Department of Periodontology, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-Sen University, Guangzhou, China.
Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, China.
Cell Biol Int. 2020 Dec;44(12):2383-2394. doi: 10.1002/cbin.11445. Epub 2020 Sep 2.
Periodontitis is a chronic inflammatory disease that results in the destruction of periodontal soft tissue and the resorption of alveolar bone. Evidence indicates that in diabetic patients, hyperglycemia suppresses periodontal ligament stem cell (PDLSC) functions and leads to difficulties in periodontal repair. The present study aimed to explore the mechanisms by which high-glucose concentrations aggravate cell viability reduction in human CD146-positive PDLCs (CD146 PDLCs) under tumor necrosis factor-alpha (TNF-alpha) induction. CD146 PDLCs were isolated from periodontal ligament tissues and treated in the absence or presence of 10 ng/ml of TNF-alpha and 30 mM glucose. Cell viability was detected using Cell Counting Kit-8 assays and Luminescent Cell Viability Assays. Western blotting and real-time polymerase chain reaction were performed to determine tumor necrosis factor-alpha receptor-1 (TNFR-1) protein and messenger RNA expression. Bisulfite and MassArray methylation analyses were used to analyze the methylation status of the TNFR-1 gene. Our results indicated that cell viability was reduced after treatment with a combination of both high-glucose concentration and TNF-alpha. Treatment with 30 mM glucose suppressed DNA methyltransferase (DNMT) activities and DNMT1 protein expression, and this was accompanied by the upregulation of TNFR-1. Additionally, we found that the CpG island located within the TNFR-1 gene was hypomethylated under 30 mM glucose conditions. S-adenosylmethionine, an established methyl donor, reversed TNFR-1 upregulation and restored cell viability against high-glucose concentration and TNF-alpha. In conclusion, the present findings suggest that high-glucose-induced CpG island hypomethylation within the TNFR-1 gene plays an essential role in TNFR-1 upregulation, and this further enhances the cell viability reduction of CD146 PDLCs caused by TNF-alpha.
牙周炎是一种慢性炎症性疾病，导致牙周软组织破坏和牙槽骨吸收。有证据表明，在糖尿病患者中，高血糖会抑制牙周韧带干细胞（PDLSC）的功能，导致牙周修复困难。本研究旨在探讨高浓度葡萄糖在肿瘤坏死因子-α（TNF-α）诱导下加重人 CD146 阳性 PDLCs（CD146 PDLCs）细胞活力降低的机制。CD146 PDLCs 从牙周韧带组织中分离出来，在无或有 10ng/ml TNF-α和 30mM 葡萄糖的情况下进行处理。使用细胞计数试剂盒-8 检测试剂盒和发光细胞活力检测试剂盒检测细胞活力。采用 Western blot 和实时聚合酶链反应检测肿瘤坏死因子-α受体-1（TNFR-1）蛋白和信使 RNA 表达。亚硫酸氢盐和 MassArray 甲基化分析用于分析 TNFR-1 基因的甲基化状态。结果表明，高糖浓度与 TNF-α联合处理后细胞活力降低。30mM 葡萄糖处理抑制 DNA 甲基转移酶（DNMT）活性和 DNMT1 蛋白表达，同时上调 TNFR-1。此外，我们发现 30mM 葡萄糖条件下 TNFR-1 基因内的 CpG 岛呈低甲基化状态。S-腺苷甲硫氨酸是一种公认的甲基供体，可逆转 TNFR-1 的上调，并恢复细胞活力，对抗高糖浓度和 TNF-α。总之，本研究结果表明，TNFR-1 基因内高糖诱导的 CpG 岛低甲基化在 TNFR-1 上调中起关键作用，进一步增强了 TNF-α引起的 CD146 PDLCs 细胞活力降低。
