Procyanidin B2 mitigates behavioral impairment and protects myelin integrity in cuprizone-induced schizophrenia in mice

Abstract

Numerous studies have suggested that neuropathological changes in schizophrenia may be related to damage to white matter or demyelination. Procyanidin B2, which is a constituent of many fruits such as grapes and strawberries, has various biological activities such as anti-inflammatory and anti-tumor activity, as has been reported. This study aimed to estimate the effects of procyanidin B2 on behavioral impairment and the protection of myelin integrity in a cuprizone-induced schizophrenia model. Mice were exposed to cuprizone (0.2% w/w in chow) for five weeks to induce schizophrenia-like behavioral changes and demyelination. Procyanidin B2 (20 or 100 mg kg^-1 day^-1) or vehicle was administered orally to mice after withdrawal from cuprizone. Behavioral impairment was detected with an open-field test, a rotarod test and a Morris water maze. Myelin integrity was assessed using LFB staining and MBP expression, including immunofluorescence and western blotting. In addition, enhancements in the expression of HO-1 and NQO1 suggested that procyanidin B2 may regulate oxidative homeostasis via promoting the translation of Nrf2 to the nucleus. Data indicated that procyanidin B2 could mitigate behavioral impairment and protect myelin integrity in the cuprizone-induced model via regulating oxidative stress by activating Nrf2 signaling.

Fig. 1. (A) Chemical structure of procyanidin B2. (B) Body weight (n = 12) in CON group, CPZ group and PB groups. Two-way ANOVA for body weight indicated a significant group-by-day interaction. (C) Schedule of experimental arrangement including the time course of the administration of cuprizone and procyanidin B2 and timeline for behavioral testing, histological studies and western blotting analysis.

Fig. 2. The locomotor influence of cuprizone intoxication and treatment with procyanidin B2 was assessed. (A–C) Open-field test. The center dwell time (A), peripheral locomotion time (B) and center crossing percentage (C) were recorded. Cuprizone-intoxicated mice spent shorter times and covered less distance in the central grids, which was significantly reversed by treatment with 100 mg kg⁻¹ procyanidin B2 (p < 0.01). (D and E) Rotarod test. Cuprizone-intoxicated mice stayed for the shortest time and had the greatest number of falls from the rotating cylinder, which was ameliorated by procyanidin B2, with a significant difference (p < 0.01). All data are expressed as the mean ± SEM (n = 8). ^#p < 0.05, ^##p < 0.01 versus the CON group; *p < 0.05, **p < 0.01 versus the CPZ group.

Fig. 3. Effect of procyanidin B2 on cuprizone-induced impairments in spatial cognition and memory assessed by the MWM task. (A) Two-way ANOVA for escape latencies shown as the mean value for each training session for four successive days. The distance covered (B) and time spent (C) in the target quadrant were measured during the task. Cuprizone increased the number of crossings (D) to the location of the removed platform, with a significant difference (p < 0.01), which was increased by procyanidin B2 (p < 0.05). In addition, procyanidin B2 increased the swimming speed of mice in the probe test (E) in comparison with mice in the CPZ group. Swimming tracks (F) were recorded via a video tracking camera system. All data are expressed as the mean ± SEM (n = 8). ^#p < 0.05, ^##p < 0.01 versus the CON group; *p < 0.05 versus the CPZ group.

Fig. 4. Protective effect of procyanidin B2 on white matter against demyelinated lesions revealed in the corpus callosum (cc) and corpus striatum (CPu) by LFB staining. LFB staining (A and C) showed the severity of demyelination in the cc and CPu. The blue areas represent intact regions, and data are given as quantified for LFB staining (B and D). All data are expressed as the mean ± SEM (n = 4). ^##p < 0.01 versus the CON group; *p < 0.05, **p < 0.01 versus the CPZ group.

Fig. 5. Immunofluorescence staining of MBP in the cc (A) and CPu (C) was performed, and merged images are shown. The immunofluorescence optical density of MBP (B and D) demonstrated that 100 mg kg⁻¹ procyanidin B2 evidently alleviated demyelination caused by cuprizone. The effect of procyanidin B2 on myelin proteins in cuprizone-intoxicated mice was analyzed by western blotting (E and F). The densified values (F) indicated that the reduction in the expression of MBP in the cc and CPu of cuprizone-intoxicated mice was significantly reversed by procyanidin B2. All data are expressed as the mean ± SEM (n = 4). ^##p < 0.01 versus the CON group; **p < 0.01 versus the CPZ group.

Fig. 6. The influence of PB on oxidative stress and phase II detoxifying enzymes was researched. The SOD activities (A), CAT activities (B) and MDA levels (C) in the experiments are shown. Representative western blots (D) for the expression of HO-1 and NQO1 in the CON, CPZ, 20 mg kg⁻¹ PB and 100 mg kg⁻¹ PB groups were evaluated. The densified values (E) indicated that procyanidin B2 facilitated recovery of the expression of HO-1 and NQO1. All data are expressed as the mean ± SEM (n = 4). ^#p < 0.05, ^##p < 0.01 versus the CON group; *p < 0.05 versus the CPZ group.

Fig. 7. The effects of cuprizone and PB on the protein expression of Nrf2 in the nuclear and cytosolic fractions were studied using western blotting analysis. Representative western blots (A) revealed that the expression of Nrf2 in the nuclear fraction was downregulated by cuprizone intoxication. Densified value (B) of Nrf2 expression normalized to that of lamin B2. Representative western blots (C) show that the expression of Nrf2 remained unchanged. Densified value (D) of Nrf2 expression normalized to that of β-actin. All data are expressed as the mean ± SEM (n = 4). ^#p < 0.05 versus the CON group; *p < 0.05 versus the CPZ group.