联合体内外实验初步探究混合植物乳杆菌的免疫调节能力

韩玉; 岳莹雪; 程杨; 刘德玉; 曹婷; 张艺凡; 王松; 李柏良; 霍贵成

doi:10.13386/j.issn1002-0306.2023120170

联合体内外实验初步探究混合植物乳杆菌的免疫调节能力

Preliminary Investigation of the Immunomodulatory Capacity of Mixed Lactobacillus plantarum by in Vitro and in Vivo Experiments

摘要

摘要: 目的：通过体外体内实验研究混合植物乳杆菌的免疫调节能力。方法：采用CCK8法观察混合植物乳杆菌对巨噬细胞的增殖能力影响。采用巨噬细胞吞噬中性红法检测混合植物乳杆菌对巨噬细胞吞噬能力的影响。采用NO、TNF-α、IL-6、IL-1β试剂盒测定混合植物乳杆菌促巨噬细胞分泌细胞因子能力。将健康的BALB/c小鼠随机分为3组，即空白组（NC组）、模型组（MC组）、混合植物乳杆菌组（Mix组）。记录实验室小鼠建模前、后和最终体重。并在小鼠处死后测定各实验组脾脏、胸腺指数，以及脾淋巴细胞增殖能力和自然杀伤细胞（NK）活性。结果：与空白组相比，MOI=1、10、100时混合植物乳杆菌无细胞毒性，并且能显著促进巨噬细胞的增殖（P<0.05）（MOI=1、10、100、1000。1，10，100，1000分别代表混合植物乳杆菌与RAW 264.7巨噬细胞感染复数（MOI）为1:1，10:1，100:1，1000:1）。当MOI=100时，混合植物乳杆菌的最高相对增殖率为176.75%。与空白组相比，各MOI值下混合植物乳杆菌均能显著提高巨噬细胞的吞噬能力（P<0.05），表现出良好的免疫活性。特别是在MOI=100时，混合植物乳杆菌组细胞的吞噬活性（A₅₄₀=0.80）显著优于LPS组（A₅₄₀=0.55）（P<0.05）。当MOI=1、10、100时混合植物乳杆菌组与空白组相比，能显著上调巨噬细胞分泌NO和细胞因子水平（P<0.05）。当MOI=100时巨噬细胞NO分泌量为27.32 µmol/L、TNF-α分泌量为116.97 pg/mL、IL-6分泌量为41.98 pg/mL、IL-1β分泌量为149.88 pg/mL。与MC组相比，混合植物乳杆菌的摄入增加了小鼠的体重、上调脾脏指数和胸腺指数。同时，对由环磷酰胺造成的脾淋巴细胞损伤有显著促增殖的作用（P<0.05）。与MC组相比，Mix组能够显著提高小鼠NK细胞活性（P<0.05），达到41.72%。结论：混合植物乳杆菌具有免疫调节活性，是一种良好的免疫调节剂。

Abstract: Objective: To study the immunomodulatory capacity of Lactobacillus plantarum mixed by in vitro and in vivo experiments. Methods: The effect of Lactobacillus plantarum mixed on the proliferative capacity of macrophages was observed using the CCK8 method. The effect of Lactobacillus plantarum mixed on macrophage phagocytosis was detected using the macrophage phagocytosis neutral red method. NO, TNF-α, IL-6, IL-1β kits were used to determine the macrophage-promoting cytokine secretion capacity of Lactobacillus plantarum mixed. Healthy BALB/c mice were randomly divided into three groups: normal group (NC group), model group (MC group), and Lactobacillus plantarum mixed group (Mix group). The body weight of laboratory mice before, after and at the end of modeling was recorded, and the spleen and thymus indexes of each experimental group, as well as the proliferation ability of spleen lymphocytes and the activity of natural killer cells (NK) after the mice died were measured. Results: Lactobacillus plantarum mixed was not cytotoxic and significantly promoted macrophage proliferation at MOI=1, 10, and 100 compared to the normal group (P<0.05) (MOI=1, 10, 100, and 1000. 1, 10, 100, and 1000 represented the number of Lactobacillus plantarum mixed infected with the RAW 264.7 macrophage complex, respectively, (MOI) of 1:1, 10:1, 100:1, and 1000:1). The highest relative proliferation rate of Lactobacillus plantarum mixed was 176.75% when MOI=100. Lactobacillus plantarum mixed significantly increased phagocytosis of macrophages at all MOI values (P<0.05) and showed excellent immune activity compared to the normal group. Especially at MOI=100, the phagocytic activity of cells in the Lactobacillus plantarum mixed group (A₅₄₀=0.80) was significantly better than that in the LPS group (A₅₄₀=0.55) (P<0.05). The Lactobacillus plantarum mixed group significantly up-regulated the levels of NO and cytokines secreted by macrophages compared to the normal group when MOI=1, 10 and 100 (P<0.05). Macrophage NO secretion was 27.32 µmol/L, TNF-α secretion was 116.97 pg/mL, IL-6 secretion was 41.98 pg/mL, and IL-1β secretion was 149.88 pg/mL when MOI=100. Lactobacillus plantarum mixed intake increased body weight, up-regulated spleen index and thymus index in mice compared to MC. Lactobacillus plantarum mixed also had a significantly pro-proliferative effect on splenic lymphocyte damage caused by cyclophosphamide (P<0.05). Compared with MC group, Mix group could significantly increase the activity of NK cells in mice (P<0.05), reached 41.72%. Conclusion: Lactobacillus plantarum mixed has immunomodulatory activity and is a good immunomodulator.