (1381 TU/Cell), and no effect on cell viability or proliferation. Above all joint treatments resulted in effective knockdown of ADAMTS4 (92.8%) and ADAMTS5 (93.4%) along with additive enrichment of aggrecan (113.9%). Treatment effects were significant for more than 56 days after transduction (P < 0.001). In conclusion, scAAV6-shRNA-mediated combined molecular therapy could be very valuable for more effective, durable, and less immunogenic treatment approaches in DDD.

Brachyury positively regulates extracellular matrix synthesis via directly promoting aggrecan transcription in nucleus pulposus.

Nucleus pulposus (NP) degeneration is characterized by the decreased cellularity of nucleus pulposus cells (NPCs) and diminished content of hydrophilic extracellular matrix (ECM). Overexpression of brachyury has been reported to reverse the degenerated NPCs into healthy phenotypes. However, the direct correlation between brachyury and ECM has not been fully elucidated. This study revealed that brachyury expression decreased in human degenerated NP tissues and Lipopolysaccharide (LPS)-induced degenerated rat NPCs model. In vitro and in vivo experiments further showed that brachyury deficiency suppressed the synthesis of aggrecan and collagen II in NP. Mechanistically, ChIP-qPCR assays demonstrated that brachyury bound to the promoter region of aggrecan in NPCs. Furthermore, luciferase reporter assays revealed that brachyury transcriptionally activated aggrecan expression through binding with a novel specific motif. In rat in vivo model, brachyury overexpression partially reversed the degenerative phenotype. In conclusion, brachyury positively regulated ECM synthesis via directly promoting aggrecan transcription in NPCs. Accordingly, it may be helpful to be developed into a promising therapeutic target for NP degeneration.

Immunohistochemical characterization of the mandibular condyle for type I and II collagen, aggrecan, MMP-9, and MMP-13 in MMP-2-deficient mice.

Mice devoid of matrix metalloproteinase (MMP)-2 due to gene targeting have been reported to show articular cartilage destruction in the knee joint; however, the phenotype of the mandibular condylar cartilage remains unknown. Thus, in the present study, we investigated the mandibular condyle in Mmp2-/- mice. We obtained and bred Mmp2-/- mice from the same source as the previous study, and performed genotyping using genomic DNA extracted from finger snips. The mandibular condyle of Mmp2-/- mice and wild-type (WT) mice was immunohistochemically examined for the localization of extracellular matrix (ECM) proteins (type I and II collagen, and aggrecan), and MMP-9 and MMP-13. No cartilage destruction was observed in the mandibular condyle of Mmp2-/- mice, and no difference was found in the localization of the ECM proteins between the Mmp2-/- mice and WT mice. However, the bone marrow cavity in the subchondral bone of the mandibular condyle was more distinct in Mmp2-/- mice than in WT mice at the age of 50 weeks. Of note, MMP-9 characteristically localized in multinucleated cells in the mandibular condyle in 50-week-old Mmp2-/- mice. MMP-2 may be involved in the regulation of osteoclast differentiation and the formation of the bone marrow cavity in aged mice.

LncRNA ZFAS1 protects chondrocytes from IL-1ß-induced apoptosis and extracellular matrix degradation via regulating miR-7-5p/FLRT2 axis.

BACKGROUND: Increasing evidence suggested that long non-coding RNAs (lncRNAs) played vital roles in osteoarthritis (OA) progression. In this study, we aimed to reveal the protective roles of lncRNA ZFAS1 in osteoarthritis (OA) and further investigated its underlying mechanism. METHODS: The chondrocytes were stimulated by IL-1ß to establish an in vitro OA model. Then, the expression of ZFAS1, miR-7-5p, and FLRT2 in chondrocytes was determined by qRT-PCR. Gain- and loss-of-function assays of ZFAS1, miR-7-5p and FLRT2 were conducted. CCK-8 assay and flow cytometry analysis were performed to detect cell viability and apoptosis rate. The expression levels of cartilage-related proteins, including MMP13, ADAMTS5, Collagen II, and Aggrecan, were measured by western blot analysis. The interaction between ZFAS1 and miR-7-5p, as well as miR-7-5p and FLRT2, was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay. RESULTS: The expression of ZFAS1 and FLRT2 was down-regulated, while the expression of miR-7-5p was up-regulated in chondrocytes exposed to IL-1ß. ZFAS1 overexpression promoted cell viability and suppressed apoptosis in IL-1ß-treated chondrocytes. Besides, ZFAS1 overexpression suppressed the expression of MMP13 and ADAMTS5, but promoted the expression of Collagen II and Aggrecan to suppress ECM degradation. The mechanistic study showed that ZFAS1 sponged miR-7-5p to regulate FLRT2 expression. Furthermore, the overexpression of miR-7-5p could neutralize the effect of ZFAS1 in IL-1ß-treated chondrocytes, and suppression of FLRT2 counteracted the miR-7-5p down-regulation role in IL-1ß-treated chondrocytes. CONCLUSIONS: ZFAS1 could promote cell viability of IL-1ß-treated chondrocytes via regulating miR-7-5p/FLRT2 axis. Trial registration Not applicable.

Aggrecan and Hyaluronan: The Infamous Cartilage Polyelectrolytes - Then and Now.

Cartilages are unique in the family of connective tissues in that they contain a high concentration of the glycosaminoglycans, chondroitin sulfate and keratan sulfate attached to the core protein of the proteoglycan, aggrecan. Multiple aggrecan molecules are organized in the extracellular matrix via a domain-specific molecular interaction with hyaluronan and a link protein, and these high molecular weight aggregates are immobilized within the collagen and glycoprotein network. The high negative charge density of glycosaminoglycans provides hydrophilicity, high osmotic swelling pressure and conformational flexibility, which together function to absorb fluctuations in biomechanical stresses on cartilage during movement of an articular joint. We have summarized information on the history and current knowledge obtained by biochemical and genetic approaches, on cell-mediated regulation of aggrecan metabolism and its role in skeletal development, growth as well as during the development of joint disease. In addition, we describe the pathways for hyaluronan metabolism, with particular focus on the role as a "metabolic rheostat" during chondrocyte responses in cartilage remodeling in growth and disease.Future advances in effective therapeutic targeting of cartilage loss during osteoarthritic diseases of the joint as an organ as well as in cartilage tissue engineering would benefit from 'big data' approaches and bioinformatics, to uncover novel feed-forward and feed-back mechanisms for regulating transcription and translation of genes and their integration into cell-specific pathways.

Metformin regulates chondrocyte senescence and proliferation through microRNA-34a/SIRT1 pathway in osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is the most common degenerative disease in joints among elderly patients. Senescence is deeply involved in the pathogenesis of osteoarthritis. Metformin is widely used as the first-line drug for Type 2 diabetes mellitus (T2DM), and has great potential for the treatment of other aging-related disorders, including OA. However, the role of metformin in OA is not fully elucidated. Therefore, our aim here was to investigate the effects of metformin on human chondrocytes. METHODS: After metformin treatment, expression level of microRNA-34a and SIRT1 in chondrocyte were detected with quantitative real-time PCR and immunofluorescence staining. Then, microRNA-34a mimic and small interfering RNA (siRNA) against SIRT1 (siRNA-SIRT1) were transfected into chondrocyte. Senescence-associated ß-galactosidase (SA-ß-gal) staining was performed to assess chondrocyte senescence. Chondrocyte viability was illustrated with MTT and colony formation assays. Western blot was conducted to detect the expression of P16, IL-6, matrix metalloproteinase-13 (MMP-13), Collagen type II (COL2A1) and Aggrecan (ACAN). RESULTS: We found that metformin treatment (1 mM) inhibited microRNA-34a while promoted SIRT1 expression in OA chondrocytes. Both miR-34a mimics and siRNA against SIRT1 inhibited SIRT1 expression in chondrocytes. SA-ß-gal staining assay confirmed that metformin reduced SA-ß-gal-positive rate of chondrocytes, while transfection with miR-34a mimics or siRNA-SIRT1 reversed it. MTT assay and colony formation assay showed that metformin accelerated chondrocyte proliferation, while miR-34a mimics or siRNA-SIRT1 weakened this effect. Furthermore, results from western blot demonstrated that metformin suppressed expression of senescence-associated protein P16, proinflammatory cytokine IL-6 and catabolic gene MMP-13 while elevated expression of anabolic proteins such as Collagen type II and Aggrecan, which could be attenuated by transfection with miR-34a mimics. CONCLUSION: Overall, our data suggest that metformin regulates chondrocyte senescence and proliferation through microRNA-34a/SIRT1 pathway, indicating it could be a novel strategy for OA treatment.

[Analysis of a Chinese pedigree affected with familial short stature due to 15q25.3q26.1 deletion involving the ACAN gene].

OBJECTIVE: To analyze the genetic etiology of a Chinese pedigree affected with short stature. METHODS: A child with familial short stature (FSS) who had presented at the Ningbo Women and Children's Hospital in July 2020 and his parents and paternal and maternal grandparents were selected as the study subject. Clinical data of the pedigree was collected, and the proband was subjected to routine growth and development assessment. Peripheral blood samples were collected. The proband was subjected to whole exome sequencing (WES), and the proband, his parents and grandparents were subjected to chromosomal microarray analysis (CMA). RESULTS: The height of the proband and his father was 87.7cm (-3 s) and 152 cm (-3.39 s) respectively. Both of them were found to harbor a 15q25.3-q26.1 microdeletion, which has encompassed the whole of the ACAN gene which is closely associated with short stature. The CMA results of his mother and grandparents were all negative, and above deletion has not been included in population database and related literature, and was rated as pathogenic based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). After 14 months of rhGH treatment, the height of the proband has increased to 98.5 cm (-2.07 s). CONCLUSION: The 15q25.3-q26.1 microdeletion probably underlay the FSS, in this pedigree. Short-term rhGH treatment can effectively improve the height of the affected individuals.

IRF1 promotes the chondrogenesis of human adipose-derived stem cells through regulating HILPDA.

BACKGROUND: Osteoarthritis is a main cause of deformity in aging people. The chondrogenesis of human adipose-derived stem cells (hADSCs) has a positive effect on the cure of osteoarthritis. However, the regulatory mechanism of hADSC chondrogenesis still needs further exploration. This research investigates the role of interferon regulatory factor 1 (IRF1) in the chondrogenesis of hADSCs. METHODS: hADSCs were purchased and cultured. The interaction between IRF1 and hypoxia inducible lipid droplet associated (HILPDA) was predicted by bioinformatics analysis, and verified through dual-luciferase reporter and chromatin immunoprecipitation assays. The expressions of IRF1 and HILPDA in osteoarthritis cartilage samples were measured through qRT-PCR. After hADSCs were transfected or further induced for chondrogenesis, the chondrogenesis was visualized by Alcian blue staining, and the expressions of IRF1, HILPDA and chondrogenesis-related factors (SOX9, Aggrecan, COL2A1, MMP13, MMP3) were determined through qRT-PCR or Western blot. RESULTS: HILPDA bound to IRF1 in hADSCs. IRF1 and HILPDA levels were up-regulated during the chondrogenesis of hADSCs. Overexpressions of IRF1 and HILPDA promoted the chondrogenesis of hADSCs with the up-regulation of SOX9, Aggrecan and COL2A1 and the down-regulation of MMP13 and MMP3; however, IRF1 silencing generated the opposite effects. Besides, HILPDA overexpression reversed the effects of IRF1 silencing on inhibiting chondrogenesis of hADSCs and regulating the expressions of chondrogenesis-related factors. CONCLUSION: IRF1 promotes the chondrogenesis of hADSCs through up-regulating HILPDA level, providing novel biomarkers for treating osteoarthritis.

Downregulation of ACAN is Associated with the Growth hormone pathway and Induces short stature.

BACKGROUND: ACAN heterozygous mutations can cause short stature in patients with or without advanced bone age and have recently attracted researchers' attention. Growth hormone can be used to treat short stature induced by ACAN mutations; however, few studies have focused on the underlying mechanism of this treatment. METHODS: Four patients with new mutations were reported based on clinical data and genetic tests. We investigated the expression and Gene Ontology biological process enrichment of ACAN and GH pathways based on GTEx databases through bioinformatics analyses. The effect of ACAN on the growth hormone response evaluated in ATDC5 cells with a growth hormone stimulation test. RESULTS: Four mutations were reported in this study: c.619C > A, c.1967A > G, c.1888G > A, and c.1308_1309del. All patients' heights were under -2.5 SD, with one had advanced bone age, and two had GH deficiency. Two individuals received growth hormone therapy acquired variable levels of height SD score improvement. ACAN and the GH pathway were strongly associated; ACAN does not affect GHR but regulates the response to GH. Downregulating ACAN inhibited ATDC5 cell proliferation induced by GH. CONCLUSION: ACAN is associated with the GH pathway, revealing the potential mechanism underlying GH-targeted treatment for ACAN mutation-induced short stature. GH-promoting therapies may increase patients' heights.