Lotus Root Residue Polysaccharide Regulate the Immune Response of Mouse Peritoneal Macrophages via MAPK/NF-κB Pathway
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Abstract:
The in vitro immunomodulatory effect of lotus root residue polysaccharide (LRP) on primary peritoneal macrophages of BALB/C mice was investigated to explore the potential signaling pathway of LRP-induced immune response. The effects of different concentrations of LRP on cell viability were determined by the MTT method; the amount of the NO released from cells was detected by the Griess method after the stimulation with LRP at different concentration gradients, and at the same concentration but at different time points; Semi-quantitative PCR was used to detect the mRNA expressions of TLR4 and TLR2 receptors as well as immune-related factors (TNF-α, IL-6, iNOS, 1L-1β, COX-2, Nfkbia) in cells; Western blotting was used to detect the phosphorylation of MAPK pathway-related factors (ERK1/2, JNK, p38) and Akt. The effect of LRP on AP-1 and NF-κB was also studied. the immunomodulatory activity of LRP towards mouse peritoneal macrophages and associated signaling mechanism were evaluated. The result showed that LRP has no toxic effect on mouse peritoneal macrophages, LRP at 25~50 μg/mL could promote the cell growth with the cell viability being 104.83% and 102.53% (p<0.05). The NO production increased significantly with an increase of LRP concentration (p<0.05). The cells stimulated by LRP at 200 μg/mL produced 36.47 μmol/L of NO. NO production increased with the prolongation of culture time after the treatment with LRP at 200 μg/mL (p<0.05), with the NO concentration reaching 44.18 μmol/L after 24 h. The studies on mRNA expressions showed that LRP regulated the expressions of TLR4 and TLR2 receptors, and modulated the expressions of immune-related genes. In addition, LRP promoted the translocation of c-Jun and p65 from outside the nucleus into the nucleus, enhanced the phosphorylation levels of ERK1/2, JNK, and p38 proteins, while having insignificant effect on the phosphorylation of Akt. Therefore, LRP can enhance the immune response of primary peritoneal macrophages in BALB/c mice through the MAPK/NF-κB pathway.
