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副干酪乳酪杆菌N1115的巨噬细胞免疫活化产物促进原代海马神经细胞发育

Macrophage-Activated Products of Lacticaseibacillus paracasei N1115 Promote the Development of Primary Hippocampal Neurons

  • 摘要:
    目的 初步探究副干酪乳酪杆菌N1115活菌介导免疫途径对体外培养的原代海马神经细胞发育的影响。
    方法 以适宜浓度的副干酪乳酪杆菌N1115活菌悬液为实验组,以肽聚糖(PGN)、脂多糖(LPS)为阳性对照,以RPMI1640培养基为空白对照,分别与RAW264.7细胞共培养,获得共培养上清液及细胞总RNA,测定细胞因子的表达及分泌情况。各组上清液经离心后以适当的比例与原代海马神经细胞共培养,收集共培养上清液,提取细胞总RNA,测定神经细胞突触发育相关基因的表达情况;原代海马神经细胞免疫荧光染色后对突触前后膜相关蛋白——突触后膜致密蛋白95(PSD95)、突触生长蛋白(SYP)及神经细胞成熟标志物——微管相关蛋白2(MAP-2)、双皮质素(DCX)进行定量分析,对神经细胞的形态发育进行测量。
    结果 与空白对照相比,N1115活菌干预RAW264.7细胞后,细胞免疫功能激活,白细胞介素(IL)-6、肿瘤坏死因子-α(TNF-α)的mRNA相对表达量分别提高7471%、926%,分泌量分别提高184.16 pg/mL、12320.76 pg/mL,差异均有统计学意义(P<0.05);N1115活菌的激活能力强于PGN,但弱于LPS,差异有统计学意义(P<0.05)。与空白对照相比,N1115活菌-RAW264.7细胞共培养上清液干预原代海马神经细胞后,10%上清干预组原代海马神经细胞活率提升19.25%,差异有统计学意义(P<0.05),突触前后膜SYPPSD95 mRNA相对表达量分别提高137%、159%,差异有统计学意义(P<0.05)。N1115活菌上清干预组神经细胞的突起总长度(489.88 μm)较空白对照组(381.51 μm)增加,胞体面积(2092.22 μm2)、轴突长度(184.78 μm)、突起总长度、平均突起长度(108.38 μm)、分支点(4.84个)较两阳性对照组升高,差异有统计学意义(P<0.05)。
    结论 副干酪乳酪杆菌N1115可激活巨噬细胞的免疫调节功能,从而促进神经细胞的形态发育以及突触功能。

     

    Abstract:
    Objective To make a preliminary investigation of the effect of the immune pathway mediated by live Lacticaseibacillus paracasei N1115 on the development of primary hippocampal neurons cultured in vitro.
    Methods Live Lacticaseibacillus paracasei N1115 suspension of an appropriate concentration was used as the experimental group. Peptidoglycan (PGN) and lipopolysaccharide (LPS) were used as positive controls, and RPMI1640 medium served as the blank control. These were co-cultured with RAW264.7 cells to obtain the co-culture mediums and the total cellular RNA, and to measure the expression and secretion of cytokines. After centrifugation, the supernatants were co-cultured with primary hippocampal neurons at appropriate ratios. The co-culture mediums were collected, and the total cellular RNA was extracted to measure the expression of genes related to synaptic development in neurons. Following immunofluorescence staining of the primary hippocampal neurons, the presynaptic and presynaptic membrane-associated proteins, including synaptophysin (SYP) and the postsynaptic density protein 95 (PSD95), and neuronal cell maturation markers, including microtubule-associated protein 2 (MAP-2), and doublecortin (DCX) were quantitatively analyzed. Additionally, the morphological development of the neurons were measured.
    Results  Compared with the blank control, the mRNA expression levels of interleukin (IL)-6 and tumor necrosis factor- α (TNF-α) increased by 7471% and 926%, respectively, after the RAW264.7 cells were treated with live Lacticaseibacillus paracasei N1115, while their secretion levels increased by 184.16 pg/mL and 12320.76 pg/mL, respectively, all showing statistically significant differences (P<0.05). The activation ability of N1115 live bacteria was stronger than that of PGN but weaker than that of LPS, showing statistically significant differences (P<0.05). Compared with the blank control, following the intervention with the supernatant from the co-culture of N1115 live bacteria and RAW264.7 cells, the viability of primary hippocampal neurons in the 10% supernatant intervention group increased by 19.25%, showing statistically significant differences (P<0.05), and the mRNA expression of SYP and PSD95 increased by 137% and 159%, respectively, showing statistically significant differences (P<0.05). The total neurite length (489.88 μm) of the neurons of the group intervened with the supernatant of N1115 live bacteria was increased compared to that of the blank control group (381.51 μm), and the cell body area (2092.22 μm2), maximum neurite length (184.78 μm), total neurite length, average neurite length (108.38 μm), and branching points (4.84 s) were higher than those in the two positive control groups, showing statistically significant differences (P<0.05).
    Conclusion Lacticaseibacillus paracasei N1115 can significantly activate the immune regulatory function of macrophages, thereby promoting the morphological development and synaptic function of nerve cells.

     

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