Quercetin Improves Hippocampal Neurogenesis in Depression by Regulating the Level of Let-7e-5p in Microglia Exosomes

Abstract

Background: Adult hippocampal neurogenesis plays a beneficial role in the treatment of depression. The precise mechanism by which let-7e-5p functions as a potential marker for depression remains unclear. Quercetin, a flavonoid compound, exhibits antidepressant effects; however, further investigation is needed to elucidate its regulatory effect and mechanism on hippocampal neurogenesis.

Methods: Chronic unpredictable mild stress (CUMS) was employed to induce depressive-like signaling and cognitive impairment in mice, while quercetin was administered via oral gavage. The symptoms of the mice were evaluated using various signaling methods. The expression levels of microglia, neural stem cells, and let-7e-5p in the dentate gyrus (DG) area of hippocampus were assessed using pathological observation methods. The expression levels of let-7e-5p and the Wnt1/β-catenin signaling pathways in the hippocampal DG of mice were assessed using qRT-PCR and Western blotting, respectively. The exosomes from peripheral blood were isolated and identified, followed by the detection of expression levels for microglia markers CD11b and TMEM119. We isolated hippocampal neural stem cells (NSCs) and co-cultured them with exosomes secreted by BV2 cells under LPS stimulation to observe the proliferation of NSCs and the generation of new neurons. The targeting relationship between let-7e-5p and Wnt1 was ultimately confirmed through the utilization of a dual luciferase reporter assay.

Results: (1) Quercetin ameliorated depression-like behaviors in mice induced by CUMS and restored neurogenesis in the DG region of the hippocampus. (2) Quercetin suppressed the secretion of microglia-derived exosomes carrying let-7e-5p in the DG, which exerted effects on NSC. (3) let-7e-5p regulates depression-related neurogenesis through targeting the Wnt1/β-catenin signaling pathway.

Conclusion: The inhibitory effect of let-7e-5p in microglial exosomes on depression-associated neurogenesis is mediated through the blockade of the Wnt1/β-catenin signaling pathway, which can be effectively reversed by Quercetin treatment.

Keywords: Wnt1/β-catenin; depression; exosome; let-7e-5p; microglia; neurogenesis; quercetin.

Graphical abstract

Figure 1

Effect of Quercetin on depression-like behavior and cognitive impairment in CUMS-induced mice. (A) Schematic diagram of the animal experiment procedure. (B) The trajectory diagram of mice in the open field test. (C) The duration of time spent in the central area of the open field and the number of crossings by mice. (D) The immobility time of mice during forced swimming test. (E) The sucrose preference rate of mice in the sucrose preference test. (F) The trajectory diagram of mice in the Morris water maze test. (G) The number of times mice crossed the escape platform and the time of escape latency. ** P < 0.01, *** P < 0.001. Abbreviations: CUMS, chronic unpredictable mild stress; Que, Quercetin; Que-L, low dose of Quercetin; Que-H, high dose of Quercetin. ** P < 0.01, *** P < 0.001.

Figure 2

Effect of Quercetin on let-7e-5p expression and Wnt1/β-catenin signaling pathway activation in the hippocampal DG of in CUMS-induced mice. (A and B) Immunofluorescence staining was used to observe the positive expression of microglia and NSCs markers (Iba-1 and Nestin) in the hippocampal DG region. The red fluorescence represents Iba-1 or Nestin, while the blue fluorescence corresponds to DAPI. Bar scale, 50 µm. (C and D) Fluorescence in situ hybridization/ immunofluorescence staining was used to observe the co-localization of let-7e-5p with Iba-1 or Nestin in the hippocampal DG region. The red fluorescence represents Iba-1 or Nestin, green fluorescence indicates let-7e-5p, while the blue fluorescence corresponds to DAPI. Bar scale, 100 µm. (E). qRT-PCR was used to detect the expression of let-7e-5p in the hippocampal DG. (F and G). Western blotting was used to determine nuclear expression level of β-catenin and protein expression of Wnt1 and GSK-3β within the hippocampal DG. ** P < 0.01, *** P < 0.001.

Figure 3

Effect of Quercetin on the expression level of let-73-5p in exosomes of CUMS-induced mice. (A) Nanoparticle tracking analysis was used to determine the concentration and particle size of exosomes in peripheral blood of mice. (B) Western blotting was used to measure the expression of exosome markers CD63 and CD81. (C) qRT-PCR was used to detect the expression of let-7e-5p in exosomes. (D) Western blotting was used to assess the expression of microglial markers CD11b and TMEM119 in exosomes. *** P < 0.001.

Figure 4

Effects of Quercetin on proliferation of NSCs and neurogenesis. (A) CCK-8 assay was used to assess the cell proliferation activity. (B) Western blotting was used to determine the protein expression of exosome markers CD63 and CD81. (C) Scanning electron microscopy was used to observe the morphology of exosomes. (D) Nanoparticle tracking analysis was used to determine the concentration and particle size of exosomes. (E) qRT-PCR was used to detect the expression of let-7e-5p in exosomes. (F) The uptake of PKH-67-labeled exosomes by NSCs was observed. The green fluorescence represents PKH-67-labeled exosomes, while the blue fluorescence corresponds to nucleus. Bar scale, 50 µm. (G) EdU incorporation assay was used to observe the proliferation ability of NSCs. The red fluorescence indicates the newly proliferating cells labeled with EdU, while the blue fluorescence corresponds to DAPI. Bar scale, 50 µm. (H) Immunofluorescence staining was used to observe the generation of new neurons. The red fluorescence indicates the newly generated neurons labeled with DCX, while the blue fluorescence corresponds to DAPI. Bar scale, 50 µm. *** P < 0.001.

Figure 5

Quercetin down-regulated let-7e-5p to promote the activation of Wnt1/β-catenin signaling pathways in NSCs. (A) qRT-PCR was used to detect the expression of let-7e-5p in NSCs. (B and C) Western blotting was used to determine nuclear expression level of β-catenin and protein expression of Wnt1 and GSK-3β in NSCs. (D) TargetScan website predicted the target binding sites between let-7e-5p and Wnt1 3’ UTR. (E) Dual-luciferase reporter assay was used to verify the targeted regulatory relationship between let-7e-5p and Wnt1. ** P < 0.01, *** P < 0.001.