Prolyl hydroxylases positively regulated LPS-induced inflammation in human gingival fibroblasts via TLR4/MyD88-mediated AKT/NF-κB and MAPK pathways
Prolyl hydroxylases positively regulated LPS-induced inflammation in human gingival fibroblasts via TLR4/MyD88-mediated AKT/NF-κB and MAPK pathways
复制标题
脯氨酰羟化酶通过 TLR4/MyD88 介导的 AKT/NF-κB 和 MAPK 途径正向调节 LPS 诱导的人牙龈成纤维细胞炎症
DOI：
10.1111/cpr.12516
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发表时间：
2018-12-01
影响因子：
8.5
通讯作者：
Ge, Shaohua
中科院分区：
文献类型：
--
作者：
Shang, Lingling;Wang, Ting;Ge, Shaohua
目的脯氨酸羟基酶(PhDS)在氧敏感系统中起重要作用，但其在炎症反应中的作用尚未完全阐明。本研究旨在探讨PHD在脂多糖(LPS)诱导的人牙龈成纤维细胞(HGFs)炎症中的作用并阐明其可能的机制。材料与方法采用PAN羟基酶抑制剂二甲氧基烯丙基甘氨酸(DMOG)和RNA干扰技术，探讨PHD在炎症反应中的作用。细胞计数试剂盒8检测DMOG的细胞毒作用，流式细胞仪检测DMOG的细胞毒性。用双抗体夹心法测定IL-6、IL-8的分泌水平。用实时定量聚合酶链式反应检测炎性细胞因子、Toll样受体(TLR)4和MyD88的mRNA水平。免疫印迹法检测核因子-kappaB、丝裂原活化蛋白激酶(MAPK)和PI3K/AKT信号通路的激活，免疫荧光法检测核转录因子-kappaBp65的核转位。通过siRNA转染法下调PhD1和PHD2的表达。结果二甲氧烯丙基甘氨酸在基因水平上抑制内毒素诱导的炎性细胞因子TLR4和MyD88的表达，下调IL-6和IL-8的分泌。此外，DMOG可取消内毒素诱导的NF-kappa B、MAPK和AKT通路的激活。重要的是，内毒素诱导的炎性细胞因子的表达仅被PHD2基因敲除所抑制。结论TLR4/MyD88通过TLR4/MyD88介导的NF-kappa B、MAPK和AKT信号转导通路在内毒素诱导的HGFs炎症反应中发挥正向调节作用，其中PHD2起主要作用。
Objectives Prolyl hydroxylases (PHDs) play essential roles in oxygen-sensing system, whereas the effects of PHDs on inflammation have not been totally uncovered. Our study aimed to investigate the role of PHDs in lipopolysaccharide (LPS)-induced inflammation of human gingival fibroblasts (HGFs) and clarify the potential mechanisms. Materials and methods A pan hydroxylase inhibitor, dimethyloxallyl glycine (DMOG), and RNA interference were used to explore the role of PHDs in inflammation. Cytotoxic effect of DMOG was determined by cell-counting kit-8 and flow cytometry respectively. The secretion levels of IL-6 and IL-8 were assessed by ELISA. The mRNA levels of inflammatory cytokines, Toll-like receptor (TLR) 4 and MyD88 were evaluated by quantitative real-time PCR. The activation of NF-kappa B, mitogen-activated protein kinase (MAPK) and PI3K/AKT pathways were detected by western blot and the nuclear translocation of NF-kappa B p65 was examined by immunofluorescence. Downregulation of PHD1 and PHD2 was performed with siRNA transfection. Results Dimethyloxallyl glycine inhibited LPS-induced inflammatory cytokine, TLR4 and MyD88 expression in gene level and the elevated secretion of IL-6 and IL-8 was also downregulated. Additionally, LPS-induced activation of NF-kappa B, MAPK and AKT pathways was abolished by DMOG treatment. Importantly, LPS-induced inflammatory cytokine expression was merely suppressed by PHD2 knockdown. Conclusions Prolyl hydroxylases acted as a positive regulator in LPS-induced inflammation of HGFs via TLR4/MyD88-mediated NF-kappa B, MAPK and AKT signalling pathways and PHD2 among three isoforms was principally responsible for the effects.