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.

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.

Altered gut microbiome profile in patients with knee osteoarthritis.

Introduction: Osteoarthritis (OA) is a kind of chronic, degenerative disorder with unknown causes. In this study, we aimed to improve our understanding of the gut microbiota profile in patients with knee OA. Methods: 16S rDNA gene sequencing was performed to detect the gut microbiota in fecal samples collected from the patients with OA (n = 32) and normal control (NC, n = 57). Then the metagenomic sequencing was used to identify the genes or functions linked with gut microbial changes at the species level in the fecal samples from patients with OA and NC groups. Results: The Proteobacteria was identified as dominant bacteria in OA group. We identified 81 genera resulted significantly different in abundance between OA and NC. The abundance of Agathobacter, Ruminococcus, Roseburia, Subdoligranulum, and Lactobacillus showed significant decrease in the OA compared to the NC. The abundance of genera Prevotella_7, Clostridium, Flavonifractor and Klebsiella were increasing in the OA group, and the families Lactobacillaceae, Christensenellaceae, Clostridiaceae_1 and Acidaminococcaceae were increasing in the NC. The metagenomic sequencing showed that the abundance of Bacteroides stercoris, Bacteroides vulgatus and Bacteroides uniformis at the species level were significantly decreasing in the OA, and the abundance of Escherichia coli, Klebsiella pneumoniae, Shigella flexneri and Streptococcus salivarius were significantly increased in OA. Discussion: The results of our study interpret a comprehensive profile of the gut microbiota in patients with knee OA and offer the evidence that the cartilage-gut-microbiome axis could play a crucial role in underlying the mechanisms and pathogenesis of OA.

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.

Detection of selenoprotein transcriptome in chondrocytes of patients with Kashin-Beck disease.

Background: Kashin-Beck disease (KBD) is a deformed osteochondral disease with a chronic progression that is restrictively distributed in eastern Siberia, North Korea, and some areas of China, and selenium deficiency has been identified as an important factor in the pathogenesis of this disease in recent years. Objective: The aim of this study is to investigate the selenoprotein transcriptome in chondrocytes and define the contribution of selenoprotein to KBD pathogenesis. Methods: Three cartilage samples were collected from the lateral tibial plateau of adult KBD patients and normal controls paired by age and sex for real-time quantitative polymerase chain reaction (RT-qPCR) to detect the mRNA expression of 25 selenoprotein genes in chondrocytes. Six other samples were collected from adult KBD patients and normal controls. In addition, immunohistochemistry was used on four adolescent KBD samples and seven normal controls (IHC) to determine the expression of proteins screened by RT-qPCR results that had different gene levels. Results: Increased mRNA expression of GPX1 and GPX3 was observed in chondrocytes, and stronger positive staining was displayed in the cartilage from both adult and adolescent patients. The mRNA levels of DIO1, DIO2, and DIO3 were increased in KBD chondrocytes; however, the percentage of positive staining decreased in the KBD cartilage of adults. Conclusion: The selenoprotein transcriptome, mainly the glutathione peroxidase (GPX) and deiodinase (DIO) families were altered in KBD and might play a vital role in the pathogenesis of KBD.

The Status of Selenium and Zinc in the Urine of Children From Endemic Areas of Kashin-Beck Disease Over Three Consecutive Years.

Selenium deficiency is one of the main risk factors for Kashin-Beck disease (KBD). This study aimed to detect the status of selenium and zinc in the urine of children from endemic areas of KBD over three consecutive years and to evaluate whether selenium and zinc levels in children in Shaanxi Province remain normal after stopping selenium supplementation. The samples of urine were collected in consecutive years (2017-2019) to detect selenium content by hydride generation atomic fluorescence spectrometry (HGAFS) and to detect zinc content by atomic absorption spectrophotometry (AAS). Generalized estimation equation (GEE) analysis was integrated to assess the comprehensive nutritional status and dietary structure of children. Data were processed in duplicate and analyzed by SPSS 18.0. This study included 30 X-ray-positive KBD cases and 123 healthy children aged 7-12 years. A total of 424 urine and 137 hair samples were collected over three consecutive years for selenium determination. The mean value of urinary selenium in all subjects was 6.86 µg/l (2017), 8.26 µg/l (2018), and 4.04 µg/l (2019), and the mean value of urinary zinc in all subjects was 0.36 mg/l (2017), 0.39 mg/l (2018), and 0.31 mg/l (2019) for the three consecutive years of 2017-2019. The mean values of urinary selenium were 6.56 and 6.94 µg/l (2017), 8.69 and 8.14 µg/l (2018), and 4.57 and 3.90 µg/l (2019) in the KBD-X and normal groups, respectively; and the mean value of urinary zinc were 0.38 and 0.35 mg/l (2017), 0.41 and 0.39 mg/l (2018), and 0.43 and 0.28 mg/l (2019) in the KBD-X and normal groups, respectively. The mean value of hair selenium in 137 subjects was 275.08 µg/kg and the mean values of hair selenium were 267.48 and 276.61 µg/kg in the KBD-X group and normal group, respectively. The level of selenium/zinc showed a trend of increasing first and then decreasing during the three consecutive years. The level of selenium in all subjects from the endemic areas was lower than normal, which reminds us to monitor the state of KBD constantly and adjust selenium salt supplementation in accordance with the changes in the KBD state.

Investigation of selenium nutritional status and dietary pattern among children in Kashin-Beck disease endemic areas in Shaanxi Province, China using duplicate portion sampling method.

BACKGROUND AND OBJECTIVES: Selenium deficiency is a primary risk factor of Kashin-Beck disease (KBD). This study aimed to investigate whether children in endemic areas could maintain sufficient selenium intake after termination of selenium supplement administration, and evaluate their comprehensive nutritional status and dietary structure. METHODS: Duplicate portion sampling combined with a questionnaire was adopted to collect data on categories and quantity of all food ingested in three consecutive days. Occipital hair was also collected to detect selenium content by hydride generation atomic fluorescence spectrometry (HGAFS). CDGSS3.0 software and factor analysis were integrated to assess the children's comprehensive nutritional status and dietary structure. RESULTS: This study included 240 sex-matched (1:1) children aged 7-12 years from KBD endemic (n = 120) and non-endemic (n = 120) areas. Overall, 720 solid food, 720 liquid, and 240 hair samples were collected for selenium determination. The mean selenium level in hair of children in endemic areas (0.38 ± 0.16 mg/kg) was significantly lower than that in children in non-endemic areas (0.56 ± 0.28 mg/kg, Z = -5.249, p < 0.001). The dietary selenium intake of children in endemic areas was 40.0% lower than that in children in non-endemic areas (Z = -9.374, p < 0.001). Children in endemic areas consumed significantly less diverse dietary items leading to significantly less intake of multiple nutrients compared to children in non-endemic areas. CONCLUSIONS: The dietary selenium intake of most children in endemic areas was less than the recommended amount. The dietary structure of children was undiversified, which limited the intake of multiple nutrients. Therefore, comprehensive nutrition rather than sole selenium intake should be the primary concern in the future.

Comparative analysis of the gut microbiota composition between knee osteoarthritis and Kashin-Beck disease in Northwest China.

BACKGROUND: Osteoarthritis (OA) and Kashin-Beck disease (KBD) both are two severe osteochondral disorders. In this study, we aimed to compare the gut microbiota structure between OA and KBD patients. METHODS: Fecal samples collected from OA and KBD patients were used to characterize the gut microbiota using 16S rDNA gene sequencing. To identify whether gut microbial changes at the species level are associated with the genes or functions of the gut bacteria between OA and KBD groups, metagenomic sequencing of fecal samples from OA and KBD subjects was performed. RESULTS: The OA group was characterized by elevated Epsilonbacteraeota and Firmicutes levels. A total of 52 genera were identified to be significantly differentially abundant between the two groups. The genera Raoultella, Citrobacter, Flavonifractor, g__Lachnospiraceae_UCG-004, and Burkholderia-Caballeronia-Paraburkholderia were more abundant in the OA group. The KBD group was characterized by higher Prevotella_9, Lactobacillus, Coprococcus_2, Senegalimassilia, and Holdemanella. The metagenomic sequencing showed that the Subdoligranulum_sp._APC924/74, Streptococcus_parasanguinis, and Streptococcus_salivarius were significantly increased in abundance in the OA group compared to those in the KBD group, and the species Prevotella_copri, Prevotella_sp._CAG:386, and Prevotella_stercorea were significantly decreased in abundance in the OA group compared to those in the KBD group by using metagenomic sequencing. CONCLUSION: Our study provides a comprehensive landscape of the gut microbiota between OA and KBD patients and provides clues for better understanding the mechanisms underlying the pathogenesis of OA and KBD.

Abnormal expression of TSG-6 disturbs extracellular matrix homeostasis in chondrocytes from endemic osteoarthritis.

Background and aims: Kashin-Beck disease (KBD) is a unique endemic osteochondropathy with unclear pathogenesis in China. T-2 toxin exposure has been identified as a significant risk factor of KBD. However, the mechanism of articular cartilage damage induced by T-2 toxin is a conundrum. We explored the role of the extracellular matrix-related gene TSG-6 in the articular chondrocyte damage process under the exposure of HT-2 toxin. Methods: TSG-6 was identified as a candidate gene by mining our previous gene expression profiling of KBD and verified by qRT-PCR and immunohistochemistry. Then, TSG-6 was silenced by RNA interference technology and overexpressed induction by TNF-α. Gradient concentrations of HT-2 toxin were added to intervene with C28/I2 chondrocytes. MTT was used to observe the proliferation and cell viability of chondrocytes, and qRT-PCR was utilized to detect the expression changes of MMP1, MMP3, MMP13, COL2A1, and proteoglycan before and after treatments for verification. Results: TSG-6 was upregulated in KBD chondrocytes at the mRNA level and upregulated in the superficial, middle, and deep zones of KBD cartilage. After TSG-6 silencing, the expression of MMP1, MMP3, MMP13, and proteoglycan was significantly decreased while COL2A1 expression was significantly increased, which was reversed after the overexpression of TSG-6 induced by TNF-α (p < 0.05). The survival rate of chondrocytes was correspondingly reduced with an increase in the HT-2 toxin concentration. Compared with the blank control group, the expression of MMPs was increased in the intervention group of HT-2 toxin, while the expression of proteoglycan and COL2A1 decreased (p < 0.05). Conclusion: The upregulation of the TSG-6 gene may play a role in promoting the damage and degradation of the extracellular matrix in KBD chondrocytes under the exposure of HT-2 toxin.

Differential expression of cyclins CCNB1 and CCNG1 is involved in the chondrocyte damage of kashin-beck disease.

The purpose of this study was clarify the relationship between the differential expression of cyclins CCNB1 and CCNG1 and chondrocyte damage in Kashin-Beck disease. Systematic review and high-throughput sequencing of chondrocytes derived from Kashin-Beck disease patients were combined to identify the differentially expressed cyclins and cyclin-dependent kinase genes. In parallel, weaned SD rats were treated with low selenium for 4 weeks and then T-2 toxin for 4 weeks. Knee cartilage was collected to harvest chondrocytes for gene expression profiling. Finally, the protein expression levels of CCNB1 and CCNG1 were verified in knee cartilage tissue of Kashin-Beck disease patients and normal controls by immunohistochemical staining. The systematic review found 52 cartilage disease-related cyclins and cyclin-dependent kinase genes, 23 of which were coexpressed in Kashin-Beck disease, including 15 upregulated and 8 downregulated genes. Under the intervention of a low selenium diet and T-2 toxin exposure, CCNB1 (FC = 0.36) and CCNG1 (FC = 0.73) showed a downward expression trend in rat articular cartilage. Furthermore, compared to normal controls, CCNB1 protein in Kashin-Beck disease articular cartilage was 71.98% and 66.27% downregulated in the superficial and middle zones, respectively, and 12.06% upregulated in the deep zone. CCNG1 protein was 45.66% downregulated in the superficial zone and 12.19% and 9.13% upregulated in the middle and deep zones, respectively. The differential expression of cyclins CCNB1 and CCNG1 may be related to articular cartilage damage in Kashin-Beck disease.

Alterations in the gut microbiota and metabolite profiles of patients with Kashin-Beck disease, an endemic osteoarthritis in China.

Kashin-Beck disease (KBD) is a severe osteochondral disorder that may be driven by the interaction between genetic and environmental factors. We aimed to improve our understanding of the gut microbiota structure in KBD patients of different grades and the relationship between the gut microbiota and serum metabolites. Fecal and serum samples collected from KBD patients and normal controls (NCs) were used to characterize the gut microbiota using 16S rDNA gene and metabolomic sequencing via liquid chromatography-mass spectrometry (LC/MS). To identify whether gut microbial changes at the species level are associated with the genes or functions of the gut bacteria in the KBD patients, metagenomic sequencing of fecal samples from grade I KBD, grade II KBD and NC subjects was performed. The KBD group was characterized by elevated levels of Fusobacteria and Bacteroidetes. A total of 56 genera were identified to be significantly differentially abundant between the two groups. The genera Alloprevotella, Robinsoniella, Megamonas, and Escherichia_Shigella were more abundant in the KBD group. Consistent with the 16S rDNA analysis at the genus level, most of the differentially abundant species in KBD subjects belonged to the genus Prevotella according to metagenomic sequencing. Serum metabolomic analysis identified some differentially abundant metabolites among the grade I and II KBD and NC groups that were involved in lipid metabolism metabolic networks, such as that for unsaturated fatty acids and glycerophospholipids. Furthermore, we found that these differences in metabolite levels were associated with altered abundances of specific species. Our study provides a comprehensive landscape of the gut microbiota and metabolites in KBD patients and provides substantial evidence of a novel interplay between the gut microbiome and metabolome in KBD pathogenesis.

Comparison of the major cell populations among osteoarthritis, Kashin-Beck disease and healthy chondrocytes by single-cell RNA-seq analysis.

Chondrocytes are the key target cells of the cartilage degeneration that occurs in Kashin-Beck disease (KBD) and osteoarthritis (OA). However, the heterogeneity of articular cartilage cell types present in KBD and OA patients and healthy controls is still unknown, which has prevented the study of the pathophysiology of the mechanisms underlying the roles of different populations of chondrocytes in the processes leading to KBD and OA. Here, we aimed to identify the transcriptional programmes and all major cell populations in patients with KBD, patients with OA and healthy controls to identify the markers that discriminate among chondrocytes in these three groups. Single-cell RNA sequencing was performed to identify chondrocyte populations and their gene signatures in KBD, OA and healthy cells to investigate their differences as related to the pathogenetic mechanisms of these two osteochondral diseases. We performed immunohistochemistry and quantitative reverse-transcription PCR (qRT-PCR) assays to validate the markers for chondrocyte population. Ten clusters were labelled by cell type according to the expression of previously described markers, and one novel population was identified according to the expression of a new set of markers. The homeostatic and mitochondrial chondrocyte populations, which were identified by the expression of the unknown markers MT1X and MT2A and MT-ND1 and MT-ATP6, were markedly expanded in KBD. The regulatory chondrocyte population, identified by the expression of CHI3L1, was markedly expanded in OA. Our study allows us to better understand the heterogeneity of chondrocytes in KBD and OA and provides new evidence of differences in the pathogenetic mechanisms between these two diseases.

Genetic Variants and Protein Alterations of Selenium- and T-2 Toxin-Responsive Genes Are Associated With Chondrocytic Damage in Endemic Osteoarthropathy.

The mechanism of environmental factors in Kashin-Beck disease (KBD) remains unknown. We aimed to identify single nucleotide polymorphisms (SNPs) and protein alterations of selenium- and T-2 toxin-responsive genes to provide new evidence of chondrocytic damage in KBD. This study sampled the cubital venous blood of 258 subjects including 129 sex-matched KBD patients and 129 healthy controls for SNP detection. We applied an additive model, a dominant model, and a recessive model to identify significant SNPs. We then used the Comparative Toxicogenomics Database (CTD) to select selenium- and T-2 toxin-responsive genes with the candidate SNP loci. Finally, immunohistochemistry was applied to verify the protein expression of candidate genes in knee cartilage obtained from 15 subjects including 5 KBD, 5 osteoarthritis (OA), and 5 healthy controls. Forty-nine SNPs were genotyped in the current study. The C allele of rs6494629 was less frequent in KBD than in the controls (OR = 0.63, p = 0.011). Based on the CTD database, PPARG, ADAM12, IL6, SMAD3, and TIMP2 were identified to interact with selenium, sodium selenite, and T-2 toxin. KBD was found to be significantly associated with rs12629751 of PPARG (additive model: OR = 0.46, p = 0.012; dominant model: OR = 0.45, p = 0.049; recessive model: OR = 0.18, p = 0.018), rs1871054 of ADAM12 (dominant model: OR = 2.19, p = 0.022), rs1800796 of IL6 (dominant model: OR = 0.30, p = 0.003), rs6494629 of SMAD3 (additive model: OR = 0.65, p = 0.019; dominant model: OR = 0.52, p = 0.012), and rs4789936 of TIMP2 (recessive model: OR = 5.90, p = 0.024). Immunohistochemistry verified significantly upregulated PPARG, ADAM12, SMAD3, and TIMP2 in KBD compared with OA and normal controls (p < 0.05). Genetic polymorphisms of PPARG, ADAM12, SMAD3, and TIMP2 may contribute to the risk of KBD. These genes could promote the pathogenesis of KBD by disturbing ECM homeostasis.