Polygonatum sibiricum Polysaccharides Alleviate Depressive-like Symptoms in Chronic Restraint Stress-Induced Mice via Microglial Regulation in Prefrontal Cortex

Abstract

Microglia respond to stressors by secreting cytokines or growth factors, playing a crucial role in maintaining brain homeostasis. While the antidepressant-like effects of Polygonatum sibiricum polysaccharides (PSPs) have been observed in mice, their potential effectiveness involving microglial regulation remains unknown. This study investigates the antidepressant-like mechanism of PSP by regulating microglial phenotype and signaling pathways in the prefrontal cortex of chronic restraint stress (CRS)-induced mice. PSP was extracted, purified, characterized, and orally administered to CRS mice. High-performance gel permeation chromatography (HPGPC) revealed that PSP has a molecular weight of 5.6 kDa. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that PSP exhibited a layered structure with densely packed, irregular surfaces. PSP treatment significantly increased sucrose preference (low: 71%, p < 0.01; medium: 69%, p < 0.05; high: 75%, p < 0.001 vs. CRS: 58%) and reduced immobility time (low: 74 s, p < 0.01; medium: 68 s, p < 0.01; high: 79 s, p < 0.05 vs. CRS: 129 s), indicating the alleviation of depressive-like behaviors. PSP inhibited microglial activation (PSP, 131/mm² vs. CRS, 173/mm², p = 0.057), reversing CRS-induced microglial hypertrophy and hyper-ramification. Furthermore, PSP inactivated microglial activation by inhibiting NLRP3/ASC/caspase-1/IL-1β signaling pathways, increasing BDNF synthesis and activating brain-derived neurotrophic factor (BDNF)-mediated neurogenesis (PSP, 80/per DG vs. CRS, 49/per DG, p < 0.01). In conclusion, PSP exerts antidepressant-like effects through the regulation of microglial activity and neuroinflammatory pathways, indicating it as a potential natural compound for depression treatment.

Keywords: BDNF; NLRP3; Polygonatum sibiricum; antidepressant; microglia; polysaccharides.

Figure 1

Characterization of Polygonatum sibiricum polysaccharides (PSP). (A) Infrared spectrum of PSP. (B) High-performance gel permeation chromatography (HPGPC) analysis of PSP. (C) SEM image of PSP at 500× magnification. (D) SEM image of PSP at 5000× magnification. (E) Two-dimensional morphology of AFM. (F) Three-dimensional morphology of AFM.

Figure 2

Behavioral assessments of mice treated with PSP in a CRS model (n = 11). (A) Sucrose preference, as anhedonia symptom of depression was reversed by PSP. (B) Immobility time, as despair symptom of depression was attenuated by PSP. ^# p < 0.05, and ^### p < 0.001 vs. normal group. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. CRS group.

Figure 3

PSP partly inactivated TLR4/NF-κB signaling and significantly inhibited NLRP3/ASC/caspase-1/IL-1β signaling pathway in the prefrontal cortex of CRS-induced mice (n = 6). (A) TLR4. (B) pNF-κB/NF-κB ratio. (C) NLRP3. (D) ASC. (E) Cleaved caspase-1. (F) Pro IL-1β. (G) Mature IL-1β. N: normal; C: CRS; F: fluoxetine; L: 100 mg/kg PSP; M: 200 mg/kg PSP; H: 400 mg/kg PSP. ^# p < 0.05 vs. normal group. * p < 0.05 and ** p < 0.01 vs. CRS group.

Figure 4

PSP attenuated microglial activation in the prefrontal cortex of CRS-induced mice (n = 5). (A) Immunofluorescence staining of microglia: representative images showing immunofluorescence staining of microglia in the prefrontal cortex. Blue indicates DAPI-stained nuclei, and red indicates Iba-1-stained microglia. Scale bar: 50 μm. (B) Quantification of microglial number per square millimeter. ^## p < 0.01 vs. normal group.

Figure 5

PSP treatment reversed CRS-induced prefrontal cortex microglial hypertrophy and hyper-ramification (n = 5). (A) Representative 3D reconstructions of microglia show increased size and complexity in the CRS group compared to the control, with PSP treatment reducing these changes. Scale bar: 5 μm. (B–J) Quantitative analysis: CRS significantly increased microglial area, length, volume, full branch depth, branch points, and Sholl intersections. PSP treatment significantly reduced area, volume, and Sholl intersections, with trends towards normalization in other parameters. These results suggest that PSP mitigates CRS-induced microglial hypertrophy and hyper-ramification, restoring more normal morphology. ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 vs. normal group. * p < 0.05 and ** p < 0.01 vs. CRS group.

Figure 6

Effects of PSP on inflammatory marker expression in prefrontal cortex microglia of CRS-induced mice. (A) Representative immunofluorescence staining images of TLR4 (green) expression in microglia (Iba-1, red) with DAPI (blue) staining. (B) Quantification shows increased TLR4 expression in the CRS group reduced by PSP treatment. (C) NLRP3 (green) expression images. (D) Quantification shows increased NLRP3 in the CRS group, reduced by PSP. (E) ASC (green) expression images. (F) Quantification shows increased ASC in the CRS group, reduced by PSP. (G) IL-1β (green) expression images. (H) Quantification shows a significant increase in IL-1β in the CRS group, significantly reduced by PSP. Scale bar: 4 μm. Within Iba-1 means the signal of the target protein was merged by the signal of the Iba-1 constructed surface by Imaris v9.0.1. Merge means blue, red, and green signals overlaying. ^## p < 0.01 and ^### p < 0.001 vs. normal group. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. CRS group.

Figure 7

PSP partly increased microglial BDNF expression in the prefrontal cortex and enhanced neurogenesis in the DG of CRS-induced mice. (A) Representative immunofluorescence staining, BDNF (green) in microglia (Iba-1, red) with DAPI-stained nuclei (blue). (B) Quantification showing a significant decrease in relative BDNF fluorescence intensity in the CRS group that is partially reversed by PSP. (C) Representative immunofluorescence staining presents DCX staining to visualize newborn neurons in the DG. (D) Quantification showing the number of newborn neurons per DG. Within Iba-1 means the signal of the target protein was merged by the signal of the Iba-1 constructed surface by Imaris. Merge means blue, red, and green signals overlaying. ^## p < 0.01 and ^### p < 0.001 vs. normal group. ** p < 0.01 vs. CRS group.