RNA-seq analysis reveals different gene ontologies and pathways in rheumatoid arthritis and Kashin-Beck disease.

AIMS: To understand the pathogenesis of cartilage damage in Kashin-Beck disease (KBD) and rheumatoid arthritis (RA) which similar clinical symptoms. METHODS: RNA sequencing (RAN-seq) analysis was used to reveal the different pathogeneses between KBD and RA. The messenger RNA expression profiles of articular cartilage isolated from KBD patients (n = 3) and RA patients (n = 3) were compared using RNA-seq analysis. Differentially expressed genes (DEGs) were determined using the Benjamini-Hochberg approach. The Database for Annotation, Visualization and Integrated Discovery (DAVID 6.7) was employed to assess functional categories and Gene Ontology (GO). The Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology Based Annotation System (KOBAS 2.0) was used to identify significantly enriched KEGG pathways. RESULTS: In the individually sequenced dataset, we identified 1568 significant DEGs in KBD compared to RA (232 up-regulated genes and 1336 down-regulated genes). GO function analysis identified nine significant biological processes (BPs), eight molecular functions (MFs), and five cell components (CCs) in KBD, and also the top ten ranked significant BPs, MFs and CCs were found in RA. The KEGG pathway enrichment analysis identified biosynthesis of amino acids involved in KBD. The chemokine signaling pathway, nuclear factor-kappa B signaling pathway, B cell receptor signaling pathway, leukocyte transendothelial migration, and osteoclast differentiation were involved in RA. CONCLUSIONS: RNA-seq revealed that proteoglycan-mediated metabolic disorders contributed to the onset of KBD, whereas immune dysregulation was apparently involved in the pathogenesis of RA.

Inflammatory cytokine of IL-1ß is involved in T-2 toxin-triggered chondrocyte injury and metabolism imbalance by the activation of Wnt/ß-catenin signaling.

Mycotoxin T-2 exerts a causative role in Kashin-Beck disease (KBD) suffering chondrocyte apoptosis and cartilage matrix homeostasis disruption. Recent research corroborated the aberrant levels of pro-inflammatory cytokine IL-1ß in KBD patients and mycotoxin environment. In the present study, we investigated the relevance of IL-1ß in T-2 toxin-evoked chondrocyte cytotoxic injury and aberrant catabolism. High levels of IL-1ß were detected in serum and cartilages from KBD patients and in T-2-stimulated chondrocytes. Moreover, knockdown of IL-1ß antagonized the adverse effects of T-2 on cytotoxic injury by enhancing cell viability and inhibiting apoptosis. However, exogenous supplementation of IL-1ß further aggravated cell damage in response to T-2. Additionally, cessation of IL-1ß rescued T-2-elicited tilt of matrix homeostasis toward catabolism by elevating the transcription of collagen II and aggrecan, promoting release of sulphated glycosaminoglycans (sGAG) and TIMP1, and suppressing matrix metalloproteinases production including MMP-1, MMP-3 and MMP-13. Conversely, IL-1ß stimulation deteriorated T-2-induced disruption of matrix metabolism balance toward catabolism. Mechanistic analysis found the high activation of Wnt/ß-catenin in KBD patients and chondrocytes upon T-2. Furthermore, this activation was mitigated after IL-1ß inhibition, but further enhanced following IL-1ß precondition. Importantly, blocking this pathway by transfection with ß-catenin alleviated the adverse roles of IL-1ß on cytotoxic injury and metabolism disorders under T-2 conditioning. Together, this study elucidates a new insight into how T-2 deteriorates the pathological progression of KBD by regulating inflammation-related pathways, indicating a promising anti-inflammation strategy for KBD therapy.

Role of inflammation in the process of clinical Kashin-Beck disease: latest findings and interpretations.

Kashin-Beck disease (KBD), a particular type of osteoarthritis (OA), and an endemic disease with articular cartilage damage and chondrocytes apoptosis, can affect many joints, and the most commonly affected joints are the knee, ankle, and hand. KBD has traditionally been classified as a non-inflammatory OA. However, recent studies have shown that inflammation has played an important role in the development of KBD. Nowadays, clinical KBD is not only an endemic disease, but also a combined result of many other non-endemic factors, which contains age, altered biomechanics, joint trauma and secondary OA. The characteristics of the developmental joint failure of advanced KBD, because of the biochemical and mechanical processes, are tightly linked with the interaction of joint damage and its immune response, as well as the subsequent state of chronic inflammation leading to KBD progression. In this review, we focus on the epidemiology, pathology, imaging, cytokines and transduction pathways investigating the association of inflammation with KBD; meanwhile, a wide range of data will be discussed to elicit our current hypotheses considering the role of inflammation and immune activation in KBD development.

Genome-wide pathway-based association study implicates complement system in the development of Kashin-Beck disease in Han Chinese.

Kashin-Beck disease (KBD) is a chronic osteochondropathy. The pathogenesis of KBD remains unknown. To identify relevant biological pathways for KBD, we conducted a genome-wide pathway-based association study (GWPAS) following by replication analysis, totally using 2743 Chinese Han adults. A modified gene set enrichment algorithm was used to detect association between KBD and 963 biological pathways. Cartilage gene expression analysis and serum complement measurement were performed to evaluate the functional relevance of identified pathway with KBD. We found that the Complement and Coagulation Cascades (CACC) pathway was significantly associated with KBD (P value=3.09×10(-5), false-discovery rate=0.042). Within the CACC pathway, the most significant association was observed at rs1656966 (P value=1.97×10(-4)) of KNG1 gene. Further replication study observed that rs1656966 (P value=0.037) was significantly associated with KBD in an independent validation sample of 1026 subjects. Gene expression analysis observed that CFD (ratio=3.39±2.68), A2M (ratio=3.67±5.63), C5 (ratio=2.65±2.52) and CD46 (ratio=2.29±137) genes of the CACC pathway were up-regulated in KBD articular cartilage compared to healthy articular cartilage. The serum level of complement C5 in KBD patients were significantly higher than that in healthy controls (P value=0.038). Our study is the first to suggest that complement system-related CACC pathway contributed to the development of KBD.

The role of mitochondria in T-2 toxin-induced human chondrocytes apoptosis.

T-2 toxin, a mycotoxin produced by Fusarium species, has been shown to cause diverse toxic effects in animals and is also a possible pathogenic factor of Kashin-Beck disease (KBD). The role of mitochondria in KBD is recognized in our recent research. The aim of this study was to evaluate the role of mitochondria in T-2 toxin-induced human chondrocytes apoptosis to understand the pathogenesis of KBD. T-2 toxin decreased chondrocytes viabilities in concentration- and time-dependent manners. Exposure to T-2 toxin can reduce activities of mitochondrial complexes III, IV and V, ΔΨm and the cellular ATP, while intracellular ROS increased following treatment with T-2 toxin. Furthermore, mitochondrial cytochrome c release, caspase-9 and 3 activation and chondrocytes apoptosis were also obviously observed. Interestingly, Selenium (Se) can partly block T-2 toxin -induced mitochondria dysfunction, oxidative damage and chondrocytes apoptosis. These results suggest that the effect of T-2 toxin on human chondrocytes apoptosis may be mediated by a mitochondrial pathway, which is highly consistent with the chondrocytes changes in KBD.

Increased levels of IL-6, IL-1ß, and TNF-α in Kashin-Beck disease and rats induced by T-2 toxin and selenium deficiency.

The objective of this study is to investigate the possible role of inflammatory mediators such as IL-6, IL-1ß, and TNF-α in Kashin-Beck disease (KBD) children and rats fed with T-2 toxin under a selenium-deficient nutrition status in order to determine possible mechanism underlying KBD. Sprague-Dawley rats were administered a selenium-deficient diet for 4 weeks prior to their exposure to T-2 toxin for 4 weeks. The morphology of joint cartilages of KBD children and rats was examined by light microscopy, and the expression of proteoglycans was determined by histochemical staining. The serum levels of IL-6, IL-1ß, and TNF-α were determined by enzyme-linked immunosorbent assay. IL-6, IL-1ß and TNF-α were localized by immunohistochemistry, and their mRNA levels were detected by real-time RT-PCR. The serum levels of IL-6 were significantly elevated in rats fed with selenium-deficient, T-2 toxin, and T-2 toxin plus selenium-deficient diets compared to those in the normal diet, while the serum levels of IL-1ß and TNF-α were significantly increased only in the T-2 toxin plus selenium-deficient diet group. IL-6, IL-1ß and TNF-α protein and mRNA levels in cartilage were significantly higher in rats with diets of T-2 toxin and T-2 toxin plus selenium deficiency than in rats fed normal or selenium-deficient diet. While staining for the cytokines in cartilages of KBD children was significantly higher than that in controls. T-2 toxin under a selenium-deficient nutritional status induces increased levels of IL-6, IL-1ß, and TNF-α in serum and cartilages, which may account for the pathological mechanism underlying the cartilage damage in 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.

Autoimmune and inflammatory responses in Kashin-Beck disease compared with rheumatoid arthritis and osteoarthritis.

To examine plasma levels of arthritis-related autoantibodies and inflammatory factors in Kashin-Beck disease (KBD) patients compared with rheumatoid arthritis (RA) patients, osteoarthritis (OA) patients, and healthy controls, the plasma levels of autoantibodies to types II, IX, and XI collagen and cyclic citrullinated peptide (CCP) and immunoglobulin (Ig)-G and IgM rheumatoid factors (IgG-RF and IgM-RF) from 45 KBD patients, 39 RA patients, 46 OA patients, and 30 healthy controls were determined by enzyme-linked immunosorbent assay. The plasma concentrations of nitric oxide (NO) and tumor necrosis factor-α (TNF-α) were measured using the Griess method and bioassay, respectively. Statistical analysis was performed using one-way analysis of variance followed by the least significant difference t test for differences among groups. Results indicated that the plasma levels of collagen IX antibodies, IgG-RF, and NO significantly increased in KBD patients compared with patients with RA and OA and the control group. The levels of collagen XI antibodies, CCP antibodies, and IgM-RF but not collagen II antibodies and TNF-α were significantly increased in the plasma of the KBD group compared with that of the control group. We conclude that autoimmunity and inflammation may be involved in the pathogenesis of KBD, in particular in the advanced stage.

Serum levels of IL-1ß, IL-6 and TNF-α in rats fed with Kashin-Beck disease-affected diet.

AIM: To investigate the serum level of interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α in rats which have been fed with Kashin-Beck disease (KBD) epidemic district food. METHOD: Two hundred and twenty Wistar rats were divided into five groups. Group A was fed with a normal diet as control; group B was fed with a normal diet and T-2 toxin; group C was fed with a low-nutrition diet and T-2 toxin; group D was fed with a low-nutrition diet; and group E was fed with a KBD-affected diet. The serum bioactivity of IL-1ß, IL-6 and TNF-α were tested by enzyme-linked immeunosorbent assay. RESULTS: After 4 weeks, the epiphyseal plate showed more obvious necrosis of chondrocytes in groups B, C and E. Among the KBD-affected feed, normal feed combined with T-2 toxin and low protein combined with T-2 toxin, KBD-affected feed rats had the highest serum levels, and normal feed combined with T-2 toxin group was the lowest. Although the IL-1ß, IL-6 and TNF-α levels were no different in the KBD-affected feed compared to low protein combined with T-2 toxin, there were significant differences compared to normal feed combined with the T-2 toxin (P < 0.05). CONCLUSIONS: Serum levels of IL-1ß, IL-6 and TNF-α metabolism are altered in the KBD model rats. This effect is relatively similar to the low-nutrition diet combined with the T-2 toxin, which means low-nutrition diet may be involved in the aetiology of KBD.