Plasma metabolites and inflammatory proteins profiling predict outcome of Fufang Duzhong Jiangu granules treating Kashin-Beck disease.

To investigate predictive biomarkers that could be used to identify patients' response to treatment, plasma metabolomics and proteomics analyses were performed in Kashin-Beck disease (KBD) patients treated with Fufang Duzhong Jiangu Granules (FDJG). Plasma was collected from 12 KBD patients before treatment and 1 month after FDJG treatment. LC-MS and olink proteomics were employed for obtaining plasma metabolomics profiling and inflammatory protein profiles. Patients were classified into responders and non-responders based on drug efficacy. Enrichment analyses of differential metabolites and proteins of the responders at baseline and after treatment were conducted to study the mechanism of drug action. Differential metabolites and proteins between the two groups were screened as biomarkers to predict the drug efficacy. The receiver operating characteristic curve was used to evaluate the prediction accuracy of biomarkers. The changes in metabolites and inflammatory proteins in responders after treatment reflected the mechanism of FDJG treatment for KBD, which may act on glycerophospholipid metabolism, d-glutamine and d-glutamate metabolism, nitrogen metabolism and NF-kappa B signaling pathway. Three metabolites were identified as potential predictors: N-undecanoylglycine, ß-aminopropionitrile and PC [18:3(6Z,9Z,12Z)/20:4(8Z,11Z,14Z,17Z)]. For inflammatory protein, interleukin-8 was identified as a predictive biomarker to detect responders. Combined use of these four biomarkers had high predictive ability (area under the curve = 0.972).

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.

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.

Effects of selenium and iodine on Kashin-Beck disease: an updated review.

Kashin-Beck disease (KBD) is an endochondral osteogenesis disorder characterised by epiphysis damage and secondary deformable arthropathy induced by multiple external factors, among which selenium (Se) and iodine deficiency are important influencing factors. Iodine deficiency is usually accompanied by a low Se content in the soil in the KBD areas of China. Se can reverse oxidative damage to chondrocytes. In addition, Se is related to the bone conversion rate and bone mineral density. Low Se will hinder growth and change bone metabolism, resulting in a decrease in the bone conversion rate and bone mineral density. Thyroid hormone imbalance caused by thyroid dysfunction caused by iodine deficiency can damage bone homeostasis. Compared with Se deficiency alone, Se combined with iodine deficiency can reduce the activity of glutathione peroxidase more effectively, which increases the vulnerability of chondrocytes and other target cells to oxidative stress, resulting in chondrocyte death. Clinical studies have shown that supplementation with Se and iodine is helpful for the prevention and treatment of KBD.

Prevalence of T-2 Toxin in the Food and Beverages of Residents Living in a Kashin-Beck-Disease Area of Qamdo, Tibet.

It has been strongly suggested that selenium deficiency and T-2 toxin contamination have a strong relationship with the occurrence and development of Kashin-Beck disease (KBD). In order to provide information for understanding the high prevalence of KBD in Tibet, this study collected the responses to a cubital venous blood and dietary questionnaire of 125 subjects including 75 KBD patients and 50 healthy controls in a KBD-prevalent county (Luolong County) in Tibet, China. A total of 10 household local families were randomly selected in this area, and local diet samples of brick tea, Zanba powder, milk residue, and hulless Barley were collected from these residents. Selenium content in blood was detected by inductively coupled plasma mass spectrometry (ICP-MS). The T-2 toxin contamination level in food sample was assayed using an ELISA kit. The selenium levels of patients and controls were 42.0 ± 19.8 and 56.06 ± 22.4 µg/L, respectively. The serum selenium level in controls was higher than that in patients, but there was no significant difference, and the serum selenium level both in patients and controls in Tibet was lower than the normal range. The results of the dietary survey showed that the number of respondents who consumed butter tea was large; 46.67% of patients indicated that they drank buttered tea every day, which was significantly higher than in controls. The contents of T-2 toxin in Zanba powder, milk residue, hulless barley and drinking water samples were below the detection limit (0.05 µg/kg); this result was labeled Tr. Unexpectedly, the contents of T-2 toxin in brick tea were higher, with average levels of 424 ± 56 µg/kg in Detong village and 396 ± 24 µg/kg in Langcuo village. For the first time, we report the presence of an extremely high concentration of T-2 toxin in brick tea of Tibet.

Whole-Transcriptome Sequencing of Knee Joint Cartilage from Kashin-Beck Disease and Osteoarthritis Patients.

The aim of this study was to provide a comprehensive understanding of similarities and differences in mRNAs, lncRNAs, and circRNAs within cartilage for Kashin-Beck disease (KBD) compared to osteoarthritis (OA). We conducted a comparison of the expression profiles of mRNAs, lncRNAs, and circRNAs via whole-transcriptome sequencing in eight KBD and ten OA individuals. To facilitate functional annotation-enriched analysis for differentially expressed (DE) genes, DE lncRNAs, and DE circRNAs, we employed bioinformatic analysis utilizing Gene Ontology (GO) and KEGG. Additionally, using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), we validated the expression levels of four cartilage-related genes in chondrocytes. We identified a total of 43 DE mRNAs, 1451 DE lncRNAs, and 305 DE circRNAs in KBD cartilage tissue compared to OA (q value < 0.05; |log2FC| > 1). We also performed competing endogenous RNA network analysis, which identified a total of 65 lncRNA-mRNA interactions and 4714 miRNA-circRNA interactions. In particular, we observed that circRNA12218 had binding sites for three miRNAs targeting ACAN, while circRNA12487 had binding sites for seven miRNAs targeting COL2A1. Our results add a novel set of genes and non-coding RNAs that could potentially serve as candidate diagnostic biomarkers or therapeutic targets for KBD patients.

The Long-term Efficacy of Total Knee Arthroplasty on End-stage Kashin-Beck Disease of the Knee in Highland Tibetan Areas Patients: A Retrospective Study with 10-Year Follow-up.

OBJECTIVE: Despite the established success of total knee arthroplasty (TKA) with end-stage osteoarthritis, there is a notable scarcity of research on its long-term outcomes in individuals suffering from end-stage Kashin-Beck disease (KBD). This retrospective study aimed to assess the long-term outcomes and effectiveness of clinical function, quality of life, and complications of TKA and end-stage KBD patients in Tibetan highland areas. METHODS: The retrospective cohort included 43 KBD patients, comprising a total of 59 knees, who had undergone TKA at West China Hospital, Sichuan University between 2008 and 2021. Patients were subsequently followed up for a minimum of 3 years, and received rigorous radiological and clinical assessments at 3, 6, and 12 months post surgery, followed by annual examinations thereafter. The evaluation included various efficacy indices, including visual analogue scale (VAS) scores, hospital for special surgery (HSS) scores, functional score for adult Tibetans with Kashin-Beck disease (FSAT-KBD), and radiographic findings. Comparison of indicators within the same group was conducted using one-way repeated-measures analysis of variance or paired sample t-tests, whereas between-group differences were compared using an independent t-test. RESULTS: Throughout the average follow-up duration of 10.8 years, patients experienced a substantial reduction in knee pain and noteworthy functional improvement. The VAS scores decreased significantly from 77.47 ± 4.12 mm before surgery to 10.91 ± 1.97 mm after surgery, indicating considerable alleviation of knee pain. The HSS scores improved markedly, increasing from 44.26 ± 4.95 preoperatively to 91.26 ± 4.37, indicating enhanced joint function. Similarly, the FSAT-KBD exhibited positive progression, increasing from 25.90 ± 3.12 to 36.95 ± 3.54. Importantly, at the last follow-up, none of the patients presented with periprosthetic infection, prosthesis loosening, or periprosthetic fracture. CONCLUSION: At long-term follow-up, compared with patients in the preoperative period, patients in Tibetan highland areas with KBD of the knee who underwent TKA benefited from a significant reduction in pain, improvement in joint function, and satisfactory improvement in quality of life.

Outcomes of ankle arthrodesis in adult patients with ankle osteoarthritis in Kashin-Beck disease.

PURPOSE: We retrospectively evaluated the characteristics of these patients and the effectiveness of ankle arthrodesis in the treatment of ankle arthritis caused by Kashin-Beck disease (KBD). METHODS: A retrospective study of KBD patients with ankle osteoarthritis who underwent ankle arthrodesis between December 2012 and January 2022 was performed. A total of 46 patients were included. The general characteristics, clinical manifestations and imaging features of the patients were recorded and summarized. measured using the VAS score, and ankle function was assessed by the AOFAS ankle-hindfoot score. RESULTS: Multiple subchondral cystic changes were found in 42(91.3%) patients. The VAS scores for both resting and weight-bearing conditions were 6.28 ± 1.30 vs. 2.09 ± 1.12 (P < .001) and 6.87 ± 1.01 vs. 2.17 ± 0.98 (P < .001), respectively. The AOFAS scores were 59.17 ± 5.50 and 88.39 ± 1.42, respectively (P < .001). CONCLUSIONS: The subchondral multiple cystic transformation of the ankle KBD has a certain suggestive role.Arthrodesis is an effective method to reduce ankle pain and improve ankle function in KBD patients with ankle osteoarthritis.

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.

Association of SOX6 gene polymorphisms with Kashin-Beck disease risk in the Chinese Han population.

Kashin-Beck disease (KBD) is an endemic osteochondropathy. A specific gene called SRY-box transcription factor 6 (SOX6) is important for forming cartilage. This study aims to explore the potential correlation between SOX6 single nucleotide polymorphisms (SNPs) and KBD risk for the first time. In the case-control study, 735 unrelated Chinese Han individuals were enrolled. The four mutation sites of the SOX6 gene (rs4539287 G/A, rs3203295 C/A, rs7928675 C/A, and rs10832681 A/G) were screened and genotyped on the Agena MassARRAY platform. The correlation between SOX6 SNPs and KBD risk was explored based on logistic regression analysis. The interaction between SNP and SNP was analyzed based on the multi-factor dimensionality reduction (MDR) method. Overall analysis revealed a remarkable correlation between rs7928675 and rs10832681 and the reduction of KBD risk (p < 0.05). Subgroup analyses further indicated that these two SNPs have a significant protective effect on KBD risk among participants aged ≤65 years, males, and non-smokers (p < 0.05). MDR displayed a marked interaction between rs3203295 and rs10832681. Our study revealed that SOX6 rs7928675 and rs10832681 are markedly correlated with a reduced risk of KBD in the Chinese Han population, providing a new direction for the prevention, diagnosis, and treatment of KBD.

Disease and Economic Burden of Kashin-Beck Disease - China, 2021 .

What is already known about this topic?: Kashin-Beck disease (KBD) is a chronic and degenerative osteoarthropathy characterized by cartilage degeneration. It is an endemic disease that is highly prevalent among the Chinese population and poses a significant health risk. What is added by this report?: This is the first national report on the economic burden of KBD in China. According to the data from 2021, KBD has caused significant disease and economic burdens. The most substantial reduction in healthy life expectancy was observed among patients with degree II severity and those aged 60 years and older, resulting in a total indirect economic burden of 112.74 million Chinese Yuan (CNY). What are the implications for public health practice?: The results of this study will contribute to informing the development of tailored prevention and control strategies by the government. These strategies will include targeted policies and recommendations for appropriate healthcare and financial subsidies, which will be based on the demographic characteristics of the endemic areas.

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.

Increased FGFR3 is involved in T-2 toxin-induced lesions of hypertrophic cartilage associated with endemic osteoarthritis.

This study evaluated the effect of fibroblast growth factor receptor 3 (FGFR3) on damaged hypertrophic chondrocytes of Kashin-Beck disease (KBD). Immunohistochemical staining was used to evaluate FGFR3 expression in growth plates from KBD rat models and engineered cartilage. In vitro study, hypertrophic chondrocytes were pretreated by FGFR3 binding inhibitor (BGJ398) for 24 h before incubation at different T-2 toxin concentrations. Differentiation -related genes (Runx2, Sox9, and Col Ⅹ) and ECM degradation -related genes (MMP-13, Col Ⅱ) in the hypertrophic chondrocytes were analyzed using RT-PCR, and the corresponding proteins were analyzed using western blotting. Hypertrophic chondrocytes death was detected by the Annexin V/PI double staining assay. The integrated optical density of FGFR3 staining was increased in knee cartilage of rats and engineered cartilage treated with T-2 toxin. Both protein and mRNA levels of Runx2, Sox9, Col Ⅱ, and Col Ⅹ were decreased in a dose-dependent manner when exposed to the T-2 toxin and significantly upregulated by 1 µM BGJ398. The expression of MMP-1, MMP-9, and MMP-13 increased in a dose-dependent manner when exposed to T-2 toxin and significantly reduced by 1 µM BGJ398. 1 µM BGJ398 could prevent early apoptosis and necrosis induced by the T-2 toxin. Inhibiting the FGFR3 signal could alleviate extracellular matrix degradation, abnormal chondrocytes differentiation, and excessive cell death in T-2 toxin-induced hypertrophic chondrocytes.

Comparative Analysis of Gut Microbiota from Rats Induced by Se Deficiency and T-2 Toxin.

The aim of this study was to analyze the differences in gut microbiota between selenium deficiency and T-2 toxin intervention rats. Knee joint and fecal samples of rats were collected. The pathological characteristics of knee cartilage were observed by safranin O/fast green staining. DNA was extracted from fecal samples for PCR amplification, and 16S rDNA sequencing was performed to compare the gut microbiota of rats. At the phylum level, Firmicutes (81.39% vs. 77.06%) and Bacteroidetes (11.11% vs. 14.85%) were dominant in the Se-deficient (SD) group and T-2 exposure (T-2) groups. At the genus level, the relative abundance of Ruminococcus_1 (12.62%) and Ruminococcaceae_UCG-005 (10.31%) in the SD group were higher. In the T-2 group, the relative abundance of Lactobacillus (11.71%) and Ruminococcaceae_UCG-005 (9.26%) were higher. At the species level, the high-quality bacteria in the SD group was Ruminococcus_1_unclassified, and Ruminococcaceae_UCG-005_unclassified in the T-2 group. Lactobacillus_sp__L_YJ and Lactobacillus_crispatus were the most significant biomarkers in the T-2 group. This study analyzed the different compositions of gut microbiota in rats induced by selenium deficiency and T-2 toxin, and revealed the changes in gut microbiota, so as to provide a certain basis for promoting the study of the pathogenesis of Kashin-Beck disease (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.

Untargeted Metabolomics Approach Correlated Enniatin B Mycotoxin Presence in Cereals with Kashin-Beck Disease Endemic Regions of China.

Kashin-Beck disease (KBD) is a multifactorial endemic disease that only occurs in specific Asian areas. Mycotoxin contamination, especially from the Fusarium spp., has been considered as one of the environmental risk factors that could provoke chondrocyte and cartilage damage. This study aimed to investigate whether new mycotoxins could be identified in KBD-endemic regions as a potential KBD risk factor. This was investigated on 292 barley samples collected in Tibet during 2009-2016 and 19 wheat samples collected in Inner Mongolia in 2006, as control, from KBD-endemic and non-endemic areas. The LC-HRMS(/MS) data, obtained by a general mycotoxin extraction technic, were interpreted by both untargeted metabolomics and molecular networks, allowing us to identify a discriminating compound, enniatin B, a mycotoxin produced by some Fusarium spp. The presence of Fusarium spp. DNA was detected in KBD-endemic area barley samples. Further studies are required to investigate the role of this mycotoxin in KBD development in vivo.

ATAC-seq reveals the roles of chromatin accessibility in the chondrocytes of Kashin-Beck disease compared with primary osteoarthritis.

Objective: This study aimed to investigate the roles of accessible chromatin in understanding the different pathogeneses between Kashin-Beck disease (KBD) and primary osteoarthritis (OA). Methods: Articular cartilages of KBD and OA patients were collected, and after tissue digestion, primary chondrocytes were cultured in vitro. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) was performed to compare the accessible chromatin differences of chondrocytes between KBD and OA groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were executed for the promoter genes. Then, the IntAct online database was used to generate networks of significant genes. Finally, we overlapped the analysis of differentially accessible region (DAR)-associated genes and differentially expressed genes (DEGs) obtained from whole-genomic microarray. Results: We obtained 2,751 total DARs, which contained 1,985 loss and 856 gain DARs and belonged to 11 location distributions. We obtained 218 motifs associated with loss DARs, 71 motifs associated with gain DARs, 30 motif enrichments of loss DARs, and 30 motif enrichments of gain DARs. In total, 1,749 genes are associated with loss DARs, and 826 genes are associated with gain DARs. Among them, 210 promoter genes are associated with loss DARs, and 112 promoter genes are associated with gain DARs. We obtained 15 terms of GO enrichment and 5 terms of KEGG pathway enrichment from loss DAR promoter genes, and 15 terms of GO enrichment and 3 terms of KEGG pathway enrichment from gain DAR promoter genes. We obtained CAPN6 and other 2 overlap genes from loss DARs-vs-down DEGs, AMOTL1 from gain DARs-vs-down DEGs, EBF3 and other 12 overlap genes from loss DARs-vs-up DEGs, and ADARB1 and other 10 overlap genes from 101 gain DARs-vs-up DEGs. These overlap genes were built into 4 gene interaction networks. Conclusion: FGF7, GPD1L, NFIB, RUNX2, and VCAM1 were the overlapped genes from the DAR-associated genes and DEGs. These genes were associated with the abnormal chondrocyte function, which may play crucial roles in different processes between KBD and OA in the way of accessible chromatin.