title: [Regulatory analysis of hypoxia on innate immunity of human corneal epithelium].
abstract: Objective:To investigate the role of hypoxia on the regulation of innate immunity of human corneal epithelium.Methods:Telomerase-immortalized human epithelial cells (THCEs) were incubated under normoxia (21% O2) or hypoxic (1% O2) conditions respectively. After 6, 12, 24, 48 h culture, the mRNA and protein levels of toll like receptor 4 (TLR4) were measured by real-time polymerase chain reaction (RT-PCR) and Western blot analysis. After 24 h culture, THCEs of each group were challenged respectively with TLR4 ligand lipopolysaccharide (LPS) (1 μg/ml) for 6 h. RT-PCR was used to assess the mRNA level of myeloid differentiation factor 88 (MyD88), interleukin(IL)6, IL-8 and tumor necrosis factor α (TNF-α). Western blot was used to examine the protein level of inhibitor of nuclear factor kappa-B α (IκBα) and phosphorylated IκBα (p-IκBα). Enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of the inflammatory cytokines IL-6, IL-8 and TNF-α.Results:The results of RT-PCR and Western blot analysis showed that the expression of TLR4 downregulated 90% and 55% respectively after hypoxic exposure for 48 h. Hypoxia also inhibited LPS-induced secretion of IL-6, IL-8, TNF-α, expression of MyD88 and activation of NF-κB. The mRNA level of MyD88 was diminished 63%, and the protein expression of p-IκBα was also lowered. Meanwhile, the secretions of IL-6, IL-8 and TNF-α under hypoxia were reduced (31%, 55% and 50% respectively).Conclusion:Hypoxia attenuated immune and inflammatory response of the cornea epithelium by suppressing TLR4 signaling, and could enhance cell susceptibility to microorganism infection.
authors.affiliation: Department of Ophthalmology, Qilu Hospital of Shandong University, Jinan 250012, China.
authors.forename: K P
authors.initials: KP
authors.lastname: Pang
authors.affiliation: Department of Ophthalmology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, China.
authors.forename: H
authors.initials: H
authors.lastname: Pan
authors.forename: X Y
authors.initials: XY
authors.lastname: Wu
mesh_terms: D015687:Cell Hypoxia; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells; D019573:Epithelium, Corneal; D006801:Humans; D007113:Immunity, Innate; D015850:Interleukin-6; D016209:Interleukin-8; D008070:Lipopolysaccharides; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D010766:Phosphorylation; D012333:RNA, Messenger; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction; D051197:Toll-Like Receptor 4; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha
chemical_list: C508606:CXCL8 protein, human; C508600:IL6 protein, human; D015850:Interleukin-6; D016209:Interleukin-8; D008070:Lipopolysaccharides; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; C498143:RELA protein, human; D012333:RNA, Messenger; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha