O-linked N-acetylglucosamine modification induced by lipopolysaccharide is involved in inflammatory signaling pathway in endothelial cells

Abstract

To explore whether the lipopolysaccharide (LPS)-induced modification of O-linked N-acetylglucosamine (O-GlcNAc) is involved in the inflammatory signaling pathway of endothelial cells. Human umbilical vein endothelial cells (HUVEC) were cultured in vitro, and cells in logarithmic growth phase were used for experiments. Cells were divided into blank control group, LPS group (2 000 mg/L LPS), O-GlcNAc transferase (OGT) overexpression (OGT-OE)+LPS group (plasmid transfection OGT+2 000 mg/L LPS), protein kinase C (PKC) inhibitor+LPS group (10 μmol/L Go 6983+2 000 mg/L LPS), RhoA inhibitor+LPS group (40 μmol/L Rhoin hydrochloride+2 000 mg/L LPS), phosphatidylinositol-3-kinase (PI3K) inhibitor+LPS group (1 μmol/L SL-2052+2 000 mg/L LPS), serine/threonine kinase (Akt) inhibitor+LPS group (10 μmol/L PP2+2 000 mg/L LPS) and small interfering RNA (siRNA) treated Akt (si-AKT)+LPS group (si-Akt+2 000 mg/L LPS). After 24 hours of LPS treatment, real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the transcription levels of inflammatory cytokines [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)]. The protein expression or phosphorylation of OGT, O-GlcNAc, Akt, extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK), nuclear factor-κB p65 (NF-κB p65), and signal transducer and activator of transcription 3 (STAT3) were determined by Western blotting. Compared with the blank control group, the expression of OGT and the modification of O-GlcNAc in the LPS group were decreased, while the expressions of phosphorylated ERK, p38MAPK, and STAT3 were increased, and the transcript levels of inflammatory cytokines were also significantly increased [IL-6 mRNA (2-ΔΔCt): 4.71±0.60 vs. 1.03±0.29, TNF-α mRNA (2-ΔΔCt): 1.89±0.11 vs. 1.04±0.35, ICAM-1 mRNA (2-ΔΔCt): 2.06±0.18 vs. 1.02±0.21, VCAM-1 mRNA (2-ΔΔCt): 2.94±0.57 vs. 1.01±0.17, all P < 0.05], indicating that LPS could decrease O-GlcNAc modification, activate inflammatory signaling pathways and increase inflammatory cytokines expression. Compared with the LPS group, the expressions of phosphorylated ERK, p38MAPK, NF-κB p65, and STAT3 in the endothelial cells of the OGT-OE+LPS group were decreased, and the expression of inflammatory factors were significantly decreased [IL-6 mRNA (2-ΔΔCt): 0.12±0.01 vs. 0.90±0.17, TNF-α mRNA (2-ΔΔCt): 0.31±0.01 vs. 0.91±0.14, ICAM-1 mRNA (2-ΔΔCt): 0.64±0.02 vs. 1.13±0.16, VCAM-1 mRNA (2-ΔΔCt): 0.11±0.01 vs. 0.93±0.11, all P < 0.05], indicating that the increase of OGT level could inhibit the partial activation of the endothelial inflammatory signal pathway under the LPS stimulation. Compared with the blank control group, the phosphorylation level of Akt in the LPS group was increased. Compared with the LPS group, both OGT expression and O-GlcNAc modification were down-regulated after pretreatment of PKC inhibitor, RhoA inhibitor, PI3K inhibitor, or Akt inhibitor. Compared with the LPS group, the transcript levels of IL-6, TNF-α and ICAM-1 in the PP2+LPS group were significantly decreased [IL-6 mRNA (2-ΔΔCt): 1.46±0.16 vs. 3.55±0.87, TNF-α mRNA (2-ΔΔCt): 0.98±0.14 vs. 1.76±0.10, ICAM-1 mRNA (2-ΔΔCt): 1.39±0.24 vs. 2.04±0.13, all P < 0.05], but there was no significant change in VCAM-1. Compared with the LPS group, the expression of OGT and O-GlcNAc modification in the si-Akt+LPS group were decreased, while the transcript levels of inflammatory cytokines were also significantly decreased [IL-6 mRNA (2-ΔΔCt): 0.75±0.03 vs. 0.99±0.09, TNF-α mRNA (2-ΔΔCt): 0.69±0.01 vs. 1.10±0.08, ICAM-1 mRNA (2-ΔΔCt): 0.76±0.01 vs. 0.99±0.02, VCAM-1 mRNA (2-ΔΔCt): 0.93±0.08 vs. 1.20±0.21, all P < 0.05], indicating that Akt participated in the action process of LPS on OGT and affected the inflammatory factor expression. The decreased level of O-GlcNAc modification in endothelial cells stimulated with LPS promotes partial activation of inflammatory signaling pathways, mainly involving ERK, p38MAPK, and STAT3, and affects the expression of inflammatory factors. AKT may be involved in the effect of LPS on the inhibition of O-GlcNAc modification.