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.

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.

Genetic association scan of 32 osteoarthritis susceptibility genes identified TP63 associated with an endemic osteoarthritis, Kashin-Beck disease.

BACKGROUND: Kashin-Beck disease (KBD) is an endemic chronic osteochondropathy. The clinical manifestations and radiographic features of adult KBD were similar to those of osteoarthritis (OA). METHODS: We first performed a genetic association scan of 32 OA susceptibility genes with KBD in 898 Han Chinese subjects. The MassARRAY genotyping system (Agena) was used for SNP genotyping. PLINK 1.9 was used for quality control and association testing. Using articular cartilage specimens from 7 adult KBD patients and 4 control subjects, lentivirus-mediated RNA interference (RNAi), qRT-PCR, Western blot and immunohistochemistry were employed to explore the functional relevance of TP63 to KBD chondrocyte. RESULTS: SNP genotyping and association analysis identified TP63 (rs12107036, P = 0.005, OR = 0.71) and OARD1 (rs11280, P = 0.004, OR = 1.51) were significantly associated with KBD. It was also found that TP63 was significantly up-regulated in KBD articular cartilage in both mRNA and protein level compared with the controls (P < 0.05). TP63 suppression by lentivirus-mediated RNAi notably decreased the abundance of Caspase3 and SOX9 in chondrocytes. Most importantly, compared with the scrambled sequence (shControl) group, the protein level of ACAN was increased in the shTP63 group. The mRNA expression of chondrocyte marker genes (COL2A1 and ACAN) was not significantly changed after TP63 knockdown relative to shControl group. CONCLUSION: Our study identifies TP63 as a novel susceptibility gene for KBD, and demonstrates that the inhibition of TP63 suppresses chondrocyte apoptosis and partly facilitates chondrogenesis. The combination of SNP genotyping and molecular biology techniques provides a useful tool for understanding the biological mechanism and differential diagnosis studies of KBD and OA.

LncRNA MIAT regulated by selenium and T-2 toxin increases NF-κB-p65 activation, promoting the progress of Kashin-Beck Disease.

LncRNA myocardial infarction associated transcript (MIAT) has been shown to be involved in osteoarthritis (OA), but its role in Kashin-Beck Disease (KBD) has rarely been reported. In this study, rats were administered with low selenium and/or T-2 toxin for 4 weeks to establish a KBD animal model. The serum selenium level, TNF-α and IL-1ß contents, phosphorylated p65 (p-p65) and MIAT expression were increased in each intervention group. Next, we isolated the primary epiphyseal chondrocytes, and found that selenium treatment reversed the effects of T-2 toxin on chondrocyte injury, p-p65 and MIAT expression. In addition, MIAT overexpression or T-2 toxin treatment led to increased cell death, apoptosis, inflammation, NF-κB-p65 pathway activation and MIAT expression, which was rescued by selenium treatment or MIAT siRNA transfection. Our results suggested that lncRNA MIAT regulated by selenium and T-2 toxin increased the activation of NF-κB-p65, thus being involved in the progress of KBD.

The Study on Polymorphism of TrxR and Nrf2/HO-1 Signaling Pathway in Kaschin-Beck Disease.

Kaschin-Beck disease (KBD) is an endemic, chronic, and degenerative osteoarthropathy, which seriously impairs the quality of patients' life. We detected the expression of TrxR by ELISA and found that TrxR was lower in KBD than in normal control group significantly (P < 0.001); this result indicated that TrxR must be related to KBD. We retrieved cSNPs in NCBI SNP database and used three bioinformatics programmers, including SIFT, PolyPhen, and SNP3d, to help select the researched nsSNP. Then, we used PCR-RFLP to analyze the relationship between the SNP site rs5746841 in TrxR2 gene and susceptibility of KBD and detected the expression of Nrf2 and HO-1 by western blot. The results showed that the genotype of rs5746841 in 93 normal controls and 103 KBD subjects were C/C totally, but A/A and A/C were not found, which indicated preliminarily that there was no correlation between rs5746841 in TrxR2 gene and susceptibility of KBD. The expression of TrxR was lower in KBD than in normal control group significantly, while the expressions of Nrf2 and HO-1 were higher in KBD than in normal control group. These results indicated that the low expression of selenoprotein TrxR may be a candidate factor of KBD, which related to Nrf2/HO-1 signaling pathway.

Expression Profiles of Selenium-Related Genes in Human Chondrocytes Exposed to T-2 Toxin and Deoxynivalenol.

The combination of excess mycotoxin exposure and selenium deficiency has been widely considered as a cause of Kashin-Beck disease (KBD). The present study aimed to investigate the expression profiles of selenium-related genes in human chondrocytes after exposure to T-2 toxin and deoxynivalenol (DON) and to preliminarily identify the potential biological functions of the identified genes. Gene expression profiling was performed on human chondrocytes treated with 0.01 µg/ml T-2 toxin and 1.0 µg/ml DON by using Affymetrix Human Gene Arrays. The 1660 selenium-related genes were derived from the Comparative Toxicogenomics Database. Gene-term enrichment analysis was conducted by the DAVID gene annotation tool. Our results showed that 69 and 191 selenium-related genes were differentially expressed after T-2 toxin and DON treatment, respectively. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these identified genes were involved in various biological functions, such as the GO terms in response to oxidative stress, cell cycle arrest, and apoptotic process and the KEGG metabolic, FoxO signaling, and p53 signaling pathways. Our results may help explain the mechanisms of interaction between mycotoxins and selenium following human chondrocyte damage and reveal the potential roles of environmental risk factors in cartilage lesions during KBD development.

The study of GPX3 methylation in patients with Kashin-Beck Disease and its mechanism in chondrocyte apoptosis.

OBJECTIVE: Selenium deficiency is a risk factor for Kashin-Beck Disease (KBD), an endemic osteoarthropathy. Although promoter hypermethylation of glutathione peroxidase 3 (GPX3) (a selenoprotein) has been identified in several cancers, little is known about promoter methylation and expression of GPX3 and their relation to selenium in KBD. The present study was thus conducted to investigate this research question. METHODS: Methylation and expressions of GPX3 in whole blood drawn from 288 KBD patients and 362 healthy controls and in chondrocyte cell line were evaluated using methylation-specific PCR and qRT-PCR, respectively. The protein levels of PI3K/Akt/c-fos signaling in the whole blood and chondrocyte cell line were determined with Western blotting. Chondrocytes apoptosis were detected by Hoechst 33342 and Annexin V-FITC/PI staining. RESULTS: GPX3 methylation was increased, GPX3 mRNA was decreased, and protein levels in the PI3K/Akt/c-fos signaling pathway were up-regulated in the whole blood collected from KBD patients as compared with healthy controls. Similar results were obtained for chondrocytes injured by oxidative stress. There was a significant, decreasing trend in GPX3 expression across groups of unmethylation, partial methylation, and complete methylation for GPX3, in sequence. Compared with unmethylation group, protein levels in PI3K/Akt/c-fos pathway were enhanced in partial and complete methylation groups. Treatment of chondrocytes with sodium selenite resulted in reduced methylation and increased expression of GPX3 as well as down-regulated level of PI3K/Akt/c-fos proteins. CONCLUSIONS: The methylation and expression of GPX3 and expression of PI3K/Akt/c-fos pathway are altered in KBD and these changes are reversible by selenium supplementation.

The expression of ERK and JNK in patients with an endemic osteochondropathy, Kashin-Beck disease.

Kashin-Beck disease (KBD) is a chronic, endemic osteochondropathy. Its etiopathogenesis is still obscure until now. Epidemiological observation has shown that low selenium play a crucial role in the pathogenesis of KBD. Extracellular signal-regulated kinases (ERKs) and C-Jun N-terminal kinase (JNK), members of the mitogen-activated protein kinase (MAPK) superfamily, play an important role in cell proliferation and differentiation. Nuclear factor-ĸB (NF-ĸB), an important signaling mediator for inflammatory and immune responses, is involved in the regulation of osteoclastogenesis. In the present study, we investigated the expression of ERK and JNK signal molecular, as well as nuclear factor-ĸB in the pathogenesis of Kashin-Beck disease, evaluated the effect of selenium on ERK signal pathway. The expression levels of ERK and JNK signal pathway, as well as nuclear factor-ĸB were investigated for 218 patients and 209 controls by immunoblot analysis in whole blood. Evaluated the effect of selenium on ERK signal pathway by Na2SeO3 treatment. The protein levels of pRaf-1, pMek1/2 and pErk1/2 decreased significantly in KBD patients, p-JNK and NF-ĸB increased in KBD patients. Furthermore, Na2SeO3 treatment improved the reduction of proteins in ERK signal pathway. These findings indicated that ERK and JNK signaling pathways, as well as the expression level of NF-κB signaling molecular are important contributor to the pathogenesis of KBD. Selenium stimulates the phosphorylation of the ERK signaling pathway.

Identified molecular mechanism of interaction between environmental risk factors and differential expression genes in cartilage of Kashin-Beck disease.

As environmental risk factors (ERFs) play an important role in the pathogenesis of Kashin-Beck disease (KBD), it is important to identify the interaction between ERFs and differentially expression genes (DEGs) in KBD. The environmental response genes (ERGs) were analyzed in cartilage of KBD in comparison to normal controls.We searched 5 English and 3 Chinese databases from inception to September 2015, to identify case-control studies that examined ERFs for KBD using integrative meta-analysis and systematic review. Total RNA was isolated from articular cartilage of KBD patients and healthy controls. Human whole genome microarray chip (Agilent) was used to analyze the amplified, labeled, and hybridized total RNA, and the validated microarray data were partially verified using real-time quantitative polymerase chain reaction (qRT-PCR). The ERGs were derived from the Comparative Toxicogenomics Database. The identified ERGs were subjected to KEGG pathway enrichment, biological process (BP), and interaction network analyses using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7, and STRING.The trace elements (selenium and iodine), vitamin E, and polluted grains (T-2 toxin/HT-2 toxin, deoxynivalenol, and nivalenol) were identified as the ERFs for KBD using meta-analysis and review. We identified 21 upregulated ERGs and 7 downregulated ERGs in cartilage with KBD compared with healthy controls, which involved in apoptosis, metabolism, and growth and development. KEGG pathway enrichment analysis found that 2 significant pathways were involved with PI3K-Akt signaling pathway and P53 signaling pathway, and gene ontology function analysis found 3 BPs involved with apoptosis, death, and cell death in KBD cartilage.According to previous results and our own research, we suggest that the trace element selenium and vitamin E induce PI3K-Akt signaling pathway and the mycotoxins (T-2 toxin/HT-2 toxin and DON) induce P53 signaling pathway, contributing to the development of KBD, and chondrocyte apoptosis and cell death.

Investigation of MMP-1 genetic polymorphisms and protein expression and their effects on the risk of Kashin-Beck disease in the northwest Chinese Han population.

BACKGROUND: The etiology of Kashin-Beck disease (KBD), an endemic osteochondropathy, is largely unknown. Matrix metalloproteinase-1 (MMP-1) plays a central role in the initiation and progression of cartilage destruction; however, no study has reported on the relationship between KBD and MMP-1. This study was to investigate the role of MMP-1 in the pathogenesis and progression of KBD. METHODS: Single nucleotide polymorphism (SNP) genotyping was conducted for 274 KBD cases and 248 healthy controls using the Sequenom MassARRAY system. Additionally, the expression of MMP-1 in the knee articular cartilage of 22 KBD patients and 21 controls was analyzed by immunohistochemistry, and the concentration of MMP-1 in their joint fluid was also measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The results showed that two SNPs (rs470221 and rs1144396) had a weak association with increased KBD risk; however, the significance of these results did not survive Bonferroni's correction. Moreover, the percentages of cells expressing MMP-1 in each layer of cartilage were significantly higher in the KBD group than in the controls (F = 11.41-28.31, P = 0.002-0.000). The concentration of MMP-1 in KBD joint fluid was significantly higher than that in the controls (t = 9.83, P < 0.0001). CONCLUSIONS: The increased expression of MMP-1 has a potential effect on the risk of KBD in the northwest Chinese Han population. However, six selected SNPs in the MMP-1 gene might not be useful as significant markers for predicting KBD susceptibility in Chinese Han population. Therefore, future studies in the association of MMP-1 with KBD should focus on other candidate SNPs.

Role of selenoprotein S (SEPS1) -105G>A polymorphisms and PI3K/Akt signaling pathway in Kashin-Beck disease.

OBJECTIVE: To investigate the relationship between SEPS1 polymorphism and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in Kashin-Beck disease (KBD) and further explore the pathogenesis of KBD. METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to detect SEPS1 -105G>A polymorphism in 232 cases and 331 controls. The protein expressions of PI3K/Akt signaling molecules in whole blood and chondrocytes were detected by Western blot. RESULTS: The frequencies of SEPS1 -105G>A genotype AA (21.1% vs 3.0%) and minor allele A (34.1% vs 16.0%) in KBD are significantly higher than those in controls (OR: 8.020, 95% confidence interval (95% CI) 6.341-10.290, P < 0.0001; OR: 2.470, 95% CI 2.001-4.463, P < 0.0001, respectively). SEPS1 AA genotype was an independent risk factor for KBD (adjusted OR: 9.345, 95% CI 4.254-20.529; P < 0.0001). The expression of Gßγ, PI3Kp110, pAkt and pGSK3ß in KBD group were higher than that in control group (all P < 0.05). Gßγ, pAkt and pGSK3ß protein expression of AA and GA increased than GG (all P < 0.05). Cell apoptosis was increasing and molecule expression of PI3K/Akt signaling pathway were up-regulated in the tert-Butyl hydroperoxide (tBHP)-injured group, the cell apoptosis and expression levels of PI3K/Akt in Na2SeO3 group were decreased. CONCLUSIONS: The SEPS1 -105G>A is associated with an increased risk of KBD and influences the expression of PI3K/Akt signaling pathway in KBD patients. Apoptosis induced by tBHP in chondrocyte might be mediated via up-regulation of PI3K/Akt, Na2SeO3 has an effect of anti-apoptosis by down-regulating of PI3K/Akt signaling pathway.

Identification of differentially expressed genes and pathways between primary osteoarthritis and endemic osteoarthritis (Kashin-Beck disease).

OBJECTIVES: Primary osteoarthritis (OA) and Kashin-Beck disease (KBD) exhibit similar clinical manifestations and common articular cartilage lesions. Revealing the pathogenetic differences between OA and KBD is helpful for differential diagnosis and may provide new insights into the pathogenesis of OA and KBD. In this study, we compared the genome-wide gene ontology (GO) and pathway expression patterns of articular cartilage derived from both OA and KBD patients. METHODS: Total RNA was isolated, amplified, labelled, and hybridized using Agilent whole genome microarray analysis. Gene set enrichment analysis (GSEA) was used to identify differentially expressed genes and pathways between OA and KBD. Nine differentially expressed GO categories and 85 differentially expressed pathways were identified by this study. RESULTS: The reactive oxygen species (ROS)-related HOUSTIS_ROS pathway and the vascular endothelial growth factor (VEGF)-related ABE_VEGFA_TARGETS_2HR pathway were significantly up-regulated in OA compared to KBD. Higher expression levels of the collagen-related COLLAGEN GO, EXTRACELLULAR_MATRIX_PART GO, and nitric oxide (NO)-related BIOCARTA_NO1_PATHWAY pathways were detected in KBD than in OA. CONCLUSIONS: ROS-induced cartilage lesions seem to be more involved in the pathogenesis of OA whereas NO-mediated chondrocyte apoptosis contributes more to the development of KBD.

Association of TNF-α and Fas gene promoter polymorphism with the risk of Kashin-Beck disease in Northwest Chinese population.

The objective of this study is to investigate the relationship between single-nucleotide polymorphisms (SNPs) of the tumor necrosis factor-α (TNF-α) and Fas genes and Kashin-Beck disease (KBD) in Shaanxi province, Northwest in China. Blood samples of 388 residents were collected from 14 KBD villages in Linyou and Yongshou counties, Shaanxi, Northern of China. One hundred eighty-six cases with KBD and 202 cases of health in KBD areas were diagnosed by "Diagnosis Criterion of Kashin-Beck disease in China (WS/T207- 2010)". The TNF-α -308G/A, TNF-α -238G/A, and Fas -670A/G SNPs were determined by polymerase chain reaction-restriction fragment length polymorphism in combination with sequence analysis in KBD and healthy control groups. The genotypes and allele frequencies distribution of these SNPs were then analyzed. TNF-α -308A allele frequency in KBD patients were significantly higher than that in healthy controls. Although TNF-α -238 genotypes and allele frequencies were not significantly different between KBD patients and the healthy controls, GA genotype and A allele frequency in KBD patients were higher than those in healthy controls. The TNF-α -308G/A SNPs were associated with the susceptibility of KBD.

Genome-wide gene expression analysis suggests an important role of suppressed immunity in pathogenesis of Kashin-Beck disease.

OBJECTIVE: To investigate the differences between the gene expression profiles in peripheral blood mononuclear cells (PBMC) from normal controls and patients with Kashin-Beck disease (KBD). METHODS: Twenty KBD patients and 12 normal subjects were selected from a KBD-endemic area and divided into four pairs of KBD vs. control (KBD, n = 5 per pair; control, n = 3 per pair). RNAs were respectively isolated from KBD PBMCs and normal PBMCs. Gene expression profiles were analyzed by oligonucleotide microarray. The gene expression profiles in PBMCs from KBD patients and normal controls were compared and the differentially expressed genes were identified. The obtained microarray data was further confirmed by using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). RESULTS: Approximately 501 genes, corresponding to 2.4% of the total probe transcripts, showed a 2-fold change in differential expression. 19.4% (97 out of 501)of the differentially expressed genes were commonly detected in all the four pairs. Among the 97 differentially expressed genes, 83 genes were up-regulated and 14 genes were down-regulated, compared with those in the normal controls. Some differentially expressed genes were found to be related to functions such as immunity, metabolism, apoptosis, cystoskeleton and cell movement, and extracellular matrix. The validity of our microarray data were supported by the results of qRT-PCR assay. CONCLUSION: Differences in the PBMC gene expression profile between the KBD patients and the normal controls exhibited a similar pattern among all the four pairs of microarrays examined, indicating that the suppressed immunity may play an important role in the pathogenesis of KBD.