Fig. 4: L. plantarum and NaB restored the level of Gln(glutamine)/Glu (glutamate) in MIA offspring and regulated gene expression in hippocampus.

(A-D) The concentrations of Gln (A), Glu (B), Taurine (C), and GABA (D) in offspring brains. n = 5 mice/group. (E) Scheme of single-cell transcriptome sequencing and analysis. Hippocampal tissues from male mice aged 8-9 weeks were pooled into one sample for every three mice, and single-cell RNA sequencing was performed using the 10× Genomic Chromium system. (F-G) Uniform manifold approximation and projection (UMAP) plot of all single cells via 10× Genomics (F), 10 clusters with different functions were identified (G). (H-I) Uniform manifold approximation and projection (UMAP) plot of all astrocytes via 10× Genomics (H), 7 clusters with different functions were identified (I). (J) Heatmap of the top 20 DEGs in astrocyte subset C4. (K) GSEA plot of neurons for L-glutamate transmembrane transport-related gene sets (up) and GSEA plot of astrocytes for glutamate transmembrane transport-related gene sets (down). (L) Scheme of NaB and Glu treatment in the neuron-astrocyte coculture. The astrocytes in transwells were treated with NaB for 24 h and transferred to the 24-well plate containing primary neurons. This co-culture system was then stimulated with glutamate for 30 min and the neuronal damage was evaluated after the co-culture system was continued for 18 h. (M) Representative immunofluorescence images of cleaved caspase-3 (red) and NeuN (green) expression. Scale bar: 50 µm. (N) Statistical analysis of Caspase3⁺/NeuN⁺ cells in the neuron-astrocyte coculture. n = 3 biological replicates /group. (O) Representative immunofluorescence images of cleaved caspase-3 (red) and NeuN (green) expression in the neuron-astrocyte co-culture. Scale bar: 50 µm. (P) Statistical analysis of Caspase3⁺/NeuN⁺ cells from the neuron-astrocyte co-culture. n = 3 biological replicates /group. (Q) The Cxcl16 level in the normal and ASD patients. n = 30–35 patients /group. Data were presented as mean ± SEM, and statistical significance was determined by Unpaired t test (P and Q) and Tukey’s multiple comparisons test (A-D, and N), significant differences were classified as follows: ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.