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

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.