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.

Involvement of Yes-Associated Protein 1 Activation in the Matrix Degradation of Human-Induced-Pluripotent-Stem-Cell-Derived Chondrocytes Induced by T-2 Toxin and Deoxynivalenol Alone and in Combination.

T-2 toxin and deoxynivalenol (DON) are two prevalent mycotoxins that cause cartilage damage in Kashin-Beck disease (KBD). Cartilage extracellular matrix (ECM) degradation in chondrocytes is a significant pathological feature of KBD. It has been shown that the Hippo pathway is involved in cartilage ECM degradation. This study aimed to examine the effect of YAP, a major regulator of the Hippo pathway, on the ECM degradation in the hiPS-derived chondrocytes (hiPS-Ch) model of KBD. The hiPS-Ch injury models were established via treatment with T-2 toxin/DON alone or in combination. We found that T-2 toxin and DON inhibited the proliferation of hiPS-Ch in a dose-dependent manner; significantly increased the levels of YAP, SOX9, and MMP13; and decreased the levels of COL2A1 and ACAN (all p values < 0.05). Immunofluorescence revealed that YAP was primarily located in the nuclei of hiPS-Ch, and its expression level increased with toxin concentrations. The inhibition of YAP resulted in the dysregulated expression of chondrogenic markers (all p values < 0.05). These findings suggest that T-2 toxin and DON may inhibit the proliferation of, and induce the ECM degradation, of hiPS-Ch mediated by YAP, providing further insight into the cellular and molecular mechanisms contributing to cartilage damage caused by toxins.

Abnormal Level of Manganese, Iron, Iodine, and Selenium in the Hair of Children Living in Kashin-Beck Disease Endemic Areas.

Biological geochemistry is a main suggested cause of Kashin-Beck disease (KBD), due to the absence or excess of elements in the environment. Initially, Se deficiency is regarded as the most key role in the etiology of KBD, and selenium supplementation effectively helps to prevent and control KBD. However, several elements are reported to be relevant to KBD or selenium in succession, which indicated selenium deficiency is not the original etiology of KBD. The study comprehensively analyzed the characteristics of the bio-element profile of KBD and further re-examined the unique role of selenium in etiology. The study measured 14 elements, including sodium, potassium, calcium, phosphorus, magnesium, copper, iron, zinc, selenium, iodine, manganese, lead, arsenic, and mercury, which were detected from hair samples collected from 150 boys. Research participants were separated based on whether they had received any preventative treatment (with and without selenium supplementation). From endemic areas, 30 KBD and 30 healthy children without any preventative treatment were selected alongside 30 KBD and 30 healthy children with selenium supplementation. The participants from endemic areas were then compared to 30 healthy children living in non-endemic areas. Compared to the non-endemic group, the levels of iron and manganese were all significantly higher in the endemic groups and were further elevated in KBD participants (p < 0.05). In contrast, selenium and iodine levels in endemic areas were much lower than those of the control group (p < 0.05). The proportions of selenium excess (p < 0.05) and iodine deficiency (p < 0.05) in endemic groups were significantly lower than participants from non-endemic areas. Meanwhile, excess levels of iron (p < 0.05) and manganese (p < 0.05) were higher in the endemic groups. Moreover, the proportions of Zn/Fe and Se/Mn were found to be significantly lower in endemic area participants than those in the control group (p < 0.05). Three pairs of elements had a correlation coefficient value of more than 0.6: 0.7423 for manganese and calcium, 0.6446 for potassium and sodium, and 0.6272 for manganese and iron. The ratios of Se/Mn and Zn/Fe were associated with a correlation coefficient value of 0.8055. Magnesium, sodium, copper, and iodine levels were meticulously examined using binary regression analysis. This was also used to determine the ratios of Ca/Mg, Ca/P, Zn/Fe, Se/Mn, and Se/I. Thus, the study largely revealed the vital role of manganese, iron, and iodine (in conjunction with selenium) in KBD etiology and pathogenesis. High manganese and iron levels with low selenium and iodine levels were identified as characteristic features of the bio-element profile of KBD. The different element ratios reflect the interaction between several elements. The most significant of these were the proportions of Se/Mn and Zn/Fe, which may be significant in the occurrence and development of 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.

Identifying discriminative features for diagnosis of Kashin-Beck disease among adolescents.

INTRODUCTION: Diagnosing Kashin-Beck disease (KBD) involves damages to multiple joints and carries variable clinical symptoms, posing great challenge to the diagnosis of KBD for clinical practitioners. However, it is still unclear which clinical features of KBD are more informative for the diagnosis of Kashin-Beck disease among adolescent. METHODS: We first manually extracted 26 possible features including clinical manifestations, and pathological changes of X-ray images from 400 KBD and 400 non-KBD adolescents. With such features, we performed four classification methods, i.e., random forest algorithms (RFA), artificial neural networks (ANNs), support vector machines (SVMs) and linear regression (LR) with four feature selection methods, i.e., RFA, minimum redundancy maximum relevance (mRMR), support vector machine recursive feature elimination (SVM-RFE) and Relief. The performance of diagnosis of KBD with respect to different classification models were evaluated by sensitivity, specificity, accuracy, and the area under the receiver operating characteristic (ROC) curve (AUC). RESULTS: Our results demonstrated that the 10 out of 26 discriminative features were displayed more powerful performance, regardless of the chosen of classification models and feature selection methods. These ten discriminative features were distal end of phalanges alterations, metaphysis alterations and carpals alterations and clinical manifestations of ankle joint movement limitation, enlarged finger joints, flexion of the distal part of fingers, elbow joint movement limitation, squatting limitation, deformed finger joints, wrist joint movement limitation. CONCLUSIONS: The selected ten discriminative features could provide a fast, effective diagnostic standard for KBD adolescents.

Dysregulation of Cells Cycle and Apoptosis in Human Induced Pluripotent Stem Cells Chondrocytes Through p53 Pathway by HT-2 Toxin: An in vitro Study.

Kashin-Beck disease (KBD) mainly damages growth plate of adolescents and is susceptible to both gene and gene-environmental risk factors. HT-2 toxin, which is a primary metabolite of T-2 toxin, was regarded as one of the environmental risk factors of KBD. We used successfully generated KBD human induced pluripotent stem cells (hiPSCs) and control hiPSCs, which carry different genetic information. They have potential significance in exploring the effects of HT-2 toxin on hiPSC chondrocytes and interactive genes with HT-2 toxin for the purpose of providing a cellular disease model for KBD. In this study, we gave HT-2 toxin treatment to differentiating hiPSC chondrocytes in order to investigate the different responses of KBD hiPSC chondrocytes and control hiPSC chondrocytes to HT-2 toxin. The morphology of HT-2 toxin-treated hiPSC chondrocytes investigated by transmission electron microscope clearly showed that the ultrastructure of organelles was damaged and type II collagen expression in hiPSC chondrocytes was downregulated by HT-2 treatment. Moreover, dysregulation of cell cycle was observed; and p53, p21, and CKD6 gene expressions were dysregulated in hiPSC chondrocytes after T-2 toxin treatment. Flow cytometry also demonstrated that there were significantly increased amounts of late apoptotic cells in KBD hiPSC chondrocytes and that the mRNA expression level of Fas was upregulated. In addition, KBD hiPSC chondrocytes presented stronger responses to HT-2 toxin than control hiPSC chondrocytes. These findings confirmed that HT-2 is an environmental risk factor of KBD and that p53 pathway interacted with HT-2 toxin, causing damaged ultrastructure of organelles, accelerating cell cycle in G1 phase, and increasing late apoptosis in KBD hiPSC chondrocytes.

Transcriptome-wide association study identifies susceptibility genes for rheumatoid arthritis.

OBJECTIVE: To identify rheumatoid arthritis (RA)-associated susceptibility genes and pathways through integrating genome-wide association study (GWAS) and gene expression profile data. METHODS: A transcriptome-wide association study (TWAS) was conducted by the FUSION software for RA considering EBV-transformed lymphocytes (EL), transformed fibroblasts (TF), peripheral blood (NBL), and whole blood (YBL). GWAS summary data was driven from a large-scale GWAS, involving 5539 autoantibody-positive RA patients and 20,169 controls. The TWAS-identified genes were further validated using the mRNA expression profiles and made a functional exploration. RESULTS: TWAS identified 692 genes with PTWAS values < 0.05 for RA. CRIPAK (PEL = 0.01293, PTF = 0.00038, PNBL = 0.02839, PYBL = 0.0978), MUT (PEL = 0.00377, PTF = 0.00076, PNBL = 0.00778, PYBL = 0.00096), FOXRED1 (PEL = 0.03834, PTF = 0.01120, PNBL = 0.01280, PYBL = 0.00583), and EBPL (PEL = 0.00806, PTF = 0.03761, PNBL = 0.03540, PYBL = 0.04254) were collectively expressed in all the four tissues/cells. Eighteen genes, including ANXA5, AP4B1, ATIC (PTWAS = 0.0113, downregulated expression), C12orf65, CMAH, PDHB, RUNX3 (PTWAS = 0.0346, downregulated expression), SBF1, SH2B3, STK38, TMEM43, XPNPEP1, KIAA1530, NUFIP2, PPP2R3C, RAB24, STX6, and TLR5 (PTWAS = 0.04665, upregulated expression), were validated with integrative analysis of TWAS and mRNA expression profiles. TWAS-identified genes functionally involved in endoplasmic reticulum organization, regulation of cytokine production, TNF signaling pathway, immune response-regulating signaling pathway, regulation of autophagy, etc. CONCLUSION: We identified multiple candidate genes and pathways, providing novel clues for the genetic mechanism of RA.

Cell cycle-related lncRNAs and mRNAs in osteoarthritis chondrocytes in a Northwest Chinese Han Population.

BACKGROUND: A group of differentially expressed long non-coding RNAs (lncRNAs) have been shown to play key roles in osteoarthritis (OA), although they represented only a small proportion of lncRNAs that may be biologically and physiologically relevant. Since our knowledge of regulatory functions of non-coding RNAs is still limited, it is important to gain better understanding of their relation to the pathogenesis of OA. METHODS: We performed mRNA and lncRNA microarray analysis to detect differentially expressed RNAs in chondrocytes from three OA patients compared with four healthy controls. Then, enrichment analysis of the differentially expressed mRNAs was carried out to define disease molecular networks, pathways and gene ontology (GO) function. Furthermore, target gene prediction based on the co-expression network was performed to reveal the potential relationships between lncRNAs and mRNAs, contributing an exploration of a role of lncRNAs in OA mechanism. Quantitative RT-PCR analyses were used to demonstrate the reliability of the experimental results. FINDINGS: Altogether 990 lncRNAs (666 up-regulated and 324 down-regulated) and 546 mRNAs (419 up-regulated and 127 down-regulated) were differentially expressed in OA samples compared with the normal ones. The enrichment analysis revealed a set of genes involved in cell cycle. In total, 854 pairs of mRNA and lncRNA were highly linked, and further target prediction appointed 12 genes specifically for their corresponding lncRNAs. The lncRNAs lncRNA-CTD-2184D3.4, ENST00000564198.1, and ENST00000520562.1 were predicted to regulate SPC24, GALM, and ZNF345 mRNA expressions in OA. INTERPRETATION: This study uncovered several novel genes potentially important in pathogenesis of OA, and forecast the potential function of lnc-CTD-2184D3.4, especially for the cell cycle in the chondrocytes. These findings may promote additional aspects in studies of OA.

ADAMTS4 and ADAMTS5 may be considered as new molecular therapeutic targets for cartilage damages with Kashin-Beck Disease.

There are a pretty number of research demonstrating that ADAMTS4 and ADAMTS5 playing primary roles in the degradation of cartilage during inflammatory joint diseases like osteoarthritis (OA). Because Kashin-Beck Disease (KBD) has been found to own the common pathological changes and symptoms with OA, and is regarded as the specific type of osteoarthritis, it's reasonable to believe that ADAMTS4 and ADAMTS5 may exert an enormous functions on the injury of cartilage of the KBD and may be potential molecular therapeutic targets for KBD.

Cytotoxic Properties of HT-2 Toxin in Human Chondrocytes: Could T3 Inhibit Toxicity of HT-2?

Thyroid hormone triiodothyronine (T3) plays an important role in coordinated endochondral ossification and hypertrophic differentiation of the growth plate, while aberrant thyroid hormone function appears to be related to skeletal malformations, osteoarthritis, and Kashin-Beck disease. The T-2 toxin, present extensively in cereal grains, and one of its main metabolites, HT-2 toxin, are hypothesized to be potential factors associated with hypertrophic chondrocyte-related osteochondropathy, known as the Kashin-Beck disease. In this study, we investigated the effects of T3 and HT-2 toxin on human chondrocytes. The immortalized human chondrocyte cell line, C-28/I2, was cultured in four different groups: controls, and cultures with T3, T3 plus HT-2 and HT-2 alone. Cytotoxicity was assessed using an MTT assay after 24-h-exposure. Quantitative RT-PCR was used to detect gene expression levels of collagen types II and X, aggrecan and runx2, and the differences in runx2 were confirmed with immunoblot analysis. T3 was only slightly cytotoxic, in contrast to the significant, dose-dependent cytotoxicity of HT-2 alone at concentrations ≥ 50 nM. T3, together with HT-2, significantly rescued the cytotoxic effect of HT-2. HT-2 induced significant increases in aggrecan and runx2 gene expression, while the hypertrophic differentiation marker, type X collagen, remained unchanged. Thus, T3 protected against HT-2 induced cytotoxicity, and HT-2 was an inducer of the pre-hypertrophic state of the chondrocytes.

Individual and combined toxicity of T-2 toxin and deoxynivalenol on human C-28/I2 and rat primary chondrocytes.

Deoxynivalenol (DON) and T-2 toxin are prevalent mycotoxin contaminants in the food and feed stuffs worldwide, with non-negligible co-contamination and co-exposure conditions. Meanwhile, they are considerable risk factors for Kashin-Beck disease, a chronic endemic osteochondropathy. The aim of this study was to investigate the individual and combined cytotoxicity of DON and T-2 toxin on proliferating human C-28/I2 and newborn rat primary costal chondrocytes by MTT assay. Four molar concentration combination ratios of DON and T-2 toxin were used, 1:1 for R1 mixture, 10:1 for R10, 100:1 for R100 and 1000:1 for R1000. The toxicological interactions were quantified by the MixLow method. DON, T-2 toxin, and their mixtures all showed a clear dose-dependent toxicity for chondrocytes. The cytotoxicity of T-2 toxin was 285-fold higher than DON was in human chondrocytes, and 22-fold higher in the rat chondrocytes. The combination of DON and T-2 toxin was significantly synergistic at middle and high level concentrations of R10 mixtures in rat chondrocytes, but significantly antagonistic at the low concentrations of R100 mixtures in both cells and at the middle concentrations of R1000 mixtures in rat chondrocytes. These results indicated that the combined toxicity was influenced by the cell sensitivity for toxins, the difference between the combination ratio and equitoxic ratio, the concentrations and other factors.

[The Effect of Disordered Glycometabolism of Kashin-Beck Disease on the Function of Chondrocytes].

OBJECTIVE: To reveal the effect of disordered glycometabolism in Kashin-Beck disease (KBD) chondrocytes,we compared changes in expressions of extracellular matrix components (collagen and aggrecan),apoptosis and oxidative stress under the condition of different concentrations of glucose. METHODS: The damage of KBD chondrocytes and normal chondrocytes under high glucose culture was measured in compared with cells under normal culture,that included the changes of proliferation and morphology; the concentrations of glucose in culture medium during the process of chondrocytes culture; the expressions of type Ⅱ collagen and aggrecan detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Toluidine blue staining; cell apoptosis and reactive oxygen species (ROS) content detected by flow cytometry and fluorescence staining. RESULTS: The growth and proliferation of KBD chondrocytes were inferior to normal chondrocytes. The glucose uptake of KBD chondrocytes and normal chondrocytes under high glucose culture were basically the same (P>0.05). Disordered glycometabolism caused by high glucose decreased the expression of type Ⅱ collagen and aggrecan in KBD chondrocytes (P<0.05),meanwhile,increased apoptosis and cellular ROS generation of cultured chondrocytes (P<0.05). CONCLUSION: The disordered glycometabolism can affect the function of KBD chondrocytes through reducing the expression of type Ⅱ collagen and aggrecan and increasing the apoptosis and the oxidative stress.

Long noncoding RNA expression profile reveals lncRNAs signature associated with extracellular matrix degradation in kashin-beck disease.

Kashin-Beck disease (KBD) is a deformative, endemic osteochondropathy involving degeneration and necrosis of growth plates and articular cartilage. The pathogenesis of KBD is related to gene expression and regulation mechanisms, but long noncoding RNAs (lncRNAs) in KBD have not been investigated. In this study, we identified 316 up-regulated and 631 down-regulated lncRNAs (≥ 2-fold change) in KBD chondrocytes using microarray analysis, of which more than three-quarters were intergenic lncRNAs and antisense lncRNAs. We also identified 232 up-regulated and 427 down-regulated mRNAs (≥ 2-fold change). A lncRNA-mRNA correlation analysis combined 343 lncRNAs and 292 mRNAs to form 509 coding-noncoding gene co-expression networks (CNC networks). Eleven lncRNAs were predicted to have cis-regulated target genes, including NAV2 (neuron navigator 2), TOX (thymocyte selection-associated high mobility group box), LAMA4 (laminin, alpha 4), and DEPTOR (DEP domain containing mTOR-interacting protein). The differentially expressed mRNAs in KBD significantly contribute to biological events associated with the extracellular matrix. Meanwhile, 34 mRNAs and 55 co-expressed lncRNAs constituted a network that influences the extracellular matrix. In the network, FBLN1 and LAMA 4 were the core genes with the highest significance. These novel findings indicate that lncRNAs may play a role in extracellular matrix destruction in KBD.

Clinical Outcomes of Wulingsan Subtraction Decoction Treatment of Postoperative Brain Edema and Fever as a Complication of Glioma Neurosurgery.

Objective. To evaluate the efficacy of Wulingsan subtraction ( WLSS) decoction in the treatment of postoperative brain edema and fever as a complication of glioma neurosurgery. Methods. This retrospective study was conducted at the Department of Neurosurgery of Liaocheng People's Hospital. Patients hospitalized between March 2011 and December 2014 were divided into three groups: Group A received WLSS oral liquid (50 mL), twice a day; Group B received an intravenous infusion of mannitol; and Group C received WLSS combined with mannitol (n = 30 patients per group). All patients were treated for 10 days continuously. Therapeutic efficacy was evaluated by measuring body temperature and indicators of renal function before and 3, 5, and 10 days after treatment. Results. Compared to the other two groups, significantly greater clinical efficacy was observed in the patients treated with mannitol (Group B; P < 0.05), although marked clinical efficacy was also observed over time in patients treated with WLSS (Group A). After 5 days, the quantifiable effects of the WLSS and mannitol combination group (Group C) were substantial (P < 0.05). The renal damage in Group B was more obvious after 5 days and 10 days. Conclusion. Compared with mannitol treatment alone, WLSS combined with mannitol induced a more rapid reduction in body temperature. Our findings suggest that patients should be started on mannitol for 3 days and then switched to WLSS to achieve obvious antipyretic effects and protect renal function. This method of treatment should be considered for clinical applications.