2022 年 31 巻 4 号 p. 245-252
We investigated the anti-inflammatory effect of hydroxyethyl starch (HES) 6% 200/0.5 on lipopolysaccharide (LPS)-induced RAW264.7 cells based on the high mobility group protein B1/nuclear transcription factor-κB (HMGB1/NF-κB) signaling pathway. The mouse monocyte macrophage RAW264.7 cells were divided into control group (no additional treatment), LPS group (0.2 pg/ml LPS), LPS+HES group (0.2 pg/ml LPS+6% HES) and LPS+TAK-242 (HMGB1/NF-κB signal pathway blocker) group (0.2 pg/ml LPS The LPS+HES+TAK-242 group (0.2 pg/ml LPS+6% HES+1 μ mol/l TAK-242) was cultured for 48 hours. Griess method and ELISA were used to detect the levels of nitric oxide (NO), tumor necrosis factor α (TNF-α), interleukin (IL)-6 and IL-1β in cell supernatant. The level of intracellular reactive oxygen species (ROS) was detected by flow cytometry. The mitochondrial membrane potential was detected by immunofluorescence. The mRNA expressions of inducible nitric oxide synthase (iNOS), HMGB1, Toll-like receptor 4 (TLR4), nuclear factor-κ b inhibitory protein α (IκBα) and NF-κB were detected by qRT-PCR. The expressions of HMGB1, TLR4, IκBα, phosphorylated IκBα (p-IκBα), NF-κB and phosphorylated NF-κB (p-NF-κB) were detected by western blotting. Compared with the control group, the expression levels of NO, TNF-α, IL-6, IL-1β, ROS, iNOS, HMGB1, TLR4, NF-κB mRNA, HMGB1, TLR4 and p-NF-κB proteins in the LPS group, LPS+HES group, LPS+TAK-242 group and LPS+HES+TAK-242 group increased (P<0.05), the expression levels of mitochondrial membrane potential, IκBα mRNA and p-IκBα protein decreased (P<0.05). Compared with LPS group, the expression levels of NO, TNF-α, IL-6, IL-1β, ROS, iNOS, HMGB1, TLR4, NF-κB mRNA, HMGB1, TLR4 and p-NF-κB proteins in the LPS+HES group, LPS+TAK-242 group and LPS+HES+TAK-242 group increased (P<0.05), the expression levels of mitochondrial membrane potential, IκBα mRNA and p-IκBα protein decreased (P<0.05). The expression levels of mitochondrial membrane potential, IκBα mRNA and p-IκBα protein increased (P<0.05). Six % HES 200/0.5 could down-regulate the expression of molecules related to HMGB1/NF-кB signaling pathway, and reduce the inflammatory response of RAW264.7 cells induced by LPS.
