Active Fraction Combination From Liuwei Dihuang Decoction Improves Adult Hippocampal Neurogenesis and Neurogenic Microenvironment in Cranially Irradiated Mice

Abstract

Background: Cranial radiotherapy is clinically used in the treatment of brain tumours; however, the consequent cognitive and emotional dysfunctions seriously impair the life quality of patients. LW-AFC, an active fraction combination extracted from classical traditional Chinese medicine prescription Liuwei Dihuang decoction, can improve cognitive and emotional dysfunctions in many animal models; however, the protective effect of LW-AFC on cranial irradiation-induced cognitive and emotional dysfunctions has not been reported. Recent studies indicate that impairment of adult hippocampal neurogenesis (AHN) and alterations of the neurogenic microenvironment in the hippocampus constitute critical factors in cognitive and emotional dysfunctions following cranial irradiation. Here, our research further investigated the potential protective effects and mechanisms of LW-AFC on cranial irradiation-induced cognitive and emotional dysfunctions in mice. Methods: LW-AFC (1.6 g/kg) was intragastrically administered to mice for 14 days before cranial irradiation (7 Gy γ-ray). AHN was examined by quantifying the number of proliferative neural stem cells and immature neurons in the dorsal and ventral hippocampus. The contextual fear conditioning test, open field test, and tail suspension test were used to assess cognitive and emotional functions in mice. To detect the change of the neurogenic microenvironment, colorimetry and multiplex bead analysis were performed to measure the level of oxidative stress, neurotrophic and growth factors, and inflammation in the hippocampus. Results: LW-AFC exerted beneficial effects on the contextual fear memory, anxiety behaviour, and depression behaviour in irradiated mice. Moreover, LW-AFC increased the number of proliferative neural stem cells and immature neurons in the dorsal hippocampus, displaying a regional specificity of neurogenic response. For the neurogenic microenvironment, LW-AFC significantly increased the contents of superoxide dismutase, glutathione peroxidase, glutathione, and catalase and decreased the content of malondialdehyde in the hippocampus of irradiated mice, accompanied by the increase in brain-derived neurotrophic factor, insulin-like growth factor-1, and interleukin-4 content. Together, LW-AFC improved cognitive and emotional dysfunctions, promoted AHN preferentially in the dorsal hippocampus, and ameliorated disturbance in the neurogenic microenvironment in irradiated mice. Conclusion: LW-AFC ameliorates cranial irradiation-induced cognitive and emotional dysfunctions, and the underlying mechanisms are mediated by promoting AHN in the dorsal hippocampus and improving the neurogenic microenvironment. LW-AFC might be a promising therapeutic agent to treat cognitive and emotional dysfunctions in patients receiving cranial radiotherapy.

Keywords: LW-AFC; adult hippocampal neurogenesis; cranial irradiation; dorsal hippocampus; neural stem cells; neurogenic microenvironment; traditional Chinese medicine; ventral hippocampus.

FIGURE 1

The HPLC fingerprint of LW-AFC. LW-AFC consists of polysaccharide fraction (LWB-B), glycoside fraction (LWD-B), and CA-30. (A) The HPLC fingerprint of LWB-B and CA30 mixture. The peak at 6.26, 6.83, 8.19, 18.30, and 21.50 min represents, respectively, fructose, glucose, sucrose, mannotriose, and stachyose. (B) The HPLC fingerprint of LWD-B. The peak S represents loganin.

FIGURE 2

Scheme of the experimental procedure and schematic representation of the dorsal and ventral hippocampus in mice. (A) Scheme of the experimental procedure. (B) The image of the dorsal and ventral hippocampus along the longitudinal axis. (C) The coronal brain section of the dorsal hippocampus. (D) The coronal brain section of the ventral hippocampus.

FIGURE 3

LW-AFC improves cognitive and emotional dysfunctions in IR mice on day 1 after irradiation. (A) Quantification of the total distances travelled by mice during the open field test. (B) Quantification of the center time of mice during the open field test. (C) Quantification of the freezing time of mice during the contextual fear conditioning test. (D) Quantification of the immobility time of mice during the tail suspension test. The values denote mean ± S.D., n = 8. *p < 0.05 and **p < 0.01, versus the control group; ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001, versus the IR group. Abbreviation: IR, irradiation.

FIGURE 4

LW-AFC promotes the proliferation of neural stem cells preferentially in the dorsal hippocampus of IR mice on day 1 after irradiation. Representative immunofluorescence images of BrdU-positive cells in the (A) dorsal hippocampus and the (B) ventral hippocampus. The area of white square frame in the left image indicates the enlarged image of BrdU-positive cells in the right. The confocal images in the left are of 10×, and the enlarged images in the right are of 40×. Quantification of BrdU-positive cells in the (C) dorsal hippocampus and the (D) ventral hippocampus. The scale bar in the left image = 300 μm, and the scale bar of the enlarged image = 100 μm. The values denote mean ± S.D., n = 4. ***p < 0.001, versus the control group; ^## p < 0.01, versus the IR group. Abbreviation: IR, irradiation.

FIGURE 5

LW-AFC increases the number of immature neurons preferentially in the dorsal hippocampus of IR mice on day 1 after irradiation. Representative immunofluorescence images of DCX-positive cells in the (A) dorsal hippocampus and (B) ventral hippocampus. The area of white square frame in the left image indicates the enlarged image of DCX-positive cells in the right. The confocal images in the left are of 10×, and the enlarged images in the right are of 40×. Quantification of DCX-positive cells in the (C) dorsal hippocampus and the (D) ventral hippocampus. The scale bar in the left image = 300 μm, and the scale bar of the enlarged image = 100 μm. The values denote mean ± S.D., n = 4. ***p < 0.001, versus the control group; ^# p < 0.05, versus the IR group. Abbreviation: IR, irradiation.

FIGURE 6

LW-AFC reduces the oxidative stress level in the hippocampus of IR mice on day 1 after irradiation. Concentrations of (A) GSH, (B) GSH-Px, (C) CAT, (D) SOD, and (E) MDA were detected. The values denote mean ± S.D., n = 6. *p < 0.05, **p < 0.01, and ***p < 0.001, versus the control group; ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001, versus the IR group. Abbreviation: IR, irradiation.

FIGURE 7

LW-AFC increases the level of neurotrophic and growth factors in the hippocampus of IR mice on day 1 after irradiation. Concentrations of (A) BDNF, (B) IGF-1, and (C) VEGF were detected. The values denote mean ± S.D., n = 4. *p < 0.05 and **p < 0.01, versus the control group. ^# p < 0.05, versus the IR group. Abbreviation: IR, irradiation.

FIGURE 8

LW-AFC increases the level of anti-inflammatory factor in the hippocampus of IR mice on day 1 after irradiation. Concentrations of (A) IL-4, (B) IL-10, and (C) G-CSF were detected. The values denote mean ± S.D., n = 4. *p < 0.05, versus the control group; ^## p < 0.01, versus the IR group. Abbreviation: IR, irradiation.

FIGURE 9

A schematic illustration of the protective mechanisms of LW-AFC against cranial irradiation–induced cognitive and emotional dysfunctions via increasing AHN in the dorsal hippocampus and modulating the neurogenic microenvironment.