Serious Selenium Deficiency in the Serum of Patients with Kashin-Beck Disease and the Effect of Nano-Selenium on Their Chondrocytes.

To investigate selenium (Se) concentrations in serum of patients with rheumatoid arthritis (RA), osteoarthritis (OA), and Kashin-Beck disease (KBD), together with the effect of Se supplement (chondroitin sulfate [CS] nano-Se [SeCS]) on CS structure-modifying sulfotransferases in KBD chondrocyte. Fifty serum samples from each group with aged-matched (40-60 years), normal control (N), RA, OA, and KBD (25 males and females, respectively) were collected to determine Se concentrations. Furthermore, the KBD chondrocytes were divided into two groups following the intervention for 24 h: (a) non-treated KBD group and (b) SeCS-treated KBD group (100 ng/mL SeCS). The ultrastructural changes in chondrocytes were observed by transmission electron microscopy (TEM). Live/dead staining was used to observe cell viability. The expression of CS-modifying sulfotransferases including carbohydrate sulfotransferase 12, 13, and 15 (CHST-12, CHST-13, and CHST-15, respectively), and uronyl 2-O-sulfotransferase (UST) were examined by quantitative real-time polymerase chain reaction and western blotting analysis after SeCS intervention. The Se concentrations in serum of KBD, OA, and RA patients were lower than those in control. In OA, RA, and control, Se concentrations were higher in male than in female, while it is opposite in KBD. In the cell experiment, cell survival rate and mitochondrial density were increased in SeCS-treated KBD groups. Expressions of CHST-15, or CHST-12, and CHST-15 on the mRNA or protein level were significantly increased. Expression of UST slightly increased on the mRNA level, but no change was visible on the protein level. Se deficiency in serum of RA, OA, and KBD was observed. SeCS supplemented in KBD chondrocytes improved their survival rate, ameliorated their ultrastructure, and increased the expression of CS structure-modifying sulfotransferases.

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.

Selenium and Iodine Levels in Subjects with Kashin-Beck Disease: a Meta-analysis.

Kashin-Beck disease (KBD) is an endemic, degenerative osteoarthropathy, and particularly seen in China. A deficiency of selenium and iodine is implicated as the main etiological factor for KBD. This meta-analysis aimed to evaluate the differences in the selenium and iodine levels between patients with KBD and healthy individuals. Eligible articles published before March 6, 2015 were searched from four electronic databases. Data extraction and quality assessment of included studies were performed by two independent reviewers. Results were summarized as standardized mean difference (SMD) with 95 % confidence intervals (CIs). Cohen's d test was used to estimate the difference of the effect size between patients with KBD and healthy controls. A total of 26 cross-sectional studies were included in the meta-analysis. The pooled SMD showed that the whole blood selenium (Cohen's d = 4.39, P < 0.001), serum selenium (Cohen's d = 2.42, P = 0.015), hair selenium (Cohen's d = 5.46, P < 0.001), and urinary selenium (Cohen's d = 4.16, P < 0.001) levels were significantly lower in patients with KBD than that in healthy controls. There was no significant difference of plasma selenium (Cohen's d = 0.08, P = 0.936) and urinary iodine (Cohen's d = 0.33, P = 0.744) levels between subjects with KBD and healthy controls. In conclusion, the levels of selenium, but not iodine were significantly lower in subjects with KBD than that in healthy controls. Selenium deficiency might be associated with the risk of KBD.

Prevalence of Selenium, T-2 Toxin, and Deoxynivalenol in Kashin-Beck Disease Areas in Qinghai Province, Northwest China.

The aim of this study was to determine the levels of selenium, T-2 toxin, and deoxynivalenol (DON) contamination in Kashin-Beck disease (KBD) areas and provide information for understanding the high prevalence of KBD in Qinghai Province. A total of 183 subjects were chosen in a KBD-prevalent county (Guide County) and a non-KBD county (Huangzhong County) in Qinghai Province, northwestern China, and the samples of wheat flour, soil, drinking water and blood, urine, and hair of children were collected from these residents. The selenium concentrations from all these sources were determined using atomic fluorescence spectrophotometry. The levels of T-2 toxin and DON contamination in the wheat flour samples were assayed using HPLC-MS/MS. The average selenium content in the soil, drinking water, and wheat flour samples from KBD areas were 26.93 ± 10.06 µg/kg, 0.097 ± 0.038 µg/L, and 9.50 ± 7.17 µg/kg, respectively. Among these, the selenium levels in the drinking water and wheat flour samples from the KBD endemic county were significantly lower than those from the non-KBD county. For the selenium nutrient status, only the hair selenium concentration of children from the KBD endemic county was significantly lower than that from the non-KBD county. The contents of T-2 toxin in all wheat samples were below the detection limit (0.4 µg/kg). The levels of DON contamination in wheat flour samples from KBD and non-KBD children's households within the KBD endemic county were relatively higher, with average levels of 302 ± 49 and 280 ± 48 µg/kg, respectively. The DON level of wheat flour samples from the children's households in the non-KBD county was significantly lower than that from the KBD endemic county. These results suggest that the lower selenium status in Guide County still remains. While the selenium nutritional status of the local children has improved to some extent, partly due to the introduction of food produce from nonlocal areas. DON contamination in the wheat flour may be involved in the fluctuating high prevalence rates of KBD in children in the KBD endemic Guide County in Qinghai Province.

Association study of polymorphisms in selenoprotein genes and Kashin-Beck disease and serum selenium/iodine concentration in a Tibetan population.

BACKGROUND: Kashin-Beck disease is a kind of degenerative osteoarthropathy. Genetic factors may play an important role in the pathogenesis of KBD. OBJECTIVE: To investigate the association of the selenoprotein genes GPX1 (rs1050450, rs1800668, and rs3811699), TrxR2 (rs5748469), and DIO2 (rs225014) with Kashin-Beck disease (KBD) in a Tibetan population and to investigate the association of these SNPs with the serum iodine/selenium concentration in the Tibetan population. DESIGN: Five SNPs including rs1050450, rs1800668, and rs3811699 in the GPX1 gene, rs5748469 in the TrxR2 gene, and rs225014 in the DIO2 gene were analyzed in Tibetan KBD patients and controls using the SNaPshot method. P trend values of the SNPs were calculated using an additive model. RESULTS: None of the five SNPs in the three genes showed a significant association with KBD. Haplotypes TCC, TTC and TTT of rs1050450, rs1800668 and rs3811699 in GPX1 showed a significant association with KBD and controls with P value of 0.0421, 5.0E-4 and 0.0066, respectively. The GPX1 gene (rs1050450) showed a potential significant association with the iodine concentration in the Tibetan study population (P = 0.02726). However, no such association was detected with the selenium concentration (P = 0.2849). CONCLUSIONS: In this study, we showed that single SNPs in the genes GPX1 (rs1050450, rs1800668 and rs3811699), TrxR2 (rs5748469), and DIO2 (rs225014) may not be significantly associated with KBD in a Tibetan population. However, haplotype analysis of SNPs rs1050450, rs1800668 and rs3811699 in GPX1 gene showed a significant association with KBD. The results suggested that GPX1 gene play a protective role in the susceptivity of KBD in Tibetans. Furthermore, the GPX1 gene (rs1050450) may be significantly associated with the serum iodine concentration in Tibetans.

An animal model of Kashin-Beck disease induced by a low-nutrition diet and exposure to T-2 toxin.

OBJECTIVE: We investigated the combined roles of a low-nutrition diet (low levels of protein, iodine, and selenium) and T-2 toxin in bone development and to establish an experimental animal model of Kashin-Beck disease (KBD) that reliably mimics the disease's pathological changes for further study of the pathogenesis and prevention of the disease. METHODS: Sprague-Dawley rats were randomly divided among four groups: group A, normal diet; group B, normal diet plus T-2 toxin; group C, low-nutrition diet; and group D, low-nutrition diet plus T-2 toxin exposure. The radiographic and histopathological changes in the tibial growth zone, plate cartilage and metaphysis were examined. RESULTS: In group D, all epiphyseal plates were blurred, thin, and irregular. Tibias were significantly shorter in group D than in groups A and B. After 4 weeks, epiphyseal plates showed chondrocyte necrosis, with the more obvious necrosis appearing in groups C and D. The positive rate of lamellar necrosis was significantly higher in group D than in groups B and A (P < 0.01). In group D, metaphyseal trabecular bone was sparse, disordered, and disrupted, and massive transverse trabecular bone appeared in the metaphysis at 12 weeks. CONCLUSIONS: A rat model of KBD induced by a low-nutrition diet and T-2 toxin exposure demonstrated radiographic and histopathological abnormalities of the proximal epiphyseal plate and the tibial metaphysis that are very similar to the bone changes found in patients with KBD. This animal model will be helpful for further study of the pathogenesis and prevention of KBD.

SNP and mRNA expression for glutathione peroxidase 4 in Kashin-Beck disease.

Kashin-Beck disease (KBD) is a chronic endemic osteoarthropathy, which mainly occurs in West and Northeast China. Epidemiological studies suggest that Se deficiency is an important environmental factor for the incidence of KBD. Glutathione peroxidase 4 (GPx4) belongs to the glutathione peroxidase family, which is crucial for optimal antioxidant defences. Our purpose is to investigate the putative association between GPx4 polymorphisms and the risk of KBD. Restriction fragment length polymorphism-PCR was used to detect two SNP (rs713041, rs4807542) in 219 cases and 194 controls in Han Chinese subjects, and quantitative analysis for the GPx4 mRNA level was performed by the real-time PCR method. The results revealed that linkage disequilibrium existed in the two SNP. A significant difference was observed in the haplotype A-T (P = 0·0066) of GPx4, which was obviously lower in the KBD cases (0·006 v. 0·032 %). Correlation analysis based on a single locus showed no association between each SNP and KBD risk. Furthermore, the GPx4 mRNA level was dramatically lower in the blood of KBD patients. Overall, our finding indicated GPx4 polymorphisms and decreased mRNA level may be related to the development of KBD in the Chinese population, suggesting GPx4 as a possible candidate susceptibility gene for KBD.

[Interaction of plasma selenium levels and D12S304 gene site in the pathogenesis of Kashin-Beck disease analyzed by multifactor dimensionality reduction software].

OBJECTIVE: To explore the interaction of plasma selenium levels and D12S304 gene site in the pathogenesis of Kashin-Beck disease (KBD). METHODS: Case-control design was taken to compare the difference of plasma selenium levels and genotype of D12S304 between KBD patients and non-patients, and the interactions were analyzed by MDR software. RESULTS: Plasma selenium levels was lower in the case group than in the control group, while the D12S304 gene site was not different between the two groups, and no interaction between plasma selenium and genotype was observed. CONCLUSION: There was no interaction between plasma selenium and genotype at D12S304. Enlarging sample size or selecting another gene site might be needed in exploring the gene-environment interactions in the pathogenesis of KBD.

Genetic variants in the HLA-DRB1 gene are associated with Kashin-Beck disease in the Tibetan population.

OBJECTIVE: To investigate the association between variants in the HLA-DRB1 gene and Kashin-Beck disease (KBD), as well as associations of selenium and iodine deficiencies with KBD in a Tibetan population. METHODS: Fourteen single-nucleotide polymorphisms (SNPs) were genotyped around the HLA-DRB1 gene, and HLA-DRB1 allele genotyping was performed in a discovery cohort, composed of 605 patients with KBD and 393 control subjects, and/or a replication cohort, composed of 290 patients with KBD and 295 controls. Plasma concentrations of selenium and iodine were measured and compared by t-test in 299 patients with KBD and 280 controls from the same villages. RESULTS: Four SNPs (rs6457617, rs6457620, rs9275295, and rs7745040) in the HLA-DRB1 gene locus were significantly associated with KBD in both the discovery cohort and replication cohort (combined cohort odds ratios [ORs] 1.307-1.402, P = 0.0039-0.0006). The protective haplotype GTCC and the risk haplotype ACGT, each generated by the 4 SNPs, showed a significant association with KBD (for GTCC, OR 0.77, P = 0.0031; for ACGT, OR 1.40, P = 0.0014). HLA-DRB1 allele genotyping revealed that the frequencies of HLA-DRB1*08 and *11 were significantly different between patients with KBD and controls (for HLA-DRB1*08, OR 0.731, P = 0.00564; for HLA-DRB1*11, OR 0.489, P = 0.000395). Moreover, plasma concentrations of selenium and iodine were significantly different between patients with KBD and controls from the same villages (P = 0.0013 and P = 1.84 × 10(-12) , respectively). CONCLUSION: These findings, obtained in plasma samples from Tibetan patients with KBD and healthy control subjects from the same regions, confirm the role of selenium and iodine deficiencies in the development of KBD. Moreover, genetic variants in the HLA-DRB1 gene significantly increase the susceptibility to KBD in this population.

[Transmission disequilibrium test for 15 short tandem repeat loci in Kashin-Beck disease and their interaction with low selenium].

OBJECTIVE: To identify the genetic susceptibility to Kashin-Beck disease (KBD) and explore the interaction between low selenium (Se) and the susceptibility gene loci in KBD. METHODS: The DNA samples collected from 23 KBD nuclear families were analyzed using PCR and GeneScan Analysis 3.7 and Genotyper3.7 software. The haplotype relative risk (HRR) and transmission disequilibrium test (TDT) were used to test the data of the genotypes. The serum selenium (Se) concentration was measured by atomic fluorescence spectrometry, and the interaction between low Se and the susceptibility loci was calculated using a binary logistic regression. RESULTS: In the 23 nuclear families, the alleles of D2S151 (248 bp), D2S305 (320 bp), and D11S4094 (194 bp) showed significant correlation to KBD (P<0.05). Serum Se concentrations in the studied individuals was 0.037 µg/ml. No significant statistical interaction was observed between low Se exposure and the susceptibility loci (P>0.05). CONCLUSION: The polymorphisms in the STR loci D2S305, D2S151, and D11S4094 or the polymorphism loci near them might been related to KBD susceptibility. Low Se exposure shows no significant interaction with the susceptibility loci.