Abstract:

Objective To investigate the induction effect of different concentrations of glucose on the polarization of Raw264.7 macrophages in vitro. Methods The Raw264.7 cells cultured in DMEM medium were divided into control group （5.5 mmol·L^-1 glucose）， different high-glucose groups （15.0， 25.0， 35.0， and 45.0 mmol·L^-1 glucose）， and positive control group ［lipopolysaccharide（LPS）］. The cells were cultured for 3， 6， and 9 h， respectively. Cell morphology was observed in various groups. Cell counting kit-8 （CCK-8） method was used to assess the survival rates of cells in various groups. Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， and interleukin-10 （IL-10） mRNA in cells in various groups. Enzyme-linked immunosorbent assay （ELISA） was used to measure the levels of IL-6， TNF-α， and IL-10 in the cell supernatants in various groups. Flow cytometry was used to determine the percentages of M1 and M2 macrophage markers， CD86+ and CD163+ cells， in various groups. Results In control group， the Raw264.7 cells adhered to the culture dish， with a predominantly round morphology. The cells in 35.0 mmol·L^-1 high-glucose group and positive control group showed elongated shapes and pseudopodium formation， indicating inflammatory changes. Compared with control group， the survival rates in different high-glucose groups at 6， 12， 24， and 48 h after treatment were increased （P<0.05）. Compared with control group， after 3 h of treatment， the expression levels of IL-6 and IL-10 mRNA in the cells in 35.0 mmol·L^-1 high-glucose group were increased （P<0.05 or P<0.01）， while no significant changes were observed in IL-6， TNF-α， and IL-10 levels in the cell supernatants （P>0.05）； after 6 h of treatment， the expression level of TNF-α mRNA in the cells in 35.0 mmol·L^-1 high-glucose group was increased （P<0.001）， and the levels of IL-6， TNF-α， and IL-10 in the cell supernatants were significantly increased （P<0.05 or P<0.001）； after 3 h of treatment， the percentages of macrophage markers CD86+ cells and CD163+ cells in 35.0 mmol·L^-1 high-glucose group were significantly increased （P<0.01 or P<0.001）. Conclusion A certain high concentration of glucose may induce the polarization of Raw264.7 macrophages towards the M1 subtype in vitro.

Key words: Raw264.7 cells, Glucose, Inflammation, Macrophage polarization, Inflammatory cytokine