Fig 2: QJD alleviated the neuroinflammation through suppressing microglial and astrocytic activation in D-gal–induced mice. (A) Protein expression of TNF-a in hippocampus. (B) Protein expression of IL-1ß in hippocampus. (C) Protein expression of IL-6 in hippocampus. (D) Representative immunofluorescence staining images of GFAP (labeled in red) in the hippocampus of CA1 and CA3 regions (magnification × 400; scale bar = 20 µm). (E) Representative immunofluorescence staining images of IBA1 (labeled in red) in the hippocampus of CA1 and CA3 regions (magnification × 400; scale bar = 20 µm). (F) Mean fluorescence intensity of GFAP in the hippocampus of CA1 region. (G) Mean fluorescence intensity of GFAP in the hippocampus of CA3 region. (H) Mean fluorescence intensity of IBA1 in the hippocampus of CA1 region. (I) Mean fluorescence intensity of IBA1 in the hippocampus of CA3 region. All data were presented as mean ± SEM (n = 4 or 6/group). **p < 0.01 vs. NC group; # p < 0.05 and ## p < 0.01 vs. D-gal group.

Fig 3: Proportion of Nestin+/Ngfr+cells differentiated into neurons was significantly higher than that of Nestin+cells.(A) Representative immunofluorescence confocal laser images in freshly intermuscular plexus tissues of gastric of GFAP/ß-tubulin/HuC/D/nNOS (green) and tdTomato-labeled Nestin+ cells (red) in BAC+BMSCs+TAM (Nestin-creERT2: tdTomato mice), the nuclei (blue). Arrows indicate double-positive cells. (B) Representative immunofluorescence confocal laser images in freshly intermuscular plexus tissues of gastric of GFAP/ß-tubulin/HuC/D/nNOS (red) and GFP-labeled Nestin+/Ngfr+ cells (green) in BAC+BMSCs+TAM (Nestin-creert2 X Ngfr-Dreert2: DTRGFP mice), the nuclei (blue). Arrows indicate double-positive cells. (C and D) Proportion of Nestin+ (red)/Nestin+Ngfr+ (blue) cells differentiated into neurons and glial cells in Control+TAM group and BAC+BMSCs+TAM group. Control, the control C57 mice; Sham, sham operation group; BAC, benzalkonium chloride treated group; TAM, tamoxifen. These results are representative of independent experiments repeated at least 3 times. Results were expressed as mean ± standard deviation. *P < .05, **P < 0.01, NS, no significance.

Fig 4: BAC (0.05%) effectively ablated the myenteric plexus in gastric tissue and remained at least 31 days.(A) Representative immunofluorescence confocal laser images in transverse sections of gastric tissue sections of PGP 9.5 (red) in the control group (upper panel) and 0.05% BAC treated group (lower panel); the a-SMA (green); the nuclei (blue). (B–D) Representative immunofluorescence confocal laser images in gastric myenteric plexus of PGP 9.5 (red), HuC/D (red), and GFAP (red) in each group; the nuclei (blue). (E and F) Representative immunoblot bands and histogram of relative expression for PGP 9.5/HuC/D/GFAP in each group (normalized to GAPDH). Control, the control C57 mice; BAC, benzalkonium chloride treated C57 mice. Results were expressed as mean ± standard deviation. ****P < .0001, n = 4–5.

Fig 5: Regenerated neurons derived from Nestin+/Ngfr+cells in denervated triple-transgenic mice (Nestin-creERT2 × Ngfr-DreERT2: DTRGFP).(A–D) Representative immunofluorescence confocal laser images in freshly intermuscular plexus tissues of gastric of GFAP/ß-tubulin/HuC/D/nNOS (red) and GFP-labeled ENPCs (green) in Control, Control+TAM, Sham+TAM, and BAC+TAM, the nuclei (blue). Arrows indicate double-positive cells. Nestin+/Ngfr+ cells are stained with GFP (green) and express GFAP/ß-tubulin/ HuC/D/nNOS (red) after TAM induction and show co-localization providing evidence that Nestin+/Ngfr+ cells can be differentiated into neurons and glial. (E and F) Representative immunofluorescence confocal laser images in freshly intermuscular plexus tissues of gastric of GFAP/ß-tubulin/HuC/D/nNOS (gray), GFP-labeled ENPCs (green), and tdTomato-labeled BMSCs (red) in Sham+BMSCs+TAM and BAC+BMSCs+TAM groups, the nuclei (blue). Control, the control C57 mice; Sham, sham operation group; BAC, benzalkonium chloride treated group; BAC+BMSCs, BAC treated mice transplanted with BMSCs; TAM, tamoxifen.