Butyrate Stimulates Histone H3 Acetylation, 8-Isoprostane Production, RANKL Expression, and Regulated Osteoprotegerin Expression/Secretion in MG-63 Osteoblastic Cells
Butyrate Stimulates Histone H3 Acetylation, 8-Isoprostane Production, RANKL Expression, and Regulated Osteoprotegerin Expression/Secretion in MG-63 Osteoblastic Cells
复制标题
DOI：
10.3390/ijms19124071
复制
发表时间：
2018-12-01
影响因子：
5.6
通讯作者：
Jeng, Jiiang-Huei
中科院分区：
文献类型：
--
作者：
Chang, Mei-Chi;Chen, Yunn-Jy;Jeng, Jiiang-Huei
丁酸是一种组蛋白去乙酰化酶（HDAC）抑制剂，由许多牙周和根管微生物（如卟啉单胞菌、梭杆菌等）产生。丁酸可能影响牙周/根尖周细胞如成骨细胞、牙周韧带细胞等的生物活性，从而影响牙周/根尖周组织的破坏和愈合。本研究旨在研究丁酸盐对MG-63成骨细胞基质及矿化标志物表达的毒性作用。采用3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四唑（MTT）法测定细胞活力。碘化丙啶/膜联蛋白V流式细胞术检测细胞凋亡和坏死。采用Western blotting和逆转录聚合酶链反应（RT-PCR）分析骨保护素（OPG）和核因子κ b配体受体激活因子（RANKL）的蛋白和mRNA表达。采用酶联免疫吸附法测定培养液中OPG、可溶性RANKL （sRANKL）、8-异前列腺素、前胶原I、基质金属蛋白酶-2 （MMP-2）、骨连接素（SPARC）、骨钙素和骨桥蛋白（OPN）的分泌。碱性磷酸酶（ALP）染色检测其活性。免疫荧光染色（IF）和Western blot检测组蛋白H3乙酰化水平。我们发现丁酸盐激活MG-63细胞的组蛋白H3乙酰化。MG-63细胞暴露于丁酸盐中部分降低了细胞活力，但没有明显增加凋亡和坏死。暴露于丁酸24小时刺激RANKL蛋白表达，而抑制OPG蛋白表达。丁酸盐对MG-63细胞的OPG分泌也有抑制作用，但对sRANKL的影响低于检测限。然而，暴露于丁酸盐（1 ~ 8mm）或其他HDAC抑制剂（如苯丁酸盐、丙戊酸和曲古霉素）3天可刺激OPG分泌。丁酸盐刺激MG-63细胞中8-异前列腺素、MMP-2和OPN的分泌，但对I型前胶原和骨钙素没有刺激作用。暴露于丁酸盐（2-4 mM） 3天显著刺激骨连接素分泌和ALP活性。综上所述，牙周和根管微生物产生的高浓度丁酸可能通过改变OPG/RANKL表达/分泌、8-异前列腺素、MMP-2和OPN分泌，诱导骨破坏和骨修复，并影响细胞活力。然而，较低浓度的丁酸盐（1-4毫米）可能会刺激ALP、骨连接素和OPG。这些影响可能与组蛋白乙酰化增加有关。这些事件在牙周和根尖周破坏的发病机制和修复中是重要的。
Butyric acid as a histone deacetylase (HDAC) inhibitor is produced by a number of periodontal and root canal microorganisms (such as Porphyromonas, Fusobacterium, etc.). Butyric acid may affect the biological activities of periodontal/periapical cells such as osteoblasts, periodontal ligament cells, etc., and thus affect periodontal/periapical tissue destruction and healing. The purposes of this study were to study the toxic effects of butyrate on the matrix and mineralization marker expression in MG-63 osteoblasts. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cellular apoptosis and necrosis were analyzed by propidium iodide/annexin V flow cytometry. The protein and mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). OPG, soluble RANKL (sRANKL), 8-isoprostane, pro-collagen I, matrix metalloproteinase-2 (MMP-2), osteonectin (SPARC), osteocalcin and osteopontin (OPN) secretion into culture medium were measured by enzyme-linked immunosorbant assay. Alkaline phosphatase (ALP) activity was checked by ALP staining. Histone H3 acetylation levels were evaluated by immunofluorescent staining (IF) and Western blot. We found that butyrate activated the histone H3 acetylation of MG-63 cells. Exposure of MG-63 cells to butyrate partly decreased cell viability with no marked increase in apoptosis and necrosis. Twenty-four hours of exposure to butyrate stimulated RANKL protein expression, whereas it inhibited OPG protein expression. Butyrate also inhibited the secretion of OPG in MG-63 cells, whereas the sRANKL level was below the detection limit. However, 3 days of exposure to butyrate (1 to 8 mM) or other HDAC inhibitors such as phenylbutyrate, valproic acid and trichostatin stimulated OPG secretion. Butyrate stimulated 8-isoprostane, MMP-2 and OPN secretion, but not procollagen I, or osteocalcin in MG-63 cells. Exposure to butyrate (2-4 mM) for 3 days markedly stimulated osteonectin secretion and ALP activity. In conclusion, higher concentrations of butyric acid generated by periodontal and root canal microorganisms may potentially induce bone destruction and impair bone repair by the alteration of OPG/RANKL expression/secretion, 8-isoprostane, MMP-2 and OPN secretion, and affect cell viability. However, lower concentrations of butyrate (1-4 mM) may stimulate ALP, osteonectin and OPG. These effects are possibly related to increased histone acetylation. These events are important in the pathogenesis and repair of periodontal and periapical destruction.