School of Public Health, Zhengzhou University, Zhengzhou 450001, China.
Sanmenxia Center for Disease Control and Prevention, Sanmenxia 472000, China.
Toxins (Basel). 2023 Aug 4;15(8):496. doi: 10.3390/toxins15080496.
T-2 toxin and selenium deficiency are considered important etiologies of Kashin-Beck disease (KBD), although the exact mechanism is still unclear. To identify differentially expressed microRNAs (DE-miRNAs) in the articular cartilage of rats exposed to T-2 toxin and selenomethionine (SeMet) supplementation, thirty-six 4-week-old Sprague Dawley rats were divided into a control group (gavaged with 4% anhydrous ethanol), a T-2 group (gavaged with 100 ng/g·bw/day T-2 toxin), and a T-2 + SeMet group (gavaged with 100 ng/g·bw/day T-2 toxin and 0.5 mg/kg·bw/day SeMet), respectively. Toluidine blue staining was performed to detect the pathological changes of articular cartilage. Three rats per group were randomly selected for high-throughput sequencing of articular cartilage. Target genes of DE-miRNAs were predicted using miRanda and RNAhybrid databases, and the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway were enriched. The network map of miRNA-target genes was constructed using Cytoscape software. The expression profiles of miRNAs associated with KBD were obtained from the Gene Expression Omnibus database. Additionally, the DE-miRNAs were selected for real-time quantitative PCR (RT-qPCR) verification. Toluidine blue staining demonstrated that T-2 toxin damaged articular cartilage and SeMet effectively alleviated articular cartilage lesions. A total of 50 DE-miRNAs (28 upregulated and 22 downregulated) in the T-2 group vs. the control group, 18 DE-miRNAs (6 upregulated and 12 downregulated) in the T-2 + SeMet group vs. the control group, and 25 DE-miRNAs (5 upregulated and 20 downregulated) in the T-2 + SeMet group vs. the T-2 group were identified. Enrichment analysis showed the target genes of DE-miRNAs were associated with apoptosis, and in the MAPK and TGF-β signaling pathways in the T-2 group vs. the control group. However, the pathway of apoptosis was not significant in the T-2 + SeMet group vs. the control group. These results indicated that T-2 toxin induced apoptosis, whereas SeMet supplementation antagonized apoptosis. Apoptosis and autophagy occurred simultaneously in the T-2 + SeMet group vs. T-2 group, and autophagy may inhibit apoptosis to protect cartilage. Compared with the GSE186593 dataset, the evidence of miR-133a-3p involved in apoptosis was more abundant. The results of RT-qPCR validation were consistent with RNA sequencing results. Our findings suggested that apoptosis was involved in articular cartilage lesions induced by T-2 toxin, whereas SeMet supplementation antagonized apoptosis, and that miR-133a-3p most probably played a central role in the apoptosis process.
T-2 毒素和硒缺乏被认为是大骨节病(KBD)的重要病因,但确切机制仍不清楚。为了鉴定 T-2 毒素和硒代蛋氨酸(SeMet)补充暴露的大鼠关节软骨中的差异表达 microRNAs(DE-miRNAs),将 36 只 4 周龄的 Sprague Dawley 大鼠分为对照组(灌胃 4%无水乙醇)、T-2 组(灌胃 100ng/g·bw/day T-2 毒素)和 T-2+SeMet 组(灌胃 100ng/g·bw/day T-2 毒素和 0.5mg/kg·bw/day SeMet),每组 3 只大鼠。采用甲苯胺蓝染色检测关节软骨的病理变化。每组随机选择 3 只大鼠进行关节软骨高通量测序。使用 miRanda 和 RNAhybrid 数据库预测 DE-miRNA 的靶基因,并对基因本体论和京都基因与基因组百科全书通路进行富集。使用 Cytoscape 软件构建 miRNA-靶基因网络图。从基因表达综合数据库(GEO)中获取与 KBD 相关的 miRNA 表达谱。此外,还进行了实时定量 PCR(RT-qPCR)验证。甲苯胺蓝染色显示 T-2 毒素损伤关节软骨,SeMet 可有效缓解关节软骨损伤。与对照组相比,T-2 组有 50 个 DE-miRNA(28 个上调和 22 个下调),T-2+SeMet 组有 18 个 DE-miRNA(6 个上调和 12 个下调),T-2+SeMet 组有 25 个 DE-miRNA(5 个上调和 20 个下调)。富集分析表明,DE-miRNA 的靶基因与凋亡有关,与 T-2 组与对照组比较,MAPK 和 TGF-β信号通路中存在凋亡。然而,T-2+SeMet 组与对照组相比,凋亡途径并不显著。这些结果表明 T-2 毒素诱导凋亡,而硒补充拮抗凋亡。T-2+SeMet 组与 T-2 组同时发生凋亡和自噬,自噬可能抑制凋亡以保护软骨。与 GSE186593 数据集相比,miR-133a-3p 参与凋亡的证据更加丰富。RT-qPCR 验证结果与 RNA 测序结果一致。我们的研究结果表明,凋亡参与了 T-2 毒素诱导的关节软骨损伤,而硒补充拮抗了凋亡,miR-133a-3p 可能在凋亡过程中发挥核心作用。