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.

Modulation of Ras signaling pathway by exosome miRNAs in T-2 toxin-induced chondrocyte injury.

This study aims to investigate the impact of T-2 toxin on the regulation of downstream target genes and signaling pathways through exosome-released miRNA in the development of cartilage damage in Kashin-Beck disease (KBD). Serum samples from KBD patients and supernatant from C28/I2 cells treated with T-2 toxin were collected for the purpose of comparing the differential expression of exosomal miRNA using absolute quantitative miRNA-seq. Target genes of differential exosomal miRNAs were identified using Targetscan and Miranda databases, followed by GO and KEGG enrichment analyses. Validation of key indicators of chondrocyte injury in KBD was conducted using Real-time quantitative PCR (RT-qPCR) and Immunohistochemical staining (IHC). A total of 20 exosomal miRNAs related to KBD were identified in serum, and 13 in chondrocytes (C28/I2). The identified exosomal miRNAs targeted 48,459 and 60,612 genes, primarily enriched in cell organelles and membranes, cell differentiation, and cytoskeleton in the serum, and the cytoplasm and nucleus, metal ion binding in chondrocyte (C28/I2). The results of the KEGG enrichment analysis indicated that the Ras signaling pathway may play a crucial role in the pathogenesis of KBD. Specifically, the upregulation of hsa-miR-181a-5p and hsa-miR-21-3p, along with the downregulation of hsa-miR-152-3p and hsa-miR-186-5p, were observed. Additionally, T-2 toxin intervention led to a significant downregulation of RALA, REL, and MAPK10 expression. Furthermore, the protein levels of RALA, REL, and MAPK10 were notably decreased in the superficial and middle layers of cartilage tissues from KBD. The induction of differential expression of chondrocyte exosomal miRNAs by T-2 toxin results in the collective regulation of target genes RALA, REL, and MAPK10, ultimately mediating the Ras signaling pathway and causing a disruption in chondrocyte extracellular matrix metabolism, leading to chondrocyte injury.

Activation of Notch Signaling Pathway is involved in Extracellular Matrix Degradation in human induced pluripotent stem cells chondrocytes induced by HT-2 toxin.

Notch signaling regulates cartilage formation and homeostasis. Kashin-Beck Disease (KBD), an endemic osteochondropathy, is characterized by severe cartilage degradation. The etiology of KBD is related to the exposure of HT-2 toxin, a mycotoxin and primary metabolite of T-2 toxin. This study aims to explore the role of HT-2 toxin in the Notch signaling regulation and extracellular matrix (ECM) metabolism of hiPSCs-Chondrocytes. Immunohistochemistry and qRT-PCR were employed to investigate the expression of Notch pathway molecules in KBD articular cartilage and primary chondrocytes. hiPSCs-Chondrocytes, derived from hiPSCs, were treated with 100 ng/mL HT-2 toxin and the γ-secretase inhibitor (DAPT) for 48h, respectively. The markers related to the Notch signaling pathway and ECM were assessed using qRT-PCR and Western blot. Notch pathway dysregulation was prominent in KBD cartilage. HT-2 toxin exposure caused cytotoxicity in hiPSCs-Chondrocytes, and activated Notch signaling by increasing the mRNA and protein levels of NOTCH1 and HES1. HT-2 toxin also upregulated ECM catabolic enzymes and downregulated ECM components (COL2A1 and ACAN), indicating ECM degradation. DAPT-mediated Notch signaling inhibition suppressed the mRNA and protein level of ADAMTS5 expression while enhancing ECM component expression in hiPSCs-Chondrocytes. This study suggests that HT-2 toxin may induce ECM degradation in hiPSCs-Chondrocytes through activating Notch signaling.

Comparative Analysis of Differentially Expressed Genes in Chondrocytes from Rats Exposed to Low Selenium and T-2 Toxin.

The aim of this study was to construct rat models of environmental risk factors for Kashin-Beck disease (KBD) with low selenium and T-2 toxin levels and to screen the differentially expressed genes (DEGs) between the rat models exposed to environmental risk factors. The Se-deficient (SD) group and T-2 toxin exposure (T-2) group were constructed. Knee joint samples were stained with hematoxylin-eosin, and cartilage tissue damage was observed. Illumina high-throughput sequencing technology was used to detect the gene expression profiles of the rat models in each group. Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis were performed and five differential gene expression results were verified by quantitative real-time polymerase chain reaction (qRT‒PCR). A total of 124 DEGs were identified from the SD group, including 56 upregulated genes and 68 downregulated genes. A total of 135 DEGs were identified in the T-2 group, including 68 upregulated genes and 67 downregulated genes. The DEGs were significantly enriched in 4 KEGG pathways in the SD group and 9 KEGG pathways in the T-2 group. The expression levels of Dbp, Pc, Selenow, Rpl30, and Mt2A were consistent with the results of transcriptome sequencing by qRT‒PCR. The results of this study confirmed that there were some differences in DEGs between the SD group and the T-2 group and provided new evidence for further exploration of the etiology and pathogenesis of KBD.

Integration of miRNA in exosomes and single-cell RNA-seq profiles in endemic osteoarthritis, Kashin-Beck disease.

Kashin-Beck disease (KBD) is an endemic, chronic degenerative joint disease in China. Exosomes miRNAs, as signaling molecules in intercellular communication, can transfer specific biological martials into target cell to regulate their function and might participate in the pathogenesis of KBD. We isolated serum and chondrocytes-derived exosomes, miRNA sequencing revealed exosomes miRNA profiles and differentially expressed miRNAs (DE-miRNAs) were identified. The target genes were predicted of known and novel DE-miRNAs with TargetScan 5.0 and miRanda 3.3a database. Single-cell RNA sequencing (scRNA-seq) was performed to identify chondrocyte clusters and their gene signatures in KBD. And we performed comparative analysis between the serum and chondrocytes-derived exosomes DE-miRNA target genes and differentially expressed genes of each cell clusters. A total of 20 DE-miRNAs were identified in serum-derived exosomes. In the miRNA expression of chondrocytes-derived exosomes, 53 DE-miRNAs were identified. 16,063 predicted targets were identified as the target genes in the serum-derived exosomes, 57,316 predicted targets were identified as the target genes in the chondrocytes-derived exosomes. Seven clusters were labeled by cell type according to the expression of previously described markers. Three hundred fifteen common genes were found among serum/chondrocytes-derived exosomes DE-miRNA target genes and DEGs identified by scRNA-seq analysis. We firstly integratly analyzed the serum and chondrocytes exosomes miRNA with single-cell RNA sequencing (scRNA-seq) data of KBD chondrocyte, the results showed that DE-miRNAs in exosomes might play a potential role in regulating genes expression in different KBD chondrocytes clusters by exosomes mediating cell-cell communications functions, which could improve the new diagnosis and treatment methods for KBD.

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.

Research Progress of Selenium-Enriched Foods.

Selenium is an essential micronutrient that plays a crucial role in maintaining human health. Selenium deficiency is seriously associated with various diseases such as Keshan disease, Kashin-Beck disease, cataracts, and others. Conversely, selenium supplementation has been found to have multiple effects, including antioxidant, anti-inflammatory, and anticancer functions. Compared with inorganic selenium, organic selenium exhibits higher bioactivities and a wider range of safe concentrations. Consequently, there has been a significant development of selenium-enriched foods which contain large amounts of organic selenium in order to improve human health. This review summarizes the physiological role and metabolism of selenium, the development of selenium-enriched foods, the physiological functions of selenium-enriched foods, and provides an analysis of total selenium and its species in selenium-enriched foods, with a view to laying the foundation for selenium-enriched food development.

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.

The alteration of urinary metabolomics profiles in Kashin-Beck disease in a three consecutive year study.

Kashin-Beck disease (KBD) is a serious, endemic chronic osteochondral disease characterized by symmetrical enlargement of the phalanges, brachydactyly, joint deformity, and even dwarfism. To investigate the urinary metabolomic profiles of KBD patients, we performed an untargeted metabolomics approach using liquid chromatography coupled with mass spectrometry (LC-MS). Adult urinary specimens were collected from 39 patients with KBD and 19 healthy subjects; the children's urinary specimens were collected from 5 patients with KBD, 25 suspected KBD cases and 123 healthy subjects in the KBD endemic area during a three consecutive year study. We identified 10 upregulated and 28 downregulated secondary level metabolites highly associated with aetiology and pathogenesis of KBD between adult KBD and adult controls. A total of 163, 967 and 795 metabolites were significantly different in the urine among children with KBD, suspected children with KBD cases and healthy child controls, respectively, for each year in three consecutive years. HT-2 toxin, Se-adenosylselenomethionine (AdoSeMet), the toxin T2 tetrol, and many kinds of amino acids were identified as differential metabolites in this study. Amino sugar and nucleotide sugar metabolism, fructose and mannose metabolism, arachidonic acid metabolism, D-glutamine and D-glutamate metabolism, ubiquinone and other terpenoid-quinone biosynthesis, and D-glutamine and D-glutamate metabolism were perturbed pathways in adult and child KBD patients. Our study provides new insight into the underlying mechanisms of KBD, and suggests that we should pay more attention to these differences in small-molecule metabolites and metabolic pathways in the environmental aetiology and pathogenesis of KBD.

The Role of Selenium-Mediated Notch/Hes1 Signaling Pathway in Kashin-Beck Disease Patients and Cartilage Injury Models.

Kashin-Beck disease (KBD) is a nutrition-related osteoarthropathy, and selenium (Se) deficiency is an environmental risk factor for KBD. Notch/Hes1 signaling pathway plays a vital role in regulating cartilage, but its exact mechanisms in KBD remain unknown. The Se contents were determined using the hydride atomic fluorescence spectrometry assay technique, and the mRNA levels were detected via quantitative real-time PCR. The chondrocyte injury models were established by Se deficiency and tert-butyl hydroperoxide (tBHP), respectively; apoptosis and necrosis rates were detected using Hoechst 33,342/PI and Annexin V-FITC/PI. The results showed that the Se levels in the flour of KBD areas were lower than that of the non-KBD areas, and the Se levels in the plasma of KBD patients were lower than that of the controls. The expressions of Notch1, Jagged1, and Hes1 were higher in the whole blood of KBD patients than those of the controls, and Notch1 was negatively correlated with the expression of BCL2, while was positively correlated with BAX. In injury, chondrocytes induced by low Se and tBHP, the expression of Notch1, Jagged1, and Hes1 increased, apoptosis and necrosis rates increased in Se deficiency and tBHP groups, while Se supplementation reversed it. Decreased plasma Se in KBD patients may be related to low dietary Se. Se deficiency might be involved in the pathological process of KBD by activating the Notch/Hes1 signaling pathway to induce excessive apoptosis of chondrocytes, the activation of Notch/Hes1 promotes oxidative injury, and Se supplementation could reverse it. The importance of Notch/Hes1 signaling pathway in KBD development will provide a new potential target for KBD.

Association of oxidative stress and Kashin-Beck disease integrated Meta and Bioinformatics analysis.

OBJECTIVE: To explore the association between oxidative stress (OS) and Kashin-Beck disease (KBD). METHODS: Terms associated with "KBD" and "OS" were searched in the six different databases up to October 2021. Stata 14.0 was used to pool the means and standard deviations using random-effect or fixed-effect model. The differentially expressed genes in the articular chondrocytes of KBD were identified, the OS related genes were identified by blasting with the GeneCards. The KEGG pathway and gene ontology enrichment analysis was conducted using STRING. RESULTS: The pooled SMD and 95% CI showed hair selenium (-4.59; -6.99, -2.19), blood selenium (-1.65; -2.86, -0.44) and glutathione peroxidases (-4.15; -6.97, -1.33) levels were decreased in KBD, whereas the malondialdehyde (1.12; 0.60, 1.64), nitric oxide (2.29; 1.31, 3.27), nitric oxide synthase (1.07; 0.81, 1.33) and inducible nitric oxide synthase (1.69; 0.62, 2.77) were increased compared with external controls. Meanwhile, hair selenium (-2.71; -5.32, -0.10) and glutathione peroxidases (-1.00; -1.78, -0.22) in KBD were decreased, whereas the malondialdehyde (1.42; 1.04, 1.80), nitric oxide (3.08; 1.93, 4.22) and inducible nitric oxide synthase (0.81; 0.00, 1.61) were elevated compared with internal controls. Enrichment analysis revealed apoptosis was significantly correlated with KBD. The significant biological processes revealed OS induced the release of cytochrome c from mitochondria. The cellular component of OS located in the mitochondrial outer membrane. CONCLUSIONS: The OS levels in KBD were significantly increased because of selenium deficiency, OS mainly occurred in mitochondrial outer membrane, released of cytochrome c from mitochondria, and induced apoptotic signaling pathway.

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.

CpG methylation of the GPX3 promoter in patients with Kashin-Beck Disease potentially promotes chondrocyte apoptosis.

OBJECTIVE: To determine the methylation levels of CpGs in the GPX3 promoter region and explore their potential effects on the apoptosis of chondrocytes. METHODS: Blood specimens were collected from 32 participants; 16 KBD patients and 16 healthy subjects. Twenty-five CpGs in the promoter region of GPX3 were identified and detected by MALDI-TOF-MS. Methylation levels of CpGs were compared between KBD patients and healthy subjects as well as among the KBD patients with different degrees. C28/I2 human chondrocytes were treated with tBHP and Na2SeO3. Apoptosis in chondrocytes was examined under a fluorescence microscope. RESULTS: The methylation levels of GPX3-1_CpG_11 and GPX3-1_CpG_16 in KBD patients were significantly higher than those of healthy subjects (P < 0.05). The methylation levels of the other CpGs were not significantly different between the two groups (P > 0.05). The methylation level of GPX3-1_CpG_24 in KBD patients was significantly higher than those of healthy subjects (P < 0.05). MSP-PCR analysis indicated that the methylation rate of KBD group (9.41%) was significantly higher than that of healthy subjects (1.18%), and that GPX3 DNA methylation increased the risk of acquiring KBD 8 fold (OR = 8.000, 95% CI: 1.023-62.580); The mRNA expression of GPX3 in whole blood of KBD patients was lower than that of healthy subjects (P＜0.05); Compared with the control group, GPX3, GPX1 and GPX4 mRNA level of the tertbutyl hydroperoxide injury group decreased significantly (P < 0.05), after supplementation with Na2SeO3. The rate of chondrocytes apoptosis was decreased with the increasing of GPX3 and GPX4 mRNA levels (P＜0.05) and GPX3 mRNA showed a similar trend without statistically significant (P＞0.05). CONCLUSION: The methylation patterns of CpGs in GPX3 varied in KBD patients. The experiments indicated that the increased methylation of CpGs within the GPX3 promoter may down-regulate the expression of GPX3, thereby reducing the antioxidant function of GPX3 and promoting chondrocyte apoptosis, both of which accelerates the occurrence of KBD. We therefore propose a new understanding of GPX3's potential epigenetic and genetic mechanisms that contribute to KBD.

An eQTL variant of ALDH1A2 is associated with Kashin-Beck disease in Chinese population.

INTRODUCTION: The aims of the study were to investigate the relationship between aldehyde dehydrogenase 1 family member A2 (ALDH1A2) and Kashin-Beck disease (KBD), explore the effects of the rs3204689 polymorphism and methylation status on the expression levels of ALDH1A2, and further clarify the pathogenesis of KBD. MATERIALS AND METHODS: The genotype of ALDH1A2 rs3204689 was detected by PCR-RFLP in 103 KBD patients and 109 healthy controls in the whole blood. The mRNA level of ALDH1A2 was measured by qRT-PCR, and the protein expression was detected using IHC staining and Western blotting. The MSP-PCR was used to identify the ALDH1A2 methylation level. RESULTS: There were significant differences in G/G, G/C, and C/C frequencies of ALDH1A2 rs3204689 between the KBD and control groups (χ2 = 7.113, P = 0.029); the minor allele G of ALDH1A2 was associated with the risk of KBD (χ2 = 5.984, P = 0.014). The mRNA and protein levels of ALDH1A2 were increased in the whole blood and cartilage of KBD patients compared with the controls (P = 0.049, P < 0.0001, P = 0.019). Meanwhile, a statistically significant difference was observed between G/G, G/C and C/C genotype on mRNA expression (P = 0.039). The methylation level of the ALDH1A2 gene promoter region showed no significant difference between the KBD and control groups (χ2 = 0.317, P = 0.573). CONCLUSION: Our case-control study indicates that the common variant rs3204689 near ALDH1A2 is associated with KBD in Chinese population. The risk allele G of rs3204689 is statistically linked to the high expression of ALDH1A2, which is up-regulated in the cartilage and whole blood of KBD patients. Our findings suggest a potential role of ALDH1A2 in the pathogenesis of KBD.

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.

Comprehensive expression profiles of mRNAs, lncRNAs and miRNAs in Kashin-Beck disease identified by RNA-sequencing.

Kashin-Beck disease (KBD) is a chronic, endemic and deforming osteochondropathy, whose basic pathological alterations include apoptosis and necrosis of chondrocytes in articular cartilage and growth plates and imbalanced extracellular matrix metabolism. Numerous studies have reported that long noncoding RNAs (lncRNAs) and microRNA (miRNAs) are aberrantly expressed in KBD. Our study was comprised of 5 KBD patients and 5 healthy individuals and we compared the expression profiles of mRNAs, lncRNAs and miRNAs through RNA-sequencing (RNA-seq). Bioinformatic analysis of GO and KEGG was employed to conduct functional annotation and pathway enriched analysis. In total, 3194 mRNAs, 4103 lncRNAs and 1550 miRNAs were detected to be differentially expressed by RNA-seq (P < 0.05; |log2FC| ≥1). The lysosome pathway, Wnt signaling pathway, TNF signaling pathway, endocytosis and mTOR signaling pathway were identified to be involved in the KBD development according to the result of the KEGG analysis. In addition, a ceRNA network based on lncRNA-miRNA-mRNA was constructed to probe the intricate regulatory mechanism and interaction between transcripts, which was visualized using the Cytoscape software. The ce-lncRNAs of four aberrantly expressed genes, FOSB, EGR3, BCAM and SOX6, were determined through the network. Among the identified DElncRNAs, we selected 8 differentially expressed lncRNAs to confirm the reliability of RNA-seq data by qRT-PCR in 11 KBD patients and 11 healthy individuals. We aimed to provide a comprehensive understanding ofmRNA, lncRNA and miRNA alterations between KBD patients and healthy individuals, and meanwhile reveal several potential causative molecular and signaling pathways involved 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.