揭示莲藕渣多糖（Lotus Root Residue Polysaccharide，LRP）对BALB/c小鼠腹腔巨噬细胞的体外免疫调节作用，探讨LRP刺激腹腔巨噬细胞产生免疫应答的信号通路。利用MTT法测定不同浓度LRP对细胞活力影响；选用不同浓度梯度及同一浓度不同时间点的LRP刺激细胞，Griess法检测细胞NO释放量；半定量PCR检测细胞TLR4、TLR2受体及免疫关联因子（TNF-α、IL-6、iNOS、1L-1β、COX-2、Nfkbia）mRNA的表达，蛋白印迹法检测其MAPK通路（ERK1/2、JNK、p38）及Akt的磷酸化，同时研究了LRP对和蛋白AP-1及NF-κB的影响，对LRP对小鼠腹腔巨噬细胞的免疫调节活性及其信号机制进行评估。研究表明，LRP对小鼠腹腔巨噬细胞无毒作用，25~50 μg/mL LRP促进细胞生长，细胞存活率为104.82%和102.53%（p<0.05）；NO浓度随LRP浓度提高而显著提高（p<0.05），200 μg/mL LRP刺激细胞产生一氧化氮（NO）为36.47 μmol/L，200 μg/mL的LRP处理细胞，NO产量随培养时间延长而增多（p<0.05），24 h时NO浓度为44.18 μmol/L；mRNA基因表达研究显示，LRP调控TLR4、TLR2受体，并调节免疫基因的表达；此外，LRP促进核蛋白c-Jun、p65由核外转向核内，增强ERK1/2、JNK、p38蛋白的磷酸化水平，但对于Akt的磷酸化没有显著影响。因此，莲藕渣多糖（LRP）可通过MAPK/NF-κB途径增强BALB/c小鼠腹腔巨噬细胞免疫应答。

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.

江苏省自然科学基金项目（BK20201480）