Tension regulates the cartilage phenotypic expression of endplate chondrocytes through the α-catenin/actin skeleton/Hippo pathway.

The study aimed to investigate the regulatory mechanism of intracellular tension signaling in endplate chondrocytes and its impact on extracellular matrix synthesis. Human endplate chondrocytes were subjected to tension load using Flexcell FX-5000™, and changes in phenotype, morphology, and the expression of Hippo signaling pathway and α-Catenin were assessed through various techniques. Through the overexpression of YAP and inhibition of α-Catenin, the study clarified the intracellular tension signaling pathway and its regulation of extracellular matrix synthesis in endplate cartilage. In vitro-cultured human endplate chondrocytes significantly suppressed phenotype-related genes and proteins, accompanied by distinct changes in cytoskeleton morphology. Tension activation resulted in the substantial activation of the Hippo pathway, increased phosphorylation of YAP, and reduced nuclear translocation of YAP. YAP overexpression alleviated the inhibitory effect of tension on extracellular matrix synthesis in endplate chondrocytes. Tension also upregulated the expression of α-Catenin in endplate chondrocytes, which was attenuated by inhibiting α-Catenin expression, thereby reducing the impact of tension on cytoskeletal morphology and YAP nuclear translocation. Taken together, the α-Catenin/actin skeleton/Hippo-coupled network is a crucial signaling pathway for tension signaling in endplate chondrocytes, providing potential therapeutic targets for the treatment of endplate cartilage degeneration.

METTL3 promotes IL-1ß-induced degeneration of endplate chondrocytes by driving m6A-dependent maturation of miR-126-5p.

METTL3 is an important regulatory molecule in the process of RNA biosynthesis. It mainly regulates mRNA translation, alternative splicing and microRNA maturation by mediating m6A-dependent methylation. Interleukin 1ß (IL-1ß) is an important inducer of cartilage degeneration that can induce an inflammatory cascade reaction in chondrocytes and inhibit the normal biological function of cells. However, it is unclear whether IL-1ß is related to METTL3 expression or plays a regulatory role in endplate cartilage degeneration. In this study, we found that the expression level of METTL3 and methylation level of m6A in human endplate cartilage with different degrees of degeneration were significantly different, indicating that the methylation modification of m6A mediated by METTL3 was closely related to the degeneration of human endplate cartilage. Next, through a series of functional experiments, we found that miR-126-5p can play a significant role in IL-1ß-induced degeneration of endplate chondrocytes. Moreover, we found that miR-126-5p can inhibit the PI3K/Akt signalling pathway by targeting PIK3R2 gene, leading to the disorder of cell vitality and functional metabolism. To further determine whether METTL3 could regulate miR-126-5p maturation, we first confirmed that METTL3 can bind the key protein underlying pri-miRNA processing, DGCR8. Additionally, when METTL3 expression was inhibited, the miR-126-5p maturation process was blocked. Therefore, we hypothesized that METTL3 can promote cleavage of pri-miR-126-5p and form mature miR-126-5p by combining with DGCR8.

circRNA_0058097 promotes tension-induced degeneration of endplate chondrocytes by regulating HDAC4 expression through sponge adsorption of miR-365a-5p.

Excessive mechanical tension can lead to the degeneration of endplate chondrocytes. The presence of tension-sensitive circRNA_0058097 molecules has been detected in human endplate chondrocytes, where it was found to be a potential competing endogenous RNA. Indeed, inhibiting the expression of circRNA_0058097 effectively enhanced the stress resistance of endplate chondrocytes, suggesting that it may be an important trigger point for the degeneration of endplate cartilage. Through a series of experiments, we reveal that circRNA_0058097 can upregulate the expression of downstream target gene histone deacetylase 4 by sponge adsorption of miR-365a-5p, which promoted morphological changes of endplate chondrocytes, and increased extracellular matrix degradation and degeneration of endplate cartilage. Therefore, circRNA_0058097 may provide a new way to prevent and treat endplate cartilage degeneration.

Melatonin protects vertebral endplate chondrocytes against apoptosis and calcification via the Sirt1-autophagy pathway.

Melatonin is reportedly associated with intervertebral disc degeneration (IDD). Endplate cartilage is vitally important to intervertebral discs in physiological and pathological conditions. However, the effects and mechanism of melatonin on endplate chondrocytes (EPCs) are still unclear. Herein, we studied the effects of melatonin on EPC apoptosis and calcification and elucidated the underlying mechanism. Our study revealed that melatonin treatment decreases the incidence of apoptosis and inhibits EPC calcification in a dose-dependent manner. We also found that melatonin upregulates Sirt1 expression and activity and promotes autophagy in EPCs. Autophagy inhibition by 3-methyladenine reversed the protective effect of melatonin on apoptosis and calcification, while the Sirt1 inhibitor EX-527 suppressed melatonin-induced autophagy and the protective effects of melatonin against apoptosis and calcification, indicating that the beneficial effects of melatonin in EPCs are mediated through the Sirt1-autophagy pathway. Furthermore, melatonin may ameliorate IDD in vivo in rats. Collectively, this study revealed that melatonin reduces EPC apoptosis and calcification and that the underlying mechanism may be related to Sirt1-autophagy pathway regulation, which may help us better understand the association between melatonin and IDD.

Apoptotic bodies from endplate chondrocytes enhance the oxidative stress-induced mineralization by regulating PPi metabolism.

This study aimed to investigate the role of apoptotic bodies (Abs) from the oxidative stressed endplate chondrocytes in regulating mineralization and potential mechanisms. Endplate chondrocytes were isolated from rats and treated with H2O2 to induce oxidative stress. The calcium deposition for matrix mineralization in the cells was examined by histological staining. The expression levels of calcification-related genes in individual groups of cells were determined by quantitative real time-PCR (qRT-PCR). Subsequently, extracellular vesicles (EVs) were purified and characterized. The effect of treatment with H2O2 and/or Abs on the mineralization, extracellular PPi metabolism and related gene expression were determined. Oxidative stress significantly increased the mineralization and promoted the generation of main Abs from endplate chondrocytes. Abs were effectively endocytosed by endplate chondrocytes and co-localized with collagen (COL)-II in the cytoplasm, which enhanced the mineralization, alkaline phosphatase (ALP), osteocalcin (OCN), Runt-related transcription factor 2 (RUNX2) and COL-I expression in endplate chondrocytes. Furthermore, treatment either H2O2 or Abs significantly decreased PPi, but increased Pi production and treatment with both further enhancing the changes in endplate chondrocytes. Similarly, treatment either H2O2 or Abs significantly decreased the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), and ankylosis protein (ANK) expression and ENPP1 promoter activity, but increased the tissue-nonspecific alkaline phosphatase (TNAP) expression and TNAP promoter activity in endplate chondrocytes. Oxidative stress promoted the generation of Abs, which might enhance the oxidative stress-mediated mineralization in endplate chondrocytes by regulating the PPi metabolism.

TGF-ß/SMAD signaling inhibits intermittent cyclic mechanical tension-induced degeneration of endplate chondrocytes by regulating the miR-455-5p/RUNX2 axis.

A mechanical stimulation plays a pivotal role in maintaining normal cartilage function. Our objective was to reveal the mechanism of action of the tension-sensitive molecule miR-455-5p in the degeneration of endplate chondrocytes and to identify whether the transforming growth factor beta (TGF-ß)/SMAD signaling pathway has a regulatory effect on it. The expression profiles of members of the TGF-ß/SMAD pathway, miR-455-5p, and RUNX2 were determined by microRNA microarray analysis, reverse transcription quantitative polymerase chain reaction, luciferase reporter assay, and Western blot analysis. Intermittent cyclic mechanical tension (ICMT) induced the degeneration of endplate chondrocytes without affecting their viability. The tension-sensitive molecule miR-455-5p specifically bound to RUNX2, a gene involved in the degeneration of endplate chondrocytes. Activation of the TGF-ß/SMAD signaling pathway upregulated miR-455-5p expression and thus inhibited RUNX2 levels. Therefore, the TGF-ß/SMAD signaling pathway inhibits the ICMT-induced degeneration of endplate chondrocytes by regulating the miR-455-5p/RUNX2 axis.

P120-Catenin Mediates Intermittent Cyclic Mechanical Tension-Induced Inflammation in Chondrocytes.

To study the role of the nuclear factor (NF)-κB signaling pathway and P120-catenin in the inflammatory effects of intermittent cyclic mechanical tension (ICMT) on endplate chondrocytes. Inflammatory reactions of endplate chondrocyte were measured by real-time reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assays, a dual-luciferase reporter assay system, immunofluorescence, and Western blot analysis. ICMT loading led to inflammatory reactions of endplate chondrocytes in both the rabbit endplate cartilage model and rat endplate chondrocytes in vitro. Inhibition of NF-κB signaling significantly ameliorated the inflammation induced by ICMT in endplate chondrocytes. Moreover, the expression of P120-catenin was decreased by ICMT. However, over-expression of P120-catenin suppressed NF-κB signaling and reversed the inflammatory effects. P120-catenin prevents endplate chondrocytes from undergoing ICMT-mediated inflammation by suppressing the expression of NF-κB. J. Cell. Biochem. 118: 4508-4516, 2017. © 2017 Wiley Periodicals, Inc.

Network Pharmacology and Experimental Validation to Elucidate the Pharmacological Mechanisms of Luteolin Against Chondrocyte Senescence.

BACKGROUND: Luteolin, a flavonoid found in various medicinal plants, has shown promising antioxidant, anti-inflammatory, and anti-aging properties. The cartilaginous endplate (CEP) represents a crucial constituent of the intervertebral disc (IVD), assuming a pivotal responsibility in upholding both the structural and functional stability of the IVD. OBJECTIVE: Exploring the precise mechanism underlying the protective effects of luteolin against senescence and degeneration of endplate chondrocytes (EPCs). METHODS: Relevant targets associated with luteolin and aging were obtained from publicly available databases. To ascertain cellular functions and signaling pathways, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were employed. Core genes were identified through the construction of a protein-protein interaction (PPI) network. Molecular docking (MD) was utilized to assess the binding affinity of luteolin to these core genes. Finally, the impact of luteolin on the senescence and degeneration of EPCs was evaluated in an in vitro cellular senescence model induced by tert-butyl hydroperoxide (TBHP). RESULTS: There are 145 overlapping targets between luteolin and senescence. Analysis using GO revealed that these targets primarily participate in cellular response to oxidative stress and reactive oxygen species. KEGG analysis demonstrated that these markers mainly associate with signaling pathways such as p53 and PI3K-Akt. MD simulations exhibited luteolin's binding affinity to P53, Cyclin-dependent kinase (CDK)2, and CDK4. Cell cycle, cell proliferation, and ß- galactosidase assays confirmed that luteolin mitigated senescence in SW1353 cells. Western blot assays exhibited that luteolin significantly suppressed the expression of Matrix Metallopeptidase (MMP) 13, P53, and P21, while concurrently promoting CDK2, CDK4, and Collagen Type II Alpha 1 (COL2A1) expression. CONCLUSION: In summary, luteolin demonstrated beneficial properties against aging and degeneration in EPCs, offering novel insights to mitigate the progression of intervertebral disc degeneration (IVDD).

miR-142-3p and HMGB1 Are Negatively Regulated in Proliferation, Apoptosis, Migration, and Autophagy of Cartilage Endplate Cells.

BACKGROUND: Cartilage endplate (CEP) degeneration plays a vital role in the pathological process of intervertebral disc degeneration. It has been previously reported that microRNAs may participate in the occurrence and development of intervertebral disc degeneration through regulating its target genes directly. The regulatory roles of miR-142-3p/HMGB1 in some orthopedic diseases have been determined successively, but there was no report about the degeneration of CEP. Therefore, we aimed to determine the regulation of miR-142-3p/HMGB1 or potential molecular mechanisms on proliferation, apoptosis, migration, and autophagy of CEP cells. METHODS: The target gene of miR-142-3p was determined by double luciferase assay. We selected ATDC5 cell lines. CCK-8 method was used to detect cell proliferation. Real-time fluorescence quantitative polymerase chain reaction was used to determine gene expression levels, and western blot analysis was used to determine protein expression levels. We chose flow cytometry to measure cell apoptosis and cell cycle. RESULTS: The result of luciferase detection showed that the target gene of miR-142-3p in CEP cells was HMGB1. Knockdown of the miR-142-3p inhibited the expression level of HMGB1, the proliferation and migration of CEP cells, but it promoted apoptosis of CEP cells. In addition, the detection results of the proteins related to apoptosis or autophagy showed that knockdown of miR-142-3p promoted apoptosis and autophagy. CONCLUSION: The negative regulation of miR-142-3p/HMGB1 can affect the proliferation, apoptosis, migration, and autophagy of CEP cells. Our results provide a new idea for the targeted treatment of CEP degeneration by inhibiting the expression of HMGB1.

Curcumin prevents tension-induced endplate cartilage degeneration by enhancing autophagy.

AIMS: Intermittent cyclic tension stimulation(ICMT) was shown to promote degeneration of endplate chondrocytes and induce autophagy. However, enhancing autophagy can alleviate degeneration partly. Studies have shown that curcumin can induce autophagy and protect chondrocytes, we speculated that regulation of autophagy by curcumin might be an effective method to improve the stress resistance of endplate cartilage. In this study, human cervical endplate cartilage specimens were collected, and expression of autophagy markers was detected and compared. MAIN METHODS: Human cervical endplate chondrocytes were cultured to establish a tension-induced degeneration model, for which changes of functional metabolism and autophagy levels were detected under different tension loading conditions. Changes in functional metabolism of endplate chondrocytes were observed under high-intensity tension loading in the presence of inhibitors, inducers, and curcumin to regulate the autophagy level of cells. In addition, a rat model of lumbar instability was established to observe the degeneration of lumbar disc after curcumin administration. KEY FINDINGS: Through a series of experiments, we found that low-intensity tension stimulation can maintain a stable phenotype of endplate chondrocytes, but high-intensity tension stimulation has a negative effect. Moreover, with increasing tension intensity, the degree of degeneration of endplate chondrocytes was gradually aggravated and the level of autophagy increased. Besides, curcumin upregulated autophagy, inhibited apoptosis, and reduced phenotype loss of endplate chondrocytes induced by high-intensity tension loading, thereby relieving intervertebral disc degeneration induced by mechanical imbalance. SIGNIFICANCE: Curcumin mediated autophagy and enhanced the adaptability of endplate chondrocytes to high-intensity tension load, thereby relieving intervertebral disc degeneration.

Parkin and Nrf2 prevent oxidative stress-induced apoptosis in intervertebral endplate chondrocytes via inducing mitophagy and anti-oxidant defenses.

AIMS: Endplate chondrocyte apoptosis is an important contributor to the pathogenesis of cartilaginous endplate (CEP) degeneration that leads to the initiation and development of intervertebral disc degeneration (IDD). In this study, we hypothesized that Parkin-mediated mitophagy and nuclear factor E2-related factor 2 (Nrf2)-mediated antioxidant system played an important role in endplate chondrocyte survival under pathological conditions. MATERIALS AND METHODS: Human endplate chondrocytes were stimulated with H2O2 to mimic pathological conditions. Western blotting, immunofluorescence staining, and flow cytometry were applied to detect the indicators related to mitochondrial dynamics, mitophagy, Nrf2 signaling, and apoptosis. The puncture-induced rat models were established to evaluate the changes in vivo. KEY FINDINGS: Our results showed that H2O2 induced oxidative stress, mitochondrial dysfunction, and apoptosis in endplate chondrocytes. These H2O2-induced detrimental effects were inhibited by pretreatment with the mitochondria-targeted antioxidant Mito-TEMPO. In addition, mitochondrial dynamics, Parkin-mediated elimination of dysfunctional mitochondria, and Nrf2-mediated antioxidant system were promoted by H2O2. Knockdown of Parkin or Nrf2 increased H2O2-induced detrimental effects. Moreover, upregulation of Parkin and Nrf2 by polydatin protected endplate chondrocytes against H2O2-induced mitochondrial dysfunction, oxidative stress, and apoptosis. Finally, puncture-induced rat models showed that polydatin exerted a protective effect on CEP and disc degeneration. SIGNIFICANCE: Targeting Parkin and Nrf2 to improve mitochondrial homeostasis, redox balance and endplate chondrocyte survival may represent a potential therapeutic strategy for preventing IDD.

MSC-Derived Exosomes Protect Vertebral Endplate Chondrocytes against Apoptosis and Calcification via the miR-31-5p/ATF6 Axis.

Apoptosis and calcification of endplate chondrocytes (EPCs) can exacerbate intervertebral disc degeneration (IVDD). Mesenchymal stem cell-derived exosomes (MSC-exosomes) are reported to have the therapeutic potential in IVDD. However, the effects and related mechanisms of MSC-exosomes on EPCs are still unclear. We aimed to investigate the role of MSC-exosomes on EPCs with a tert-butyl hydroperoxide (TBHP)-induced oxidative stress cell model and IVDD rat model. First, our study revealed that TBHP could result in apoptosis and calcification of EPCs, and MSC-exosomes could inhibit the detrimental effects. We also found that these protective effects were inhibited after miroRNA (miR)-31-5p levels were downregulated in MSC-exosomes. The target relationship between miR-31-5p and ATF6 was tested. miR-31-5p negatively regulated ATF6-related endoplasmic reticulum (ER) stress and inhibited apoptosis and calcification in EPCs. Our in vivo experiments indicated that sub-endplate injection of MSC-exosomes can ameliorate IVDD; however, after miR-31-5p levels were downregulated in MSC-exosomes, these protective effects were inhibited. In conclusion, MSC-exosomes reduced apoptosis and calcification in EPCs, and the underlying mechanism may be related to miR-31-5p/ATF6/ER stress pathway regulation.

Intermittent cyclic mechanical tension altered the microRNA expression profile of human cartilage endplate chondrocytes.

Previous studies have identified the association between cartilage endplate (CEP) degeneration and abnormal mechanical loading. Several studies have reported that intermittent cyclic mechanical tension (ICMT) regulates CEP degeneration via various biological processes and signaling pathways. However, the functions of microRNAs in regulating the cellular responses of CEP chondrocytes to ICMT remain to be elucidated. The current study determined the differentially expressed microRNAs in human CEP chondrocytes exposed to ICMT using microarray analysis. A total 21 significantly upregulated and 62 downregulated miRNAs were identified compared with the control. The findings were subsequently partially validated by reverse transcription­quantitative polymerase chain reaction. Potential target genes of the significantly differentially expressed miRNAs were predicted using bioinformatics analysis and were used for Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The present study revealed that the significantly differentially expressed microRNAs were involved in various signaling pathways and biological processes that are crucial to regulating the responses of CEP chondrocytes to ICMT. The current study provided a global view of microRNA expression in CEP chondrocytes under mechanical stimulation, suggesting that microRNAs are important for regulating the mechanical response of CEP chondrocytes. Additionally, it provided a novel insight into the association between mechanical stress and the establishment and progression of intervertebral disc degeneration.

Degree of endplate chondrocyte degeneration in different tension regions during mechanical stimulation.

The aim of this study was to explore the degree of degeneration of endplate chondrocytes in different tension regions induced by intermittent cyclic mechanical tension (ICMT) in vitro. Rat endplate chondrocytes were harvested and treated with 10% ICMT for 8 h/day with a frequency of 0.5 Hz. A cartilage degeneration model was induced using an FX­5000T cell strain­loading system. The experiment was divided into the central region and the peripheral region, according to the contact area between the loading post and the six­well flexible silicone rubber BioFlex plates. Toluidine blue and phalloidin staining were used to observe the morphological changes of cells following mechanical stimulation. Apoptosis was detected by flow cytometry and the mRNA and protein expression levels of collagen type II α1, aggrecan, SRY­box 9 and matrix metalloproteinase 13 were detected by reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blotting, respectively. Endplate chondrocytes exhibited degenerative alterations under mechanical conditions of 10% ICMT and 0.5 Hz at 8 h/day. Toluidine blue and phalloidin staining demonstrated that the cells in the peripheral region were more slender compared with cells in the central region, but RT­qPCR and western blotting results demonstrated that the degree of cell degeneration between the two groups was not statistically differences. So that cell morphological alteration does not imply that cells have undergone degeneration.

[Expression and significance of hypoxia-inducible factor 1α in endplate chondrocytes of rats].

Objective: To explore the expression and significance of hypoxia-inducible factor 1α (HIF-1α) in endplate chondrocytes, and to study the relations between HIF-1α expression and endplate chondrocytes apoptosis. Methods: Eight Sprague Dawley rats were selected to obtain the L 1-5 intervertebral disc endplate; the endplate chondrocytes were isolated by enzyme digestion method, and the endplate chondrocytes at passage 3 were cultured under 20% O 2 condition (group A), and under 0.5% O 2 condition (group B). Cell morphology was observed by inverted phase contrast microscope and cell apoptosis was detected using flow cytometry after cultured for 24 hours; the mRNA expression of HIF-1α was detected by real-time fluorescent quantitative PCR, the protein expressions of HIF-1α, Bax, and Bcl-2 by Western blot. Gene clone technology to design and synthesize two siRNAs based on the sequence of HIF-1α mRNA. HIF-1α specific RNAi sequence compound was constructed and transfected into cells. The transfected endplate chondrocytes at passage 3 were cultured under 0.5% O 2 condition in group C and group D (HIF-1α gene was silenced). After cultured for 24 hours, cells were observed via immunofluorescence staining of HIF-1α, and cell apoptosis was detected using flow cytometry. Meanwhile, the mRNA expressions of HIF-1α, collagen type II (COL II), Aggrecan, and SOX9 were detected by real-time fluorescent quantitative PCR, and the protein expressions of HIF-1α, Bax, and Bcl-2 by Western blot. Results: At 24 hours after culture, small amount of vacuoles necrotic cells could be observed in group A and group B; there was no significant difference in apoptosis rate between groups A and B ( t=1.026, P=0.471), and HIF-1α mRNA and protein expressions in group B were significantly higher than those in group A ( t=22.672, P=0.015; t=18.396, P=0.013), but, there was no significant difference in protein expressions of Bax and Bcl-2 between groups A and B ( t=0.594, P=0.781; t=1.251, P=0.342). The number of vacuolar necrosis cells in group D was significantly higher than that in group C, and HIF-1α positive cells were observed in group D. The apoptosis rate of group D was significantly higher than that of group C ( t=27.143, P=0.002). The mRNA expressions of HIF-1α, COL II, Aggrecan, and SOX9 in group D were significantly lower than those in group C ( t=21.097, P=0.015; t=34.829, P=0.002; t=18.673, P=0.022; t=31.949, P=0.007). The protein expressions of HIF-1α and Bcl-2 in group D were significantly lower than those in group C ( t=37.648, P=0.006; t=16.729, P=0.036), but the protein expression of Bax in group D was significantly higher than that in group C ( t=25.583, P=0.011). Conclusion: HIF-1α mRNA expression is up-regulated under hypoxia condition, which will increase the hypoxia tolerance of endplate chondrocytes. Cell apoptosis is suppressed by the activation of HIF-1α in endplate chondrocytes under hypoxia condition.

[Effect of Melittin on collagen type II expression of rat endplate chondrocytes induced by interleukin 1ß].

Objective: To observe the effect of Melittin on collagen type II (Col-II) expression of rat endplate chondrocytes (EPCs) induced by interleukin 1ß (IL-1ß). Methods: Primary EPCs from the lumbar vertebra of 4-week-old Sprague Dawley rats were cultured in vitro and identified by morphological observation, toluidine blue staining and Col-II immunofluorescence staining. Then, MTT assay was used to determine the optimal concentration of IL-1 and Melittin. Next, EPCs at passage 3 were randomly divided into 4 groups: no treatment was done in group A as control group; the optimal concentration of IL-1ß, Melittin, and both IL-1ß and Melittin were used in groups B, C, and D respectively. The expression of Col-II was detected by Western blot after 48 hours intervention. Results: Under inverted microscope, the first generation EPCs were polygonal; cell proliferation decreased after fifth generation, and cell morphology changed into fusiform. The acidic mucosubstance in the cytoplasm (such as Aggrecan) was stained dark blue by toluidine blue. After marking Col-II by immunofluorescence, the positive expression of cytoskeleton (green fluorescence) could be observed. MTT assay showed that IL-1ß and Melittin could inhibit the EPCs in a dose-dependent manner after intervention of 24 and 48 hours, and the optimal concentrations of IL-1ß and Melittin intervention were 10 ng/mL and 1.0 µg/mL respectively. Compared with group A, the expression of Col-II was significantly reduced in group B, and was significantly increased in group C by Western blot assay, but there was no significant difference between group D and group A. The Col-II expression levels of groups A, B, C, and D were 0.991±0.024, 0.474±0.127, 1.913±0.350, and 1.159±0.297 respectively, showing significant difference between the other groups ( P<0.05) except between group A and group D ( P>0.05). Conclusion: Melittin has a protective effect on endplate cartilage, and the research results provide experimental basis for the prevention and treatment of spinal degenerative disease.

Involvement of acid-sensing ion channel 1a in matrix metabolism of endplate chondrocytes under extracellular acidic conditions through NF-κB transcriptional activity.

Acidic conditions are present in degenerated intervertebral discs and are believed to be responsible for matrix breakdown. Acid-sensing ion channel 1a (ASIC1a) is expressed in endplate chondrocytes, and its activation is associated with endplate chondrocyte apoptosis. However, the precise role of ASIC1a in regulating the matrix metabolic activity of endplate chondrocytes in response to extracellular acid remains poorly understood. Aggrecan (ACAN), type II collagen (Col2a1), and matrix metalloproteinase (MMP) expressions were determined using reverse transcription (RT)-PCR and Western blot. ASIC1a was knocked down by transfecting endplate chondrocytes with ASIC1a siRNA. MMP activity and NF-κB transcriptional activity were measured. NF-κB transcriptional activity was assessed by examining cytosolic phosphorylated IκBα and nuclear phosphorylated p65 levels. Extracellular acidic solution (pH 6.0) resulted in a decrease in ACAN and Co12a1 expressions and an increase in MMP-1, MMP-9, and MMP-13 expressions, as well as in MMP activity; while ASIC1a siRNA blocked these effects. In addition, acid-induced increase in cytosolic levels of phosphorylated IκBα and nuclear levels of phosphorylated p65 in endplate chondrocytes were inhibited by ASIC1a siRNA. ASIC1a is involved in matrix metabolism of endplate chondrocytes under extracellular acidic conditions via NF-κB transcriptional activity.

The evidence and the possible significance of autophagy in degeneration model of human cervical end-plate cartilage.

The aim of this study was to observe autophagy in chondrocytes from degenerative human cervical vertebral end-plates and to investigate the significance of variations in autophagy in the degeneration of cervical vertebral end-plate chondrocytes. Cartilage end-plates were obtained from 48 inpatients admitted to hospital between February 2011 and August 2012. The patients were divided into the control group (n=17) with cervical vertebral fracture or dislocation and the cervical spondylosis group (n=31) with cervical spondylotic myelopathy. End-plate chondrocytes were isolated via enzyme digestion and then cultured in vitro. The cells were stained with toluidine blue and hematoxylin-eosin (H&E). A laser scanning confocal microscope and monodansylcadaverine (MDC) were used to reveal autophagy in the end-plate chondrocytes. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect mRNA expression of type II collagen and aggrecan. Western blotting was conducted to detect LC3 proteins. The chondrocytes isolated from the degenerative human cervical end-plates were cultured successfully in vitro. The morphology of the cells from the cervical spondylosis group tended to exhibit changes in spindle morphology compared with the control group. Autophagic bodies were stained with MDC. LC3 proteins were visible in the intracellular and perinuclear regions under the laser scanning confocal microscope. The mRNA expression levels (relative to those of ß-actin) of aggrecan (0.715±0.194) and type II collagen (0.628±0.254) in the cervical spondylosis group were markedly decreased compared with those in the control group (0.913±0.254 and 0.845±0.186, respectively; both P<0.05). The LC3-II/LC3-I ratio was observed to be significantly reduced in the cervical spondylosis group by Western blot analysis. Autophagy has an important role in human cervical disc degeneration. The regulation of autophagy may prevent disc degeneration in cartilage end-plate cells.

Ovarian cancer G protein-coupled receptor 1 is involved in acid-induced apoptosis of endplate chondrocytes in intervertebral discs.

Ovarian cancer G protein-coupled receptor 1 (OGR1) has been shown to be a receptor for protons. We investigated the role of proton-sensing G protein-coupled receptors in the apoptosis of endplate chondrocytes induced by extracellular acid. The expression of proton-sensing G protein-coupled receptors was examined in rat lumbar endplate chondrocytes. Knockdown of OGR1 was achieved by transfecting chondrocytes with specific short hairpin RNA (shRNA) for OGR1. Apoptotic changes were evaluated by DNA fragmentation ELISA, electron microscopy, and flow cytometry. Intracellular calcium ([Ca(2+) ]i) was analyzed with laser scanning confocal microscopy. The mechanism of OGR1 in acid-induced apoptosis of endplate chondrocytes was also investigated. We found that OGR1 was predominantly expressed in rat endplate chondrocytes, and its expression was highly upregulated in response to acidosis. Knocking down OGR1 with shRNAs effectively attenuated acid-induced apoptosis of endplate chondrocytes and increased [Ca(2+) ]i. Blocking OGR1-mediated [Ca(2+) ]i elevation inhibited acid-induced calcium-sensitive proteases such as calpain and calcineurin, and also inhibited the activation of Bid, Bad, and Caspase 3 and cleavage of poly (ADP-ribose) polymerase (PARP). OGR1-mediated [Ca(2+) ]i elevation has a crucial role in apoptosis of endplate chondrocytes by regulating activation of calcium-sensitive proteases and their downstream signaling.