Animal models of Kashin-Beck disease exposed to environmental risk factors: Methods and comparisons.

The main etiological mechanism for Kashin-Beck disease (KBD) is deep chondrocyte necrosis induced by environmental risk factors (ERFs). The scholars have conducted several epidemiological, cellular, and animal model studies on ERFs. Gradually, four etiological hypotheses have been formed, including water of organic poisoning hypothesis represented by fulvic acid (FA), biogeochemical hypothesis represented by selenium (Se) deficiency, food mycotoxin poisoning hypothesis represented by T-2 toxin poisoning and compound etiology theory hypothesis. The animal models of KBD have been replicated based on the previous etiological hypotheses. The different species of animals (monkey, rat, dog, pig, chicken, and rabbit) were treated with different ERFs interventions, and the clinical manifestations and pathological changes of articular cartilages were observed. The animals in the experimental group were fed with endemic water, endemic grain, low nutrition, thallium sulfate, FA, Se, T-2 toxin, and iodine. The dose of thallium sulfate was 1154 µg/d; the doses range of FA were 5, 50, 150, 200, and 211 mg/kg; the doses range of Se were 0.00035, 0.00175, 0.005, 0.02, 0.031, 0.1, 0.15, 0.314, 0.5, and 10 mg/kg; the doses range of T-2 toxin were 40, 100, 200, 600, 1000, 1500, 3000, 6000, and 9000 ng/g; and the doses range of iodine were 0.04, 0.18, and 0.4-0.5 µg/g. The sample size ranged from 9 to 230 depending on the interventions and grouping; the follow-up duration ranged from 1 week to 18 months. Moreover, the methods and comparisons of different animal models of KBD had been summarized to provide a useful basis for studying the pathogenesis of KBD. In conclusion, the rhesus monkeys administrated endemic water and grain were susceptible animals to replicate KBD. The rats treated with T-2 toxin combined with Se/nutrition deficiency could be a suitable and widely used animal model.

Role of the hippo signaling pathway in the extracellular matrix degradation of chondrocytes induced by fluoride exposure.

To identify the role of the Hippo signaling pathway in the extracellular matrix degradation of chondrocytes induced by fluoride exposure. Environmental response genes (ERGs) of bone injury induced by fluoride exposure were obtained from the Comparative Toxicogenomics Database, and annotated by STRING for KEGG pathway enrichment analysis. The CCK-8 kit was used to measure the proliferation of ATDC5 cells. The malondialdehyde (MDA), total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-PX) levels in ATDC5 cells were measured using oxidative stress detection kit. Western blot analysis was used to measure the p-MST1/2, p-LATS1/2, and p-YAP/YAP1 expression levels in the Hippo pathway and the COL2A1, ACAN and MMP13 expression levels in the cartilage matrix. Localizations of YAP1 and COL2A1 proteins in chondrocytes were performed using cell immunofluorescence. Continuous data from the multiple groups were compared using one-way analysis of variance, and then the differences between groups were tested with Dunnett's t-test, with the test level α = 0.05. The 145 ERGs of bone injury induced by fluoride exposure were identified, and KEGG enrichment analysis revealed Hippo signaling pathways significantly related to bone injury. A CCK-8 assay revealed that the viability of the ATDC5 cells was significantly decreased with increased fluorine concentration. The MDA content in 20 mg/L sodium fluoride (NaF) exposure group was significantly higher than that in the control group, the T-SOD, T-AOC and GSH-PX activities in 15 and 20 mg/L NaF exposure groups were significantly lower than those in the control group (P < 0.05). Western blot results showed the protein levels of p-MST1/2, p-LATS1/2 and p-YAP1 in 15 and 20 mg/L NaF exposure groups were significantly lower than those in the control group, while the YAP1 protein level in 20 mg/L NaF group was significantly higher than that in the control group. The COL2A1 and ACAN proteins in 20 mg/L NaF group were significantly decreased, while the MMP13 protein level in 15 and 20 mg/L NaF groups were significantly increased (P < 0.05). It was observed that the expression of YAP1 protein expression level in the cytoplasm decreased with the increased fluoride exposure, whereas that the expression level of YAP1 protein in the nucleus increased. Fluoride inhibited the proliferation of ATDC5 cells, induced oxidative stress, inhibited the activity of the Hippo pathway, and eventually led to cartilage matrix degradation.

T-2 toxin induces mitochondrial dysfunction in chondrocytes via the p53-cyclophilin D pathway.

Kashin-Beck disease is an endemic joint disease characterized by deep chondrocyte necrosis, and T-2 toxin exposure has been confirmed its etiology. This study investigated mechanism of T-2 toxin inducing mitochondrial dysfunction of chondrocytes through p53-cyclophilin D (CypD) pathway. The p53 signaling pathway was significantly enriched in T-2 toxin response genes from GeneCards. We demonstrated the upregulation of the p53 protein and p53-CypD complex in rat articular cartilage and ATDC5 cells induced by T-2 toxin. Transmission electron microscopy showed the damaged mitochondrial structure of ATDC5 cells induced by T-2 toxin. Furthermore, it can lead to overopening of the mitochondrial permeability transition pore (mPTP), decreased mitochondrial membrane potential, and increased reactive oxygen species generation in ATDC5 cells. Pifithrin-α, the p53 inhibitor, alleviated the increased p53-CypD complex and mitochondrial dysfunction of chondrocytes induced by T-2 toxin, suggesting that p53 played an important role in T-2 toxin-induced mitochondrial dysfunction. Mechanistically, T-2 toxin can activate the p53 protein, which can be transferred to the mitochondrial membrane and form a complex with CypD. The increased binding of p53 and CypD mediated the excessive opening of mPTP, changed mitochondrial membrane permeability, and ultimately induced mitochondrial dysfunction and apoptosis of chondrocytes.

Proteomics Sequencing Reveals the Role of TGF-ß Signaling Pathway in the Peripheral Blood of Offspring Rats Exposed to Fluoride.

The underlying mechanism of fluorosis has not been fully elucidated. The purpose of this study was to explore the mechanism of fluorosis induced by sodium fluoride (NaF) using proteomics. Six offspring rats exposed to fluoride without dental fluorosis were defined as group A, 8 offspring rats without fluoride exposure were defined as control group B, and 6 offspring rats exposed to fluoride with dental fluorosis were defined as group C. Total proteins from the peripheral blood were extracted and then separated using liquid chromatography-tandem mass spectrometry. The identified criteria for differentially expressed proteins were fold change > 1.2 or < 0.83 and P < 0.05. Gene Ontology function annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using the oeCloud tool. The 177 upregulated and 22 downregulated proteins were identified in the A + C vs. B group. KEGG pathway enrichment analysis revealed that transforming growth factor-ß (TGF-ß) signaling pathway significantly enriched. PPI network constructed using Cytoscape confirmed RhoA may play a crucial role. The KEGG results of genes associated with fluoride and genes associated with both fluoride and inflammation in the GeneCards database also showed that TGF-ß signaling pathway was significantly enriched. The immunofluorescence in HPA database showed that the main expression sites of RhoA are plasma membrane and cytosol, while the main expression site of Fbn1 is the Golgi apparatus. In conclusion, long-term NaF intake may cause inflammatory response in the peripheral blood of rats by upregulating TGF-ß signaling pathway, in which RhoA may play a key role.

Association of AGTR1 gene methylation and its genetic variant in Chinese farmer with hypertension: A case-control study.

The objective was to determine the potential associations of the angiotensin II receptor type 1 (AGTR1) gene polymorphism, methylation, and lipid metabolism in Chinese farmers with hypertension. A case-control study was conducted in Wuzhi county of Henan province in China in 2013 to 2014. A total of 1034 local residents (35-74 years, 386 hypertensive cases, and 648 normotensive subjects) were enrolled in this study. Triglyceride (TG), total cholesterol (TC), high-density lipoprotein, and low-density lipoprotein were measured using automatic chemistry analyzer. The AGTR1 gene promoter methylation level was measured using quantitative methylation-specific polymerase chain reaction method. The single nucleotide polymorphism rs275653 was genotyped with TaqMan probe assay at an applied biosystems platform. The gender, body mass index (BMI), TG, TC, and family history of hypertension in the hypertension group were significantly higher than those in control group (P < .05). No significant difference was observed in the distribution of AGTR1 rs275653 polymorphism in the hypertension and controls (P > .05). The AGTR1 gene methylation in subjects carrying different genotypes was not significantly observed (P > .05). The logistic regression analysis found the AGTR1 gene methylation level was negative correlation with hypertension in the present study (odds ratio, 0.946, 95% confidence interval, 0.896-0.999) through adjusting for age, gender, BMI, education, smoking, alcohol drinking, fruit and vegetable intake, pickles intake, and family history of hypertension. The association of AGTR1 gene hypomethylation and essential hypertension was observed in Chinese farmers; no significant difference was observed in the distribution of AGTR1 rs275653 polymorphism.