PGC-1α attenuates TNF-α-induced inflammatory responses in OCCM-30 cells.
PGC-1α attenuates TNF-α-induced inflammatory responses in OCCM-30 cells.
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DOI：
10.1111/jre.13042
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发表时间：
2022-07
影响因子：
3.5
通讯作者：
Yihui Fu;Mingyuan Du;Z. Cao;Hong He
中科院分区：
文献类型：
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作者：
Yihui Fu;Mingyuan Du;Z. Cao;Hong He
背景与目的：过氧化物酶体增殖物激活受体-γ共激活因子-1α是线粒体生物发生和氧化代谢的主要调节因子，与多种炎症性疾病有关。然而，PGC-1α在牙周炎成牙本质细胞中的作用和机制尚不清楚。本研究旨在探讨pGC-1α在肿瘤坏死因子-α刺激下对永生化成牙本质细胞系OCCM-30的作用。材料与方法体外培养OCCM-30细胞，加入肿瘤坏死因子-α，用实时定量聚合酶链式反应和免疫印迹法检测α的表达。使用针对不同信号通路的化学抑制剂，包括NF-κB、p38MAPK、Akt和P53，以确定涉及的调控机制。用ZLN005上调PGC-1α表达，用定量逆转录聚合酶链式反应和酶联免疫吸附试验检测炎性细胞因子在肿瘤坏死因子-α刺激下的表达变化。采用pGC-1siRNA进一步验证pGC-1αα在炎症反应中的作用。用双报告基因检测p65的转录活性，用免疫印迹法检测p65的磷酸化水平。采用免疫荧光分析、胞核和胞浆提取法检测p65的核转位。免疫共沉淀法检测p65与pGC-1α之间是否存在直接结合。结果肿瘤坏死因子α可抑制OCCM-30细胞PGC-1α的表达。阻断p38MAPK通路可恢复pGC-1α的表达。ZLN005能上调OCCM-30细胞pGC-1α的表达。ZLN005上调pGC-1α表达可抑制α诱导的促炎细胞因子的表达，这种作用可被pGC-1αsiRNA所抑制。敲除pGC-1α也部分恢复了ZLN005降低的p65转录活性。但pGC-1α对p65的磷酸化水平和核转位无明显影响。在肿瘤坏死因子-α刺激的OCCM-30细胞中，p65与pGC-1α结合，经ZLN005处理后，p65与pGC-1 DNA结合增强。结论PGC-1α可减轻肿瘤坏死因子-α诱导的OCCM-30细胞炎症反应。
BACKGROUND AND OBJECTIVES Peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, a master regulator of mitochondrial biogenesis and oxidative metabolism, has been associated with many inflammatory diseases. However, little is known about the function and mechanism of PGC-1α in cementoblasts under periodontitis. Our study aimed to investigate the effects of PGC-1α in immortalized cementoblast cell line OCCM-30 under TNF-α stimulation. MATERIALS AND METHODS OCCM-30 cells were cultured and exposed to TNF-α, and PGC-1α expression was assessed by Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Chemical inhibitors targeting various signaling pathways including NF-κB, p38 MAPK, Akt, and p53 were used to identify the regulatory mechanism involved. ZLN005 was used to upregulate PGC-1α and the subsequent alteration of inflammatory cytokines expression under TNF-α stimulation were examined by qRT-PCR and Elisa. PGC-1α siRNA was employed to further verify the role of PGC-1α in inflammatory response. Dual-reporter gene assays were performed to examine the transcriptional activity of p65, and the phosphorylation level of p65 was evaluated by western blotting. Immunofluorescence assays and nuclear and cytoplasmic extractions were performed to check the nuclear translocation of p65. Coimmunoprecipitation studies were also performed to check whether there is direct binding between p65 and PGC-1α. RESULTS TNF-α suppressed PGC-1α expression in OCCM-30 cells. Blocking p38 MAPK pathways restored the expression of PGC-1α. ZLN005 can upregulate PGC-1α in OCCM-30 cells. The upregulation of PGC-1α by ZLN005 inhibited TNF-α-induced proinflammatory cytokine expression, which was impaired by the transfection of PGC-1α siRNA. Knocking down PGC-1α also partially restored the ZLN005-decreased transcriptional activity of p65. However, the phosphorylation level and nuclear translocation of p65 were not significantly affected by PGC-1α. It was found that p65 was bound to PGC-1α in OCCM-30 cells stimulated by TNF-α, and the binding was increased upon ZLN005 treatment. CONCLUSIONS PGC-1α can attenuate TNF-α-induced inflammatory responses in OCCM-30 cells.