Low selenium and T-2 toxin may be involved in the pathogenesis of Kashin-Beck disease by affecting AMPK/mTOR/ULK1 pathway mediated autophagy.

Kashin-Beck disease (KBD) is an endemic, environmentally associated cartilage disease. Previous studies have shown that the environmental suspected pathogenic factors of KBD, T-2 toxin and low selenium, are involved in the regulation of inflammation, oxidative stress and autophagy in some tissues and organs. In cartilage diseases, the level of cellular autophagy determines the fate of the chondrocytes. However, whether autophagy is involved in KBD cartilage lesions, and the role of low selenium and T-2 toxins in KBD cartilage injury and autophagy are still unclear. This work took the classical AMPK/mTOR/ULK1 autophagy regulatory pathway as the entry point to clarify the relationship between the environmental suspected pathogenic factors and chondrocyte autophagy. Transmission electron microscopy was used to observe the autophagy of chondrocytes in KBD patients. qRT-PCR and western blot were used to analyze the expression of AMPK/mTOR/ULK1 pathway and autophagy markers. The rat model of KBD was established by low selenium and T-2 toxin, the autophagy in rat cartilage was detected after 4- and 12-week interventions. Chondrocyte autophagy was found in KBD, and the AMPK/mTOR/ULK1 pathway was down-regulated. In the rat model, the pathway showed an up-regulated trend when low selenium and T-2 toxin, were treated for a short time or low concentration, and autophagy level increased. However, when low selenium and T-2 toxin were treated for a long time or at high concentrations, the pathway showed a down-regulated trend, and the autophagy level was reduced and even defective. In conclusion, in the process of KBD cartilage lesion, chondrocyte autophagy level may increase in the early stage, and decrease in the late stage with the progression of lesion. Low selenium and T-2 toxins may affect autophagy by AMPK/mTOR/ULK1 pathway.

WISP1 Is Involved in the Pathogenesis of Kashin-Beck Disease via the Autophagy Pathway.

OBJECTIVE: Kashin-Beck disease (KBD) is a kind of endemic and chronic osteochondropathy in China. This study aims to explore the functional relevance and potential mechanism of Wnt-inducible signaling pathway protein 1 (WISP1) in the pathogenesis of KBD. DESIGN: KBD and control cartilage specimens were collected for tissue section observation and primary chondrocyte culture. Firstly, the morphological and histopathological observations were made under a light and electron microscope. Then, the expression levels of WISP1 as well as molecular markers related to the autophagy pathway and extracellular matrix (ECM) synthesis were detected in KBD and control chondrocytes by qRT-PCR, Western blot, and immunohistochemistry. Furthermore, the lentiviral transfection technique was applied to make a WISP1 knockdown cell model based on KBD chondrocytes. In vitro intervention experiments were conducted on the C28/I2 human chondrocyte cell line using human recombinant WISP1 (rWISP1). RESULTS: The results showed that the autolysosome appeared in the KBD chondrocytes. The expression of WISP1 was significantly higher in KBD chondrocytes. Additionally, T-2 toxin, a risk factor for KBD onset, could up-regulate the expression of WISP1 in C28/I2. The autophagy markers ATG4C and LC3II were upregulated after the low-concentration treatment of T-2 toxin and downregulated after the high-concentration treatment. After knocking down WISP1 expression in KBD chondrocytes, MAP1LC3B decreased while ATG4C and COL2A1 increased. Moreover, the rWISP1 protein treatment in C28/I2 chondrocytes could upregulate the expression of ATG4C and LC3II at the beginning and downregulate them then. CONCLUSIONS: Our study suggested that WISP1 might play a role in the pathogenesis of KBD through autophagy.

A Metabolomics Study of Feces Revealed That a Disturbance of Selenium-Centered Metabolic Bioprocess Was Involved in Kashin-Beck Disease, an Osteoarthropathy Endemic to China.

Background: Kashin-Beck disease (KBD) is a distinct osteoarthropathy in China with an unclear pathogenesis. This study aims to explore whether perturbations in the intestine metabolome could be linked to KBD individuals. Methods: An investigation was conducted in KBD endemic villages and fecal samples were collected. After applying inclusion and exclusion criteria, a total of 75 subjects were enrolled for this study, including 46 KBD (including 19 Grade I KBD and 27 Grade II KBD) and 29 controls. Untargeted metabolomics analysis was performed on the platform of UHPLC-MS. PLS-DA and OPLS-DA were conducted to compare the groups and identify the differential metabolites (DMs). Pathway analysis was conducted on MPaLA platform to explore the functional implication of the DMs. Results: Metabolomics analysis showed that compared with the control group, KBD individuals have a total of 584 differential metabolites with dysregulated levels such as adrenic acid (log2FC = -1.87, VIP = 4.84, p = 7.63 × 10-7), hydrogen phosphate (log2FC = -2.57, VIP = 1.27, p = 1.02 × 10-3), taurochenodeoxycholic acid (VIP = 1.16, log2FC = -3.24, p = 0.03), prostaglandin E3 (VIP = 1.17, log2FC = 2.67, p = 5.61 × 10-4), etc. Pathway analysis revealed several significantly perturbed pathways associated with KBD such as selenium micronutrient network (Q value = 3.11 × 10-3, Wikipathways), metabolism of lipids (Q value = 8.43 × 10-4, Reactome), free fatty acid receptors (Q value = 3.99 × 10-3, Reactome), and recycling of bile acids and salts (Q value = 2.98 × 10-3, Reactome). Subgroup comparisons found a total of 267 differential metabolites were shared by KBD vs. control, KBD II vs. control, and KBD I vs. control, while little difference was found between KBD II and KBD I (only one differential metabolite detected). Conclusions: KBD individuals showed distinct metabolic features characterized by perturbations in lipid metabolism and selenium-related bioprocesses. Our findings suggest that the loss of nutrients metabolism balance in intestine was involved in KBD pathogenesis. Linking the nutrients metabolism (especially selenium and lipid) to KBD cartilage damage should be a future direction of KBD study.

Analysis of N-glycosylation protein of Kashin-Beck disease chondrocytes derived from induced pluripotent stem cells based on label-free strategies with LC-MS/MS.

We aimed to compare N-glycosylation proteins in Kashin-Beck disease (KBD) chondrocytes and normal chondrocytes derived from induced pluripotent stem cells (iPSCs). KBD and normal iPSCs were reprogrammed from human KBD and normal dermal fibroblasts, respectively. Subsequently, chondrocytes were differentiated from KBD and normal iPSCs separately. Immunofluorescence was utilized to assay the protein markers of iPSCs and chondrocytes. Differential N-glycosylation proteins were screened using label-free strategies with LC-MS/MS. Bioinformatics analyses were utilized to interpret the functions of differential N-glycosylation proteins. Immunofluorescence staining revealed that both KBD-iPSCs and normal-iPSCs strongly expressed pluripotency markers OCT4 and NANOG. Meanwhile, chondrocyte markers collagen II and SOX9 are presented in KBD-iPSC-chondrocytes and normal-iPSC-chondrocytes. We obtained 87 differential N-glycosylation sites which corresponded to 68 differential proteins, which were constructed into 1 cluster. We obtained collagen type I trimer and 9 other biological processes; polysaccharide binding and 9 other molecular functions; regulation of transcription by RNA polymerase II and 9 other cellular components from GO; the Pl3K-Akt signaling pathway and 9 other KEGG pathways; peroxisome and 7 other subcellular locations; and integrin alpha chain, C-terminal cytoplasmic region, conserved site and 9 other classifications of domain annotations, and 2 networks. FGFR3 and LRP1 are expressed at higher levels in KBD-iPSC-chondrocytes, while the expressions of COL2A1, TIMP1, UNC5B, NOG, LEPR, and ITGA1 were down-regulated in KBD-iPSC-chondrocytes. The differential expressions of these N-glycosylation proteins may lead to the abnormal function of KBD chondrocytes.

A method for Kashin-Beck disease auxiliary diagnosis based on the features in regions of the potential lesion.

BACKGROUND: Kashin-Beck disease (KBD) is a severe arthropathy that causes deformity. Patients with advanced stages of KBD often show symptoms, such as short stature. Early-stage diagnosis and treatment can effectively prevent the disease from worsening. Diagnosis of early-stage patients is usually made by X-ray examination. However, the time-consuming image recognition and the lack of professional doctors may delay the patient's condition. Therefore, a method that can efficiently complete the auxiliary diagnosis is necessary. PURPOSE: This study presents a KBD auxiliary diagnosis method based on radiographs, which uses deep learning to locate potential lesion regions and extract features for accurate diagnosis. METHODS: This work presents a method that relies on hand radiographs to locate eight regions of the potential lesion (RoPL) and finally make the KBD auxiliary diagnosis. The localization of RoPL is achieved through a two-step model, with the first step predicting a rough location and a deep convolutional neural network (DCNN) with attention mechanism used to generate precise center coordinates based on the previous step's results. Based on the localization result, regional features are extracted, which provides information about the joints and textures of RoPL from a finer granularity. Another DCNN is utilized to obtain general features from hand radiographs, which provide morphological and structural information about the entire hand bone These features offer a concatenated feature for categorization to raise accuracy. A doctor-like approach is adopted to diagnose based on regional features to enhance performance, and a final decision is made using a vote that considers diagnostic outcomes from all aspects. The dataset used in our study was collected by our research team in KBD-endemic areas of Tibet since 2017, containing 373 diseased and 764 normal images. RESULT: Our model guarantees that over 95% of the predicted coordinates are within five pixels of the real coordinates according to Euclidean distance. The accuracy of the diagnostic network achieved 91.3%, with precision and recall achieving 83% and 87%, respectively. Compared to the approach without exact localization of the illness region on the same test set, our method achieved a roughly 6% increase in accuracy and nearly 30% increase in recall rate. CONCLUSIONS: Based on hand radiographs, this study suggests a novel method for KBD diagnosis. The high-precision localization network guarantees precise extraction of lesion-prone regional features, and the multi-scale features and innovative classification method further enhance model performance compared to related approaches.

Identification of N-Glycoproteins of Knee Cartilage from Adult Osteoarthritis and Kashin-Beck Disease Based on Quantitative Glycoproteomics, Compared with Normal Control Cartilage.

Glycoproteins are involved in the development of many diseases, while the type and content of N-glycoproteins in the cartilage of osteoarthritis (OA) and Kashin-Beck disease (KBD) are still unclear. This research aims to identify N-glycoproteins in knee cartilage patients with OA and KBD compared with normal control (N) adults. The cartilage samples were collected from gender- and age-matched OA (n = 9), KBD (n = 9) patients, and N (n = 9) adults. Glycoproteomics and label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) obtained N-glycoproteins of KBD and OA. A total of 594 N-glycoproteins and 1146 N-glycosylation peptides were identified. The identified data were further compared and analyzed with Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Protein-Protein Interactions (PPI). Pairwise comparison of the glycoproteins detected in the three groups showed that integrin beta-1 (ITGB1), collagen alpha-1 (II) chain (COL2A1), collagen alpha-1 (VII) chain (COL7A1), carbohydrate sulfotransferase 3 (CHST-3), carbohydrate sulfotransferase 4 (CHST-4), thrombospondin 2 (THBS2), bone morphogenetic protein 8A (BMP8A), tenascin-C (TNC), lysosome-associated membrane protein (LAMP2), and beta-glucuronidase (GUSB) were significantly differentially expressed. GO results suggested N-glycoproteins mainly belonged to protein metabolic process, single-multicellular and multicellular organism process, cell adhesion, biological adhesion, and multicellular organism development. KEGG and PPI results revealed that key N-glycoproteins were closely related to pathways for OA and KBD, such as phagosome, ECM-receptor interaction, lysosome, focal adhesion, protein digestion, and absorption. These results reflected glycoprotein expression for OA and KBD in the process of ECM degradation, material transport, cell-cell or cell-ECM interaction, and information transduction. These key significantly differentially expressed N-glycoproteins and pathways lead to the degeneration and degradation of the cartilage of OA and KBD mainly by disrupting the synthesis and catabolism of basic components of ECM and chondrocytes and interfering with the transfer of material or information. The key N-glycoproteins or pathways in this research are potential targets for pathological mechanisms and therapies of OA and KBD.

Serum proteomic analysis of differentially expressed proteins and pathways involved in the mechanism of endemic osteoarthritis.

Kashin-Beck disease (KBD) is a chronic and endemic osteochondral disease and the etiology and pathogenic mechanism of KBD are still unknown. This study aimed to elucidate and screen KBD-associated proteins, which were differentially expressed between KBD patients and healthy controls. We combined protein fractionation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a high-resolution mass spectrometer coupled with tandem mass tags (TMTs) to quantitatively analyze and screen KBD-associated proteins, which were differentially expressed between KBD patients and healthy controls. In addition, we used parallel reaction monitoring (PRM) to quantify proteins in serum from patients with KBD and healthy controls in order to verify the differentially expressed proteins in patients with KBD. We identified 224 differentially expressed proteins, including 11 up-regulated and 213 down-regulated proteins. Catalase (CAT) was observed to be significantly elevated in patients with KBD compared with control patients. Further, the fold difference of CAT is significantly elevated in PRM compared with label-free quantification. The results in this study suggest that CAT may be the reflection of the dynamic nature of KBD and could be considered as a novel pathogenic indicator for patients with KBD.

Identification of proteins and N-glycosylation sites of knee cartilage in Kashin-Beck disease compared with osteoarthritis.

The aim of this study was to identify crucial proteins and N-glycosylated sites in the pathological mechanism of Kashin-Beck disease (KBD) compared with osteoarthritis (OA). Nine KBD knee subjects and nine OA knee subjects were selected for the study. Quantitative proteomics and N-glycoproteomics data of KBD and OA were obtained by protein and N-glycoprotein enrichment and LC-MS/MS analysis. Differentially expressed proteins or N-glycosylation sites were examined with a comparative analysis between KBD and OA. Total 2205 proteins were identified in proteomic analysis, of which 375 were significantly different. Among these, 121 proteins were up-regulated and 254 were down-regulated. In N-glycoproteomic analysis, 278 different N-glycosylated sites that were related to 187 N-glycoproteins were identified. Proteins and their N-glycosylated sites are associated with KBD pathological process including ITGB1, LRP1, ANO6, COL1A1, MXRA5, DPP4, and CSPG4. CRLF1 and GLG1 are proposed to associate with both KBD and OA pathological processes. Key pathways in KBD vs. OA proteomic and N-glycoproteomic analysis contained extracellular matrix receptor interaction, focal adhesion, phagosome, protein digestion, and absorption. N-glycosylation may influence the pathological process by affecting the integrity of chondrocytes or cartilage. It regulated the intercellular signal transduction pathway, which contributes to cartilage destruction in KBD.

The first human induced pluripotent stem cell line of Kashin-Beck disease reveals involvement of heparan sulfate proteoglycan biosynthesis and PPAR pathway.

Kashin-Beck disease (KBD) is an endemic osteochondropathy. Due to a lack of suitable animal or cellular disease models, the research progress on KBD has been limited. Our goal was to establish the first disease-specific human induced pluripotent stem cell (hiPSC) cellular disease model of KBD, and to explore its etiology and pathogenesis exploiting transcriptome sequencing. HiPSCs were reprogrammed from dermal fibroblasts of two KBD and one healthy control donor via integration-free vectors. Subsequently, hiPSCs were differentiated into chondrocytes through three-week culture. Gene expression profiles in KBD, normal primary chondrocytes, and hiPSC-derived chondrocytes were defined by RNA sequencing. A Venn diagram was constructed to show the number of shared differentially expressed genes (DEGs) between KBD and normal. Gene oncology and Kyoto Encyclopedia of Genes and Genomes annotations were performed, and six DEGs were further validated in other individuals by RT-qPCR. KBD cellular disease models were successfully established by generation of hiPSC lines. Seventeen consistent and significant DEGs present in all compared groups (KBD and normal) were identified. RT-qPCR validation gave consistent results with the sequencing data. Glycosaminoglycan biosynthesis-heparan sulfate/heparin; PPAR signaling pathway; and cell adhesion molecules (CAMs) were identified to be significantly altered in KBD. Differentiated chondrocytes derived from KBD-origin hiPSCs provide the first cellular disease model for etiological studies of KBD. This study also provides new sights into the pathogenesis and etiology of KBD and is likely to inform the development of targeted therapeutics for its treatment.

Mechanism of DNA methylation-mediated downregulation of O6-methylguanine-DNA methyltransferase in cartilage injury of Kashin-Beck disease.

OBJECTIVE: Kashin-Beck disease (KBD) is an endemic osteoarthropathy, in which excessive apoptosis of chondrocytes occurs. O6-methylguanine-DNA methyltransferase (MGMT), a DNA damage repair gene, plays an important role in apoptosis, but the mechanism is unclear in KBD cartilage injury. This study was to investigate the expression and promoter methylation of MGMT in KBD patients and its role in DNA damage and apoptosis of chondrocytes. METHODS: MGMT mRNA and protein level were detected by quantitative real-time PCR and immunohistochemistry. Demethylation of MGMT was carried out using 5-Aza-2'-deoxycytidine, and the methylation level of MGMT promoter was measured by quantitative methylation specific PCR. Next, small hairpin RNA was used to knockdown the expression of MGMT. Cell viability, apoptosis and DNA damage were determined by MTT assay, flow cytometry, Hoechst 33342 staining and alkaline comet assay following T-2 toxin and selenium treatment. RESULTS: MGMT protein expression and mRNA levels were decreased (P = 0.02, P = 0.007) and promoter methylation was increased (P = 0.008) in KBD patients. Meanwhile, MGMT level was upregulated by 5-Aza-2'-deoxycytidine in chondrocytes (P = 0.0002). DNA damage and apoptosis rates were increased in MGMT-silenced chondrocytes (all P < 0.0001). Furthermore, DNA damage and apoptosis were increased in chondrocytes treated with T-2 toxin (all P < 0.0001), but were decreased after selenium treatment (P < 0.0001, P = 0.01). Decreased mRNA level and increased methylation of MGMT were found in the T-2 toxin group (P = 0.005, P = 0.002), while selenium reversed it (P = 0.02, P = 0.004). CONCLUSIONS: MGMT might play a crucial part in the pathogenesis of KBD cartilage injury, which could provide a therapeutic target for KBD.

Downregulation of Insulin-Like Growth Factor-1 Receptor Mediates Chondrocyte Death and Matrix Degradation in Kashin-Beck Disease.

PURPOSE: To explore the relationship between insulin-like growth factor (IGF)-1R expression and the pathological progression of Kashin-Beck disease (KBD). DESIGN: KBD cartilage samples were collected from 5 patients. Additionally, T-2 toxin was administered to rats fed a selenium (Se)-deficient diet, and their knee joints were collected. Human C28/I2 chondrocytes and mouse hypertrophic ATDC5 chondrocytes were cultured in vitro and treated with T-2 toxin and Se supplementation. Subsequently, the cultured human and mouse chondrocytes were treated with the IGF-1R inhibitor, picropodophyllin. Chondrocyte death and caspase-3 activity were analyzed using flow cytometry and a specific kit, respectively. Protein and mRNA expression levels of IGF-1R and matrix molecules were measured using immunohistochemistry, western blotting, and quantitative real-time reverse transcription-polymerase chain reaction analyses. RESULTS: The cartilages from patients with KBD and T-2 toxin-treated rats on a Se-deficient diet showed significantly decreased expression of IGF-1R compared to cartilages from controls. T-2 toxin decreased IGF-1R mRNA and protein levels in both C28/I2 and hypertrophic ATDC5 chondrocytes in a dose-dependent manner; however, Se supplementation reduced the decrease of IGF-1R induced by T-2 toxin. Furthermore, inhibition of IGF-1R resulted in chondrocyte death of C28/I2 and hypertrophic ATDC5 chondrocytes, as well as decreased type II collagen expression and increased MMP-13 expression at the mRNA and protein levels. CONCLUSION: Downregulation of IGF-1R was associated with KBD cartilage destruction. Therefore, inhibition of IGF-1R may mediate chondrocyte death and extracellular matrix degeneration related to the pathological progression of KBD.

Effects of methylation of deiodinase 3 gene on gene expression and severity of Kashin-Beck disease.

Kashin-Beck disease (KBD) is a complex endemic osteoarthropathy, which mainly occurs in the northeast to southwest China. Iodothyronine deiodinases 3 (DIO3) is one of the selenoproteins, which is closely related to bone metabolism and unclear to KBD. This study aims to investigate the role and associated mechanisms of methylation and expression of DIO3 with disease severity in patients with KBD. We performed a bioinformatics analysis first to identify the biological mechanisms involved in selenoproteins. The methylation status of the DIO3 gene and DIO3 gene expression, as well as DIO3-related regulatory genes in patients with KBD, were analyzed. We found that 15 CpG sites of six selenoproteins were hypomethylated with 5-azacytidine treatment. DIO3 hypermethylation was associated with an increased risk of KBD and may lead to downregulation of DIO3 gene expression as well as be an indicator of the severity of KBD, which may provide a new insight for gene-environment correlations and interactions in etiology and pathogenesis of KBD.

Proteomic analysis of knee cartilage reveals potential signaling pathways in pathological mechanism of Kashin-Beck disease compared with osteoarthritis.

The pathological mechanism of Kashin-Beck disease (KBD), an endemic osteoarthritic disease, remains to be poorly understood. This study was designed to identify signaling pathways and crucial proteins involved in the pathological mechanism of KBD compared with osteoarthritis (OA). The knee cartilage samples were collected from gender- and age-matched KBD (n = 9) and OA (n = 9) patients. After pre-processing, samples were labeled with Tamdem Mass Tags 6plex multiplex kit, and analyzed by liquid chromatography-tandem mass spectrometry. Proteomic results were analyzed with gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interactions (PPI). The differential abundance proteins from KBD and OA were validated using western blot analysis. As a result, A total number of 375 proteins were identified to have differential abundance between KBD and OA, of which 121 and 254 proteins were observed to be up-regulated or down-regulated in KBD group. GO analysis shows that the differential abundant proteins are associated with cell junction and signal transducer activity from extracellular to intracellular. KEGG pathways enrichment and PPI network indicate four major pathways, including extracellular matrix -receptor interaction, focal adhesion, phosphatidylinositol 3-kinase (PI3K)-Protein kinase B (Akt), and Ras signaling pathways were involved in the degeneration of cartilage. Moreover, integrins, laminins, NF-κB and other regulative molecules were found as crucial proteins. In conclusion, our results demonstrated that compared with OA, the differential abundance proteins and signaling pathways may contribute to the occurrence and development of joint damage in KBD. Further investigation of their regulative roles and interaction may provide new insights into the pathological mechanisms and therapeutic targets for KBD.

Abnormal expression of chondroitin sulfate sulfotransferases in the articular cartilage of pediatric patients with Kashin-Beck disease.

The objective of this study is to investigate the expression of enzymes involved in the sulfation of articular cartilage from proximal metacarpophalangeal (PMC) joint cartilage and distal metacarpophalangeal (DMC) joint cartilage in children with Kashin-Beck disease (KBD). The finger cartilage samples of PMC and DMC were collected from KBD and normal children aged 5-14 years old. Hematoxylin and eosin staining as well as immunohistochemical staining were used to observe the morphology and quantitate the expression of carbohydrate sulfotransferase 3 (CHST-3), carbohydrate sulfotransferase 12 (CHST-12), carbohydrate sulfotransferase 13 (CHST-13), uronyl 2-O-sulfotransferase (UST), and aggrecan. In the results, the numbers of chondrocyte decreased in all three zones of PMC and DMC in the KBD group. Less positive staining cells for CHST-3, CHST-12, CHST-13, UST, and aggrecan were observed in almost all three zones of PMC and DMC in KBD. The positive staining cell rates of CHST-12 were higher in superficial and middle zones of PMC and DMC in KBD, and a significantly higher rate of CHST-13 was observed only in superficial zone of PMC in KBD. In conclusion, the abnormal expression of chondroitin sulfate sulfotransferases in chondrocytes of KBD children may provide an explanation for the cartilage damage, and provide therapeutic targets for the treatment.

Expression and localization of the small proteoglycans decorin and biglycan in articular cartilage of Kashin-Beck disease and rats induced by T-2 toxin and selenium deficiency.

Kashin-Beck disease (KBD) is an endemic degenerative osteoarthropathy of uncertain etiology. Our study sought to identify a correlation between small proteoglycans decorin and biglycan expression and Kashin-Beck Disease. Immunohistochemistry was used to assess the decorin and biglycan levels in cartilage specimens from both child KBD patients, and rats fed with T-2 toxin under a selenium-deficient condition. Real-time PCR and Western blot were used to assess mRNA and protein levels of decorin and biglycan in rat cartilages, as well as in C28/I2 chondrocytes stimulated by T-2 toxin and selenium in vitro. The result showed that decorin was reduced in all zones of KBD articular cartilage, while the expression of biglycan was prominently increased in KBD cartilage samples. Increased expression of biglycan and reduced expression of decorin were observed at mRNA and protein levels in the cartilage of rats fed with T-2 toxin and selenium- deficiency plus T-2 toxin diet, when compared with the normal diet group. Moreover, In vitro stimulation of C28/I2 cells with T-2 toxin resulted in an upregulation of biglycan and downregulation of decorin, T-2 toxin induction of biglycan and decorin levels were partly rescued by selenium supplement. This study highlights the focal nature of the degenerative changes that occur in KBD cartilage and may suggest that the altered expression pattern of decorin and biglycan have an important role in the onset and pathogenesis of KBD.

Genetic variants in the ITPR2 gene are associated with Kashin-Beck Disease in Tibetan.

BACKGROUND: Kashin-Beck Disease (KBD) is a chronic, endemic osteoarthropathy. Inositol 1,4,5-triphosphate receptor type 2 (ITPR2) gene is involved in chondrocytes. We speculated that single-nucleotide polymorphisms (SNPs) in ITPR2 gene may have an association with KBD in Tibetan. METHODS: To prove this hypothesis, a total of eight SNPs (rs1049376, rs11048526, rs11048556, rs11048585, rs16931011, rs10842759, rs2230372, and rs7134213) were selected, and genotyped in 316 KBD patients and 320 controls. The association between ITPR2 variants and KBD risk was assessed by logistic regression analysis. RESULTS: The study identified significant association (p = 0.019) between KBD and a novel locus, ITPR2 SNP rs11048526 (OR = 1.49, 95% CI = 1.07-2.08). The variant A/G genotype frequency in rs11048526 had a significantly increased risk of KBD in co-dominant model (OR = 3.70, 95% CI = 1.26-10.89, p = 0.018), dominant model (OR = 3.56, 95% CI = 1.22-10.40, p = 0.020), log-additive model (OR = 3.00, 95% CI = 1.12-8.00, p = 0.029) after adjusted for age and gender. CONCLUSION: The results indicate a potential association between ITPR2 and KBD risk in Tibetan. Further work is required to confirm these results and explore the mechanisms of these effects.

Biological Analysis of Gene Expression and Clinical Variables Suggest FZD1 as a Novel Biomarker for Patients with Kashin-Beck Disease, an Endemic Osteoarthritis in China.

Clinical variables contribute to the severity of Kashin-Beck disease (KBD). However, it is unclear if there is a correlation between gene expression and clinical variables. Peripheral blood samples were collected from 100 patients with KBD and 100 healthy controls from KBD-endemic areas to identify differentially expressed genes in KBD. Correlation analysis and multiple logistic regression analysis were performed using gene expression and clinical parameters. Immunohistochemistry (IHC) was used to detect the expression of related proteins in articular cartilage tissues. Thirty-nine differentially expressed genes were identified in patients with KBD. Nine differentially expressed genes were correlated with the metacarpal length/metacarpal breadth index. FZD1 was identified as having statistical significance in establishing the regression model of clinical parameters and gene expression. FZD1 expression levels were remarkably reduced in patients with KBD. Our results indicate that FZD1 could be involved in the pathological process of phalanges tuberositas and brachydactylia and may provide new insight into the pathogenesis of articular cartilage destruction observed in patients with KBD.

The Importance of Se-Related Genes in the Chondrocyte of Kashin-Beck Disease Revealed by Whole Genomic Microarray and Network Analysis.

Kashin-Beck disease (KBD) is an endemic, chronic, and degenerative osteoarthropathy. Selenium (Se) deficiency plays important role in the pathogenesis of KBD. We aimed to screen Se-related gene from chondrocytes of patients with KBD. Whole-genome oligonucleotide microarrays were used to detect differentially expressed genes. qRT-PCR was used to confirm the microarray results. Comparative Toxicogenomics Database (CTD) was used to screen Se-related genes from differentially expressed genes. Gene Ontology (GO) classifications and network analysis of Se-related genes were constituted by STRING online system. Three hundred ninety-nine differentially expressed genes were obtained from microarray. Among them, 54 Se-related genes were identified by CTD. The qRT-PCR validation showed that four genes expressed similarly with the ones in the microarray transcriptional profiles. The Se-related genes were categorized into 6 cellular components, 8 molecular functions, 44 biological processes, 10 pathways, and 1 network by STRING. The Se-related gene insulin-like growth factor binding protein 2 (IGFBP2), insulin-like growth factor binding protein 3 (IGFBP3), interleukin 6 (IL6), BCL2, apoptosis regulator (BCL2), and BCL2-associated X, apoptosis regulator (BAX), which involved in many molecular functions, biological processes, and apoptosis pathway may play important roles in the pathogenesis of KBD.

3-morpholinosydnonimine (SIN-1)-induced oxidative stress leads to necrosis in hypertrophic chondrocytes in vitro.

Chondrocyte is targeted for disruption in Osteoarthritis (OA) and Kashin-Beck Disease (KBD), and chondrocyte death in cartilage may contribute to the progression of OA and KBD. Oxidative stress leads to increased risk for OA. Previous work in our laboratory implicates oxidative stress as a potential mediator in children with KBD. While these studies suggest a role for oxidative stress in the modulation of OA and KBD, the direct effects of reactive oxygen species/reactive nitrogen species (ROS/RNS) on the stability of this domain remain unclear. Here, we demonstrate that oxidative stress, as induced through treatment with 3-morpholinosydnonimine (SIN-1), a spontaneous ROS/RNS generator, decreased the cell viability in hypertrophic chondrocytes in a dose- and time- dependent manner. SIN-1 induced necrosis in hypertrophic chondrocytes, whereas triggered apoptosis in non-hypertrophic cells of non-differentiated ATDC5 cells and C28/I2 cells. Ultrastructural analysis of hypertrophic chondrocyte treated with SIN-1 revealed morphological changes, such as plasma membrane breakdown, generalized swelling of the cytoplasm and organelles, even to disappearance. Moreover, SIN-1 induced chondronecrosis in the deep zone of engineered cartilage tissue, such as cell-free vacancy and "red ghost" cells. Overall, we demonstrate for the first time that oxidative stress, as induced through exogenous ROS/RNS, leads to necrosis in hypertrophic chondrocytes. Oxidative stress-mediated necrotic cell death contributes to chondronecrosis in the deep zone of cartilage in both OA and KBD.

FGF8 and FGFR3 are up-regulated in hypertrophic chondrocytes: Association with chondrocyte death in deep zone of Kashin-Beck disease.

OBJECTIVE: The aim of this study was to investigate FGF8 and FGFR3 expression in clinical samples of Kashin-Beck disease (KBD), an endemic osteochondropathy found in China, as well as in pre-clinical models of this disease. METHOD: Cartilage was collected from the hand phalanges of five patients with KBD and from five healthy children. Sprague-Dawley rats were administered a selenium-deficient diet for four weeks prior to exposure to the T-2 toxin. ATDC5 cells were differentiated into hypertrophic chondrocytes for twenty-one days, and then treated with 3-morpholinosydnonimine (SIN-1) (0, 1, 3, or 5 mM) for 24 h. FGF8 and FGFR3 were visualized using immunohistochemistry; protein levels were assessed by western blotting, and mRNA levels were determined by real-time RT-PCR. RESULTS: Increased staining of FGF8 and FGFR3 was observed in the cartilage of children with KBD compared to normal children. Both increased FGF8 and FGFR3 staining, as well as protein levels, were also observed in the cartilage of rats fed normal or Se-deficient diets plus T-2 toxin exposure, compared to those in rats fed with normal or Se-deficient diets alone. SIN-1 treatment of hypertrophic chondrocytes (ATCD5 cells) increased FGF8 and FGFR3 protein and mRNA levels in a dose-dependent manner. CONCLUSION: Our data indicate that SIN-1 induces FGF8 and FGFR3 overexpression and this is involved in the abnormal terminal differentiation and degradation of the ECM in cartilage. FGF8 and FGFR3 may therefore play an important role in the onset of deep zone necrosis and pathogenesis in KBD in adolescent children.