Lingling Shang1,2, Ting Wang1,2, Dongdong Tong3, Wenyan Kang1,2, Qianyu Liang1,2, Shaohua Ge1,2. 1. Shandong Provincial Key Laboratory of Oral Tissue Regeneration, School of Stomatology, Shandong University, Shandong, Jinan, China. 2. Department of Periodontology, School of Stomatology, Shandong University, Shandong, Jinan, China. 3. Department of Oral maxillofacial Surgery, School of Stomatology, Shandong University, Shandong, Jinan, China.
Abstract
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-κB, mitogen-activated protein kinase (MAPK) and PI3K/AKT pathways were detected by western blot and the nuclear translocation of NF-κB 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-κB, 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-κB, MAPK and AKT signalling pathways and PHD2 among three isoforms was principally responsible for the effects.
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-κB, mitogen-activated protein kinase (MAPK) and PI3K/AKT pathways were detected by western blot and the nuclear translocation of NF-κB 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-κB, 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-κB, MAPK and AKT signalling pathways and PHD2 among three isoforms was principally responsible for the effects.
Authors: Emily Hams; Sean P Saunders; Eoin P Cummins; Aisling O'Connor; Murtaza T Tambuwala; William M Gallagher; Annette Byrne; Antonio Campos-Torres; Paul M Moynagh; Christian Jobin; Cormac T Taylor; Padraic G Fallon Journal: Shock Date: 2011-09 Impact factor: 3.454
Authors: Jonathan Michael Harnoss; Moritz Johannes Strowitzki; Praveen Radhakrishnan; Lisa Katharina Platzer; Julian Camill Harnoss; Thomas Hank; Jun Cai; Alexis Ulrich; Martin Schneider Journal: Hypoxia (Auckl) Date: 2015-01-30