To investigate whether lactic acid could inhibit the LPS-activation of NF-kB p65 in rat intestinal mucosa microvascular endothelial cells (RIMMVECs), RIMMVECs, cultured in vitro, were pretreated with different concentrations of lactic acid and then exposed to lipopolysaccharide (LPS). Cells and cell culture media were then collected at different time intervals. Production of tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) was examined at the protein level by enzyme-linked immunosorbent assay. The influence of lactic acid on the LPS-activation of NF-kB was examined at mRNA and protein levels by real-time quantitative PCR and Western blot analysis, respectively. TNF-a and IL-6 protein levels were significantly decreased after pretreatment with lactic acid compared with cells exposed to LPS only. After pretreatment with 7.5, 5.0, and 2.5 μL mL-1 lactic acid, NF-kB mRNA levels were increased by 1.51-, 2.62- and 3.00-fold, respectively, compared with levels in control cells without LPS treatment. Western blot analysis indicated that the level of NF-kB p65 in the lactic acid-pretreated group was significantly lower than that in the group treated with LPS only (positive control) and was unchanged compared with the group without LPS treatment (blank control). These results suggest that lactic acid may inhibit LPS-activation of NF-kB, leading to the down-regulation of TNF-a and IL-6.
LIU Jing XUE Jiu-zhou ZHU Zhi-ning HU Ge REN Xiao-ming
This study explored the effects over time of lactic acid (LA) on I?Bα phosphorylation and nuclear factor-kappa B (NF-?B) p65 protein expression, and on tumor necrosis factor a (TNF-α) and interleukin-6 (IL-6) mRNA levels in rat intestinal mucosa microvascular endothelial cells (RIMMVECs) stimulated by lipopolysaccharide (LPS). I?Ba, phosphorylated I?Ba (p-I?Ba) and p65 protein levels were monitored by Western blot analysis, and TNF-a and IL-6 mRNA levels were analyzed using real-time PCR. LA treatment reduced TNF-a and IL-6 mRNA levels in LPS-stimulated RIMMVECs, with the greatest effect being after 3 h. The highest inhibitory effect of LA on I?Ba phosphorylation to prevent activation of NF-?B was after 6 h. These results suggest that LA reduces TNF-a and IL-6 mRNA levels through decreasing I?Ba phosphorylation and blocking the dissociation of IKK complex, which prevents activation of NF-?B.
