Apoptosis Rate Of Chondrocytes Research Articles

The role of cytokine receptor-like factor 1 (CRLF1) in facet joint osteoarthritis pathogenesis

BackgroundFacet joint osteoarthritis (FJOA) is a prevalent condition contributing to low back pain, particularly in the elderly population. This study aimed to investigate the potential role of Cytokine Receptor-like Factor 1 (CRLF1) in FJOA pathogenesis and its therapeutic implications. MethodsBioinformatics analysis was utilized to identify CRLF1 as the target gene, followed by quantification of CRLF1 expression levels and joint degeneration degree using immunohistochemistry (IHC). In primary chondrocytes, the inhibition of CRLF1 expression by siRNA was performed, and Western blot analysis was conducted to evaluate the involvement of the extracellular matrix and MAPK/ERK signaling pathway. Flow cytometry was employed to assess the apoptosis rate of chondrocytes, while immunofluorescence (IF) was utilized to evaluate the localization of CRLF1, cleaved-caspase3, MMP13, COL2A1, and ERK. ResultsThe expression of CRLF1 was found to be significantly elevated in FJOA tissues compared to normal tissues. Through the use of loss-of-function assays, it was determined that CRLF1 not only enhanced the rate of apoptosis in chondrocytes, but also facilitated the degradation of the extracellular matrix in vitro. Furthermore, CRLF1 was found to activate the ERK1/2 pathways. The pro-arthritic effects elicited by CRLF1 were mitigated by treatment with the MEK inhibitor U0126 in chondrocytes. ConclusionThese results suggest that CRLF1 enhances chondrocytes apoptosis and extracellular matrix degration in FJOA and thus may therefore be a potential therapeutic target for FJOA.

The expression of SOCS1 is regulated by promoter DNA methylation and is associated with mitochondria-mediated apoptosis of T-2 induced chondrocytes

At present, the function of SOCS1 in Kashin-Beck disease (KBD) has not been reported. This study aims to explore the expression and mechanism of SOCS1 in KBD, and provide theoretical basis for the prevention and treatment of KBD. The expression of SOCS1 were measured by qRT-PCR and Western blot. ELISA was used to detect the content of SOCS1 in serum and synovial fluid. CCK-8 kits were selected to measure the cell viability. Methylation Specific PCR (MSP) assay is used to detect the methylation level of SOCS1 in chondrocytes. Flow cytometry was used to analyze the apoptosis rate of chondrocytes in different groups. The expression of apoptosis related proteins (caspase-3 and caspase-9) and Cytochrome c were detected using Western blot. The mitochondrial ROS, ATP and the activity of mitochondrial respiratory chain complexes were detected using commercial kits. The results showed that the expression of SOCS1 significantly increases in KBD patients and T-2 induced chondrocytes. Further research has found that the methylation levels of SOCS1 were significantly reduced in KBD patients and T-2 induced chondrocytes. Functional studies have found that SOCS1 silencing inhibited chondrocyte apoptosis and mitochondrial dysfunction. More importantly, SOCS1 regulated mitochondrial mediated chondrocyte apoptosis through the IGF-1/IGF-1R/FAK/Drp1 pathway. In conclusion, SOCS1 expression is increased and methylation levels are decreased in KBD, and is involved in regulating mitochondrial mediated apoptosis in T-2 induced chondrocytes through IGF-1/IGF-1R/FAK/Drp1 signaling. This study provides new theoretical basis for the treatment and prevention of KBD in clinical practice.

HIF-1α in cartilage homeostasis, apoptosis, and glycolysis in mice with steroid-induced osteonecrosis of the femoral head.

With the prevalence of coronavirus disease 2019, the administration of glucocorticoids (GCs) has become more widespread. Treatment with high-dose GCs leads to a variety of problems, of which steroid-induced osteonecrosis of the femoral head (SONFH) is the most concerning. Since hypoxia-inducible factor 1α (HIF-1α) is a key factor in cartilage development and homeostasis, it may play an important role in the development of SONFH. In this study, SONFH models were established using methylprednisolone (MPS) in mouse and its proliferating chondrocytes to investigate the role of HIF-1α in cartilage differentiation, extracellular matrix (ECM) homeostasis, apoptosis and glycolysis in SONFH mice. The results showed that MPS successfully induced SONFH in vivo and vitro, and MPS-treated cartilage and chondrocytes demonstrated disturbed ECM homeostasis, significantly increased chondrocyte apoptosis rate and glycolysis level. However, compared with normal mice, not only the expression of genes related to collagens and glycolysis, but also chondrocyte apoptosis did not demonstrate significant differences in mice co-treated with MPS and HIF-1α inhibitor. And the effects observed in HIF-1α activator-treated chondrocytes were similar to those induced by MPS. And HIF-1α degraded collagens in cartilage by upregulating its downstream target genes matrix metalloproteinases. The results of activator/inhibitor of endoplasmic reticulum stress (ERS) pathway revealed that the high apoptosis rate induced by MPS was related to the ERS pathway, which was also affected by HIF-1α. Furthermore, HIF-1α affected glucose metabolism in cartilage by increasing the expression of glycolysis-related genes. In conclusion, HIF-1α plays a vital role in the pathogenesis of SONFH by regulating ECM homeostasis, chondrocyte apoptosis, and glycolysis.

PG545 Prevents Osteoarthritis Development by Regulating PI3K/AKT/mTOR Signaling and Activating Chondrocyte Autophagy

Introduction: Osteoarthritis (OA) is a degenerative disease common in the elderly and is characterized by joint pain, swelling, and restricted movement. In recent years, heparanase has been reported to play an important role in the development of osteoarthritic cartilage. PG545 is a heparan sulfate mimetic with heparanase inhibitory activity. In this study, the therapeutic effects and possible mechanisms of PG545 were investigated in a chondrocyte injury model induced by interleukin-1β (IL -1β). Methods: Following treatment with PG545 or the autophagy inhibitor 3-methyladenine (3-MA), chondrocyte viability was detected using Cell Counting Kit-8 and fluorescein diacetate/propidium iodide double staining. The apoptosis rate of chondrocytes was determined by flow cytometry. Expression of light chain 3 and P62 was monitored by immunofluorescence labeling. Western blot, lentivirus infection with red fluorescent protein and green fluorescent protein, and quantitative real-time polymerase chain reaction were used to determine the expression levels of chondrocyte markers, apoptosis-related factors, autophagy proteins, and key proteins of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway. The expression and activity of stress-specific enzymes such as malondialdehyde, superoxide dismutase, and catalase (CAT) were investigated. Chondrocytes with ATG5 knockdown were used to investigate the relationship between the therapeutic effect of PG545 and autophagy. The therapeutic effect of PG545 was verified in vivo. Results: PG545 had a significant protective effect on chondrocytes by reducing oxidative stress, apoptosis, and degradation of chondrocytes and increasing chondrocyte proliferation. PG545 was effective in inducing autophagy in IL-1β-treated cells, while 3-MA attenuated the effect. The PI3K/Akt/mTOR pathway may be involved in the promotion of autophagy and OA treatment by PG545. Conclusion: PG545 was able to restore impaired autophagy and autophagic flux via the PI3K/Akt/mTOR pathway, thereby delaying the progression of OA, suggesting that PG545 may be a novel therapeutic approach for OA.

Oxymatrine Protects Chondrocytes against IL-1β-triggered Apoptosis in Vitro and Inhibits Osteoarthritis in Mice Model.

Background Osteoarthritis (OA) is a multifactorial disease with various risk factors, resulting in the degeneration of articular cartilage and whole joints. However, to date, no effective disease-modifying therapy for OA has been developed. Oxymatrine (OMT) is associated with many pharmacological effects, including anti-inflammatory, antiapoptotic, and antioxidative properties. However, the role of OMT in OA remains unclear. Materials and Methods An IL-1β-induced chondrocyte model and anterior cruciate ligament transection (ACLT)-induced murine model of OA were constructed. The effect of OMT on chondrocyte viability was assessed using the CCK-8 assay. The protein level was assessed by Western blot analysis, and the apoptosis rate was assessed by flow cytometry in vitro and TUNEL staining in OA model mice. The effect of OMT on the degradation of articular cartilage in ACLT-induced OA mice was assessed by histological analysis. Results OMT at 0–2 mg/mL showed no conspicuous cytotoxicity on chondrocytes after 24 hours of incubation. OMT at 0.5, 1, and 2 mg/mL inhibited IL-1β-triggered apoptosis, upregulated MMP13, MMP9, and Col X, and upregulated Col II in chondrocytes in vitro. OMT represses the NF-κB signaling cascade in IL-1β-triggered chondrocytes in vitro. In an in vivo study, OMT decreased the apoptosis rate of chondrocytes and exerted a protective effect against the degradation of articular cartilage in ACLT-triggered OA mice. Conclusion OMT plays a protective role against chondrocyte injury induced by IL-1β in vitro or ACLT in vivo. OMT may play a role in chondrocytes during OA by inhibiting NF-κB signaling by decreasing the phosphorylation of p65 and IκB. OMT treatment may be a promising chondroprotective approach to delay OA cartilage progression.

Preparation of epigallocatechin gallate decorated Au-Ag nano-heterostructures as NIR-sensitive nano-enzymes for the treatment of osteoarthritis through mitochondrial repair and cartilage protection

Osteoarthritis (OA), a widespread degenerative disease characterized by cartilage destruction, has emerged as a public health challenge in the current aging society. In addition to applied steroids and surgery, near-infrared (NIR) sensitive nano-enzyme for the treatment of osteoarthritis through mitochondrial repair and cartilage protection is attractive and promising. In this study, a NIR sensitive multifunctional heterostructure (EGCG (Epigallocatechin gallate) decorated Au-Ag nano-jars (E@Au-Ag)) was introduced as an enzyme-sensitive active nanoplatform for the treatment of osteoarthritis. Molecular biology results indicated that E@Au-Ag possesses intrinsic properties of anti-oxidative stress and was able to reduce the apoptosis rate of chondrocytes by 83.3%. The area of the intra-articular joint cavity injected with E@Au-Ag can be elevated to 46.6 °C under NIR to promote the release of EGCG further to induce cartilage regeneration. X-ray radiography and section staining showed that E@Au-Ag reduced cartilage damage and decreased OARSI scores by approximately 52% after 8 weeks of treatment in a surgically induced OA model. The results demonstrated that this multifunctional enzyme-like nanoplatform with a synergistic NIR sensitive property to facilitate cartilage migration and regeneration repair provides a promising OA treatment strategy. Statement of significance1. NIR-sensitive nano-enzyme is gaining much attention in the field of biomedical materials.2. EGCG decorated Au-Ag nano-heterostructures were utilized as NIR-sensitive nano-enzymes for the treatment of osteoarthritis through mitochondrial repair and cartilage protection.3. The obtained multifunctional Au-Ag nano-heterostructures are promising for osteoarthritis treatment.

Charge‐Guided Micro/Nano‐Hydrogel Microsphere for Penetrating Cartilage Matrix

AbstractThe dense structure and the high‐density negative charge of the cartilage matrix impede drug penetration into the cartilage. Effectively breaking through these barriers and transporting target drugs to the cartilage is currently difficult. Herein, an adhesive hydrogel microsphere characterized by its positively charged nanosized secondary structure that allows it to penetrate deep into the cartilage through charge guidance is introduced as an efficient drug delivery system for cartilage adhesion, cartilage matrix penetration, and chondrocytes targeting and solves the problem of drug‐penetration impedance due to cartilage. The dopamine‐modified structure on the surface of the microspheres can attach the microspheres onto the cartilage surface, while the charge‐guided secondary structure can carry drugs to penetrate the cartilage matrix, release drugs under reactive oxygen species stimulation, act on chondrocytes. The experiments show that the injectable microspheres successfully deliver antioxidants to chondrocytes in osteoarthritis environments. The apoptosis rate of chondrocytes under oxidative stress decreases from 38.36 ± 5.48% to 12.86 ± 4.27%, which is significantly better than that in the direct drug treatment group (28.43 ± 5.87%). The micro/nano‐hydrogel microsphere effectively penetrates the cartilage matrix and releases the drug in ROS response, realizing the improvement of drug utilization and efficacy and thereby demonstrating the efficient delivery of cartilage drugs.

Intra-articular low-dose parathyroid hormone (1-34) improves mobility and articular cartilage quality in a preclinical age-related knee osteoarthritis model.

AimsOsteoarthritis (OA) is prevalent among the elderly and incurable. Intra-articular parathyroid hormone (PTH) ameliorated OA in papain-induced and anterior cruciate ligament transection-induced OA models; therefore, we hypothesized that PTH improved OA in a preclinical age-related OA model.MethodsGuinea pigs aged between six and seven months of age were randomized into control or treatment groups. Three- or four-month-old guinea pigs served as the young control group. The knees were administered 40 μl intra-articular injections of 10 nM PTH or vehicle once a week for three months. Their endurance as determined from time on the treadmill was evaluated before kill. Their tibial plateaus were analyzed using microcalculated tomography (μCT) and histological studies.ResultsPTH increased the endurance on the treadmill test, preserved glycosaminoglycans, and reduced Osteoarthritis Research Society International score and chondrocyte apoptosis rate. No difference was observed in the subchondral plate bone density or metaphyseal trabecular bone volume and bone morphogenetic 2 protein staining.ConclusionSubchondral bone is crucial in the initiation and progression of OA. Although previous studies have shown that subcutaneous PTH alleviates knee OA by improving subchondral and metaphyseal bone mass, we demonstrated that intra-articular PTH injections improved spontaneous OA by directly affecting the cartilage rather than the subchondral or metaphyseal bone in a preclinical age-related OA model.Cite this article: Bone Joint Res 2021;10(8):514–525.

Effects of Androgen Receptor Overexpression on Chondrogenic Ability of Rabbit Articular Chondrocytes.

The role of sex hormones and their receptors has drawn much attention in the process of cartilage regeneration. This study aimed to investigate the effect of androgen receptor (AR) on the chondrogenic ability of articular chondrocytes and the related mechanism. Articular chondrocytes were isolated, cultured, identified by toluidine blue staining and then transduced with lentivirus carrying the AR gene. The cell viability was determined using Cell Counting Kit-8, and cell apoptosis was assessed by flow cytometry analysis. The effects of AR overexpression on the expression of cartilage-specific proteins and some signalling molecules were evaluated by real-time PCR and Western blotting. Using 24 New Zealand rabbits, the regeneration of rabbit articular cartilage defects was further investigated in vivo and evaluated histologically. The overexpression of AR significantly reduced the apoptosis rate of chondrocytes but did not affect their proliferation. The overexpression of AR also promoted the expression of Sry-related HMG box 9, collagen II and aggrecan, decreased the expression of matrix metalloproteinase-13, and downregulated p-S6 and RICTOR. The experimental group with AR-overexpressing chondrocytes exhibited superior regeneration of cartilage defects. AR overexpression can maintain the phenotype of chondrocytes and promote chondrogenesis in vitro and in vivo. mTOR-related signalling was inhibited.

Dysregulation of Transcription Profile of Selenoprotein in Patients with Kashin-Beck Disease and Its Effect on Se Deficiency-Induced Chondrocyte Apoptosis.

Kashin-Beck disease (KBD) is a chronic, degenerative osteoarthropathy related to selenium (Se) deficiency. Se participates in the synthesis of selenoprotein in the form of selenocysteine. In total, 25 selenoproteins, encoded by 25 genes, are currently found in humans; however, the effects of selenoprotein genes on chondrocyte apoptosis, particularly in apoptosis-related genes, remain poorly elucidated. Therefore, in the current study, the expression of selenoprotein genes and apoptosis-related genes were determined by RT-qPCR in patients and chondrocytes and the correlations between them were analyzed using Pearson and Spearman's rank correlation, and the chondrocyte apoptosis rate was detected by Annexin V-FITC/PI. The results showed that the mRNA levels of 17 selenoprotein genes were downregulated, whereas two genes were upregulated in patients with KBD. The BAX/BCL2 ratio and the mRNA levels of BAX and P53 were increased, but the mRNA levels of BCL2 and NF-κB p65 were decreasedin patients with KBD. The mRNA levels of GPX2, GPX3, DIO1, TXNRD1, TXNRD3, and SPS2 were most closely associated with apoptosis-related genes in patients with KBD. Moreover, in the Se deficiency group, the mRNA levels of GPX3, DIO1, and TXNRD1 were downregulated and GPX activity was decreased, but the late apoptosis rate, the mRNA levels of BAX and P53, and the BAX/BCL2 ratio were increased; the opposite trend was observed in the Se supplement group. Collectively, these results indicate that selenoprotein transcription profile is dysregulated in patients with KBD. Furthermore, the expression of GPX3, DIO1, and TXNRD1 genes might be involved in the development of chondrocyte apoptosis by affecting antioxidant capacity.

BNIP3 decreases the LPS-induced inflammation and apoptosis of chondrocytes by promoting the development of autophagy

BackgroundInflammation and apoptosis of chondrocytes are the pathological bases of osteoarthritis. Autophagy could alleviate the symptoms of inflammation and apoptosis. Previous study has shown that BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) can induce the occurrence and development of autophagy. However, it is unknown whether autophagy induced by BNIP3 can alleviate the inflammation and apoptosis of chondrocytes.MethodsWe used the lentivirus to construct the overexpression BNIP3 chondrocytes. Next, the lipopolysaccharide (LPS) was used to stimulate these cells to simulate the physiological environment of osteoarthritis. After that, the enzyme-linked immunosorbent assays (ELISA) were performed to determine the levels of tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) and the flow cytometry was performed to detect the apoptosis rates of chondrocytes. At last, the expression of autophagy-related proteins was detected with the western blotting.ResultsThe expression of BNIP3 was suppressed after treatment with LPS. However, overexpression of BNIP3 inhibited the secretion of proinflammatory factors (TNF-α, IL-1β, and IL-6) and decreased the apoptosis of chondrocytes. Furthermore, overexpression of BNIP3 led to the upregulation of autophagy-related protein expression including little computer 3 (LC3), autophagy-related protein 7 (ATG7), and Beclin-1. Application of autophagy inhibitor recovered the expression of proinflammatory factors and apoptosis rates of chondrocytes.ConclusionsBNIP3 decreased the LPS-induced inflammation and apoptosis of chondrocytes by activating the autophagy.

Loss of autophagy in condylar chondrocytes causes increased apoptosis rate in temporomandibular joint osteoarthritis of rats

Objective: To observe the effect of autophagy of condylar chondrocytes on apoptosis in temporomandibular joint osteoarthritis (TMJOA) of rats. Methods: Fourty male 2-month-old SPF SD rats were equally divided into sham group (n=20) and experimental group (n=20). UAC metal prosthesis was cemented to the left incisors of maxilla and mandible of the rats in experimental group rats. After 8 weeks, all rats were sacrificed and the temporomandibular joint was taken. Two groups of rat condylar chondrocytes were extracted and cultured in vitro to the third generation. Immunofluorescence technique was used to detect the levels of collagen Ⅱ and matrix metalloproteinase-13 (MMP-13) in chondrocytes. The level of light chain-3 (LC-3), an autophagy marker of chondrocytes, was detected. Immunohistochemical technique was used to detect the level glycogenin-1, a glycogen formation marker of chondrocyte, was detected. The level of caspase-3, an apoptosis marker of chondrocyte, was also detected. Tunel technique was used to detect the apoptosis rate of the two groups at 72 h. Cracking cell extraction of total protein, Western-blotting (WB) technology to detect the levels of collagen Ⅱ, MMP -13, LC-3, glycogenin-1, caspase-3 and make gray analysis. Results: Compared with sham group, the level of collagen Ⅱ decreased, MMP-13 increased, LC-3 decreased, glycogenin-1 increased and caspase-3 increased in experimental group. The apoptosis rate of chondrocytes in experimentaal group [ (17.3±4.4) %] at 72h was higher than that in control group [ (5.6±2.1) %](t=10.732, P<0.001) .WB bands gray statistical results show that the level of collagen Ⅱ in chondrocytes of experimental group (0.43±0.21) was lower than that of control group (0.71±0.26) (t=2.409, P=0.043) , the level of MMP-13 in chondrocytes of experimental group (0.73±0.31) was higher than that of control group (0.24±0.10) (t=3.364, P=0.010) , the level of LC-3 in chondrocytes of experimental group (0.09±0.04) was lower than that of control group (0.39±0.18) (t=3.638, P=0.007) , the level of glycogenin-1 in chondrocytes of experimental group (0.68±0.30) was higher than that of control group (0.29±0.17) (t=2.529, P=0.035) , the level of caspase-3 in chondrocytes of experimental group (0.19±0.08) was higher than that of control group (0.05±0.02) (t=3.796, P=0.005) . Conclusions: The level of autophagy of condylar chondrocytes in temporomandibular joint of rats decreased, glycogen accumulation increased, the rate of chondrocyte apoptosis increased, and the number of chondrocytes decreased, resulting in degeneration of condylar cartilage tissue.

MicroRNA-375 exacerbates knee osteoarthritis through repressing chondrocyte autophagy by targeting ATG2B.

Objective: This study aimed to explore the underlying mechanism of miR-375 in exacerbating osteoarthritis (OA).Results: MiR-375 expression were upregulated in OA cartilage tissues, whereas ATG2B expression was decreased. MiR-375 targeted ATG2B 3’ UTR and inhibited its expression in the chondrocytes, and then suppressed autophagy and promoted endoplasmic reticulum stress (ERs). The apoptosis rate of chondrocytes was increased after being transfected with miR-375 mimics. In vivo results further verified that inhibition of miR-375 could relieve OA-related symptoms.Conclusion: miR-375 can inhibit the expression of ATG2B in chondrocytes, suppress autophagy and promote the ERs. It suggests that miR-375 could be considered to be a key therapy target for OA.Methods: Differential expression analyses for mRNA and miRNA microarray datasets from ArrayExpress were performed. MiR-375 and ATG2B expressions in cartilage tissues were detected by qRT-PCR. Dual luciferase assay was applied to verify the targeting relationship between ATG2B and miR-375. In vitro, the role of miR-375 on chondrocyte autophagy and ERs was investigated by western blot and immunofluorescence. The apoptotic rate was quantified by flow cytometry. In vivo, OA mice model was established, HE and Safranin O and Fast Green staining, as well as the OARSI and modified Mankin scores, were applied to measure the OA cartilage damage severity.

Nesfatin-1 suppresses interleukin-1β-induced inflammation, apoptosis, and cartilage matrix destruction in chondrocytes and ameliorates osteoarthritis in rats

Osteoarthritis (OA) is a chronic degenerative joint disease, related to the overexpression of matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), inflammation, and chondrocyte apoptosis. Nesfatin-1 is an adipokine, which plays an important role in the development of OA, especially in obese people. In the present study, cartilage degradation and apoptosis observed in OA patients was evaluated. Furthermore, the anti-inflammatory and anti-apoptotic effects of nesfatin-1, and its underlying in vitro and in vivo mechanisms were investigated. The results showed that nesfatin-1 increased significantly the expression of collagen type II alpha 1 chain (Col2a1), and reduced the expression of MMPs, ADAMTS5, cyclooxygenase (COX)-2, caspase-3, nitric oxide (NO), inducible nitric oxide synthase (iNOS), prostaglandin E2 (PGE2), interleukin (IL)-6, and chondrocyte apoptosis rate, which may be induced by IL-1β in rat chondrocytes. Furthermore, nesfatin-1 treatment prevented cartilage degeneration in the rat OA model. It was found that nesfatin-1 suppressed the IL-1β-induced activation of NF-κB, the mitogen-activated protein kinase (MAPK), and the Bax/Bcl-2 signal pathway in chondrocytes. These results suggest that in vivo nesfatin-1 could play a protective role in the development of OA and can be potentially used for its treatment.

Malfunction of autophagy in tibial growth plate chondrocytes causes increased apoptosis rate in chronic renal insufficiency rats

Objective: To observe the effect of autophagy of tibial growth plate chondrocyte on apoptosis in chronic renal insufficiency (CRI) rats. Methods: Male 4-week-old SD rats were randomly divided into two groups: (1) Sham group: only the left ureter was exposed (n=10); (2) CRI group: the left ureter was ligated to cause CRI (n=10). The urine from all the rats was collected 6 weeks after the operation and the total protein content was measured. Then all the rats were sacrificed and the concentrations of creatinine and urea nitrogen in intracardiac blood were detected. The proximal tibia were fixed and decalcified to prepare histological sections, and the number of chondrocytes of column cells in the proliferative area of tibia growth plate was observed by saffron O staining. The expression rate of protein Light Chain-3, an autophagy marker of chondrocytes, was detected by immunofluorescence. The apoptosis rate of chondrocytes was detected by the method of TUNEL assay. The level of glycogenin-1, a glycogen formation marker of chondrocyte was detected by immunohistochemistry in chondrocytes. Results: The 24 h urine total protein was higher in CRI group [(163.5±11.3) mg vs (38.6±9.8) mg, t=25.620, P<0.001]. The levels of blood creatinine [(67.3±16.2) μmol/L vs (28.4±11.5) μmol/L, t=5.974, P<0.001] and urea nitrogen [(16.4±6.4) mmol/L vs (4.8±2.0) mmol/L, t=5.198, P<0.001] were higher in CRI group. The number of chondrocytes of column cells in the proliferating area of tibia growth plate was lower in CRI group (4.2±2.1 vs 9.1±3.8, t=3.109, P=0.006). The expression rate of LC-3 protein in chondrocytes of CRI group was lower [(27.2±12.6)% vs (51.4±18.2)%, t=3.457, P=0.003]. The level of glycogenin-1 of chondrocytes in CRI group increased significantly (6.1±2.5 vs 3.5±1.8, t=2.669, P=0.016). The apoptosis rate of chondrocytes in CRI group also increased [(17.2±4.8)% vs (5.1±3.4)%, t=6.505, P<0.001]. Conclusion: Malfunction of autophagy in tibial growth plate chondrocytes causes increased apoptosis rate in CRI rats, which might be caused by the failure of glycogen degradation in chondrocytes.

FSTL1 promotes nitric oxide-induced chondrocyte apoptosis via activating the SAPK/JNK/caspase3 signaling pathway

ObjectivePrevious studies have shown that follistatin-like protein 1 (FSTL1) is elevated in the synovial fluid of osteoarthritis and is associated with disease activity. The experiment was performed to stuy the effect and mechanism of FSTL1 on chondrocyte apoptosis in osteoarthritis. DesignAfter the isolation of human normal and osteoarthritis (OA) chondrocytes, the expression of FSTL1 was detected by Q-PCR and western blot analyses. Chondrocytes were pre-transfected with FSTL1 overexpression plasmids then treated with SNP, and chondrocyte viability and apoptosis levels were detected by MTS and flow cytometry, respectively. Cartilage matrix gene expression was measured by Q-PCR and signal pathway-related proteins were assessed by western blot. ResultsThe expression of FSTL1 in OA chondrocytes was markedly up-regulated compared with normal human chondrocytes (P < 0.05). The apoptosis rate of chondrocytes in the FSTL1 overexpression groups was highly elevated in the comparison with the negative control groups (P < 0.05). Additionally, FSTL1 potentiated protein abundances of MMP1, MMP3, MMP-9, and Bax as well as reduced Coll2a1 and Aggrecan and Bcl-2 expression. Furthermore, western blot results showed that the SAPK/JNK/Caspase3 signal pathway was significantly activated and the Ac-DEVD-FMK impaired FSTL1 induced chondrocyte apoptosis. ConclusionFSTL1 promoted SNP-induced chondrocytes apoptosis by activating the SAPK/JNK/Caspase3 signal pathway.

Comparison of Apoptosis and Autophagy in Human Chondrocytes Induced by the T-2 and HT-2 Toxins.

In this report, we have investigated the apoptosis and autophagy of chondrocytes induced by the T-2 and HT-2 toxins. The viability of chondrocytes was measured by the MTT assay. Malondialdehyde (MDA) and superoxide dismutase (SOD) kits were used to measure the oxidative stress of chondrocytes. The apoptosis of chondrocytes was measured using flow cytometry. Hoechst 33258 and MDC staining agents were introduced to analyze apoptosis and autophagy induction in chondrocytes, respectively. Protein expression of Bax, caspase-9, caspase-3, and Beclin1 was examined by western blotting analysis. The T-2 and HT-2 toxins significantly decreased the viability of chondrocytes in a time-dependent manner. The level of oxidative stress in chondrocytes induced by the T-2 toxin was significantly higher when compared with that of the HT-2 toxin. The apoptosis rate of chondrocytes induced by the T-2 toxin increased from 3.26 ± 1.03%, 18.38 ± 1.28%, 34.5 ± 1.40% to 49.67 ± 5.31%, whereas apoptosis rate of chondrocytes induced by the HT-2 toxin increased from 3.82 ± 1.03%, 11.61 ± 1.27%, 25.72 ± 2.95% to 36.28 ± 2.81% in 48 h incubation time. Hoechst 33258 staining confirmed that apoptosis of chondrocytes induced by the T-2 toxin was significantly higher than that observed when the chondrocytes were incubated with the HT-2 toxin. MDC staining revealed that the autophagy rate of chondrocytes induced by the T-2 toxin increased from 6.38% to 63.02%, whereas this rate induced by the HT-2 toxin changed from 6.08% to 53.33%. The expression levels of apoptosis and autophagy related proteins, Bax, caspase-9, caspase-3, and Beclin1 in chondrocytes induced by the T-2 toxin were significantly higher when compared with those levels induced by the HT-2 toxin.

LncRNA‑p21 promotes chondrocyte apoptosis in osteoarthritis by acting as a sponge for miR‑451.

Osteoarthritis (OA) is the most common type of arthritis, and remains to be social and medical challenge. Thus, identifying novel molecular targets is important for the prevention and treatment of OA. Long noncoding RNAs (lncRNAs) have been reported to modulate various biological and pathological processes. The aim of the present study was to investigate the role of lncRNA‑p21 in OA and its underlying mechanism, in order to better understand the development of OA and its treatment. Chondrocytes were isolated from cartilage samples obtained from OA and normal patients. Chondrocytes were transfected with microRNA (miRNA/miR)‑451 mimics, miR‑451 inhibitor, pcDNA3.1(+)‑p21 or small interfering RNA‑p21. Flow cytometry was performed to analyze cell apoptosis and reverse transcription‑quantitative polymerase chain reaction was conducted to detect the expression of mRNAs and miRNAs. Cell Counting Kit‑8 assay was performed to detect cell viability. The results revealed that the level of lncRNA‑p21 was significantly upregulated in OA cartilage when compared with the normal cartilage. Silencing of lncRNA‑p21 increased cell viability and inhibited the apoptosis rate of chondrocytes in OA, while lncRNA‑p21 overexpression decreased cell viability and increased the apoptosis rate of chondrocytes in OA. Overexpression of lncRNA‑p21 suppressed the expression of miR‑451 while the silencing of lncRNA‑p21 reversed this effect. MiR‑451 inhibitor effectively inhibited the upregulatory effect of si‑p21 on miR‑451. The increased cell viability and decreased apoptosis rate induced by lncRNA‑p21 silencing was abolished by the miR‑451 inhibitor. MiR‑451 mimic effectively increased the downregulatory effect of pcDNA3.1‑lncRNA‑p21 on miR‑451. The decreased cell viability and increased apoptosis rate induced by the overexpression of lncRNA‑p21 was abolished by the miR‑451 mimic. Investigation into the underlying mechanism revealed that lncRNA‑p21 interacted with miRNA‑451. In addition, lncRNA‑p21 negatively regulated the expression of miR‑451. Furthermore, lncRNA‑p21 promoted the apoptosis of chondrocytes in OA by acting as a sponge for miR‑451. Thus, lncRNA‑p21 was proposed as a promising target for the treatment of OA.

Marked alterations in the structure, dynamics and maturation of growth plate likely explain growth retardation and bone deformities of young Hyp mice.

Mechanisms underlying growth impairment and bone deformities in X-linked hypophosphatemia are not fully understood. We here describe marked alterations in the structure, dynamics and maturation of growth plate in growth-retarded young Hyp mice, in comparison with wild type mice. Hyp mice exhibited reduced proliferation and apoptosis rates of chondrocytes as well as severe disturbance in the process of chondrocyte hypertrophy disclosed by abnormal expression of proteins likely involved in cell enlargement, irregular chondro-osseous junction and disordered bone trabecular pattern and vascular invasion in the primary spongiosa. (Hyp mice had elevated circulating FGF23 levels and over activation of ERK in the growth plate.) All these findings provide a basis to explain growth impairment and metaphyseal deformities in XLH. Hyp mice were compared with wild type mice serum parameters, nutritional status and growth impairment by evaluation of growth cartilage and bone structures. Hyp mice presented hyphosphatemia with high FGF23 levels. Weight gain and longitudinal growth resulted reduced in them with numerous skeletal abnormalities at cortical bone. It was also observed aberrant trabecular organization at primary spongiosa and atypical growth plate organization with abnormal proliferation and hypertrophy of chondrocytes and diminished apoptosis and vascular invasion processes. The present results show for the first time the abnormalities present in the growth plate of young Hyp mice and suggest that both cartilage and bone alterations may be involved in the growth impairment and the long bone deformities of XLH.

Smad2/3 is Negatively Associated with the Expression of Caspase-3 Protein and Apoptosis Rate of Chondrocytes in the Cartilage Tissues of the Patients with Osteoarthritis

Smad2/3 is Negatively Associated with the Expression of Caspase-3 Protein and Apoptosis Rate of Chondrocytes in the Cartilage Tissues of the Patients with Osteoarthritis