Polygalasaponin XXXII from Polygala tenuifolia root improves hippocampal-dependent learning and memory

Abstract

Aim: The aim of this study was to investigate the cognition-enhancing activity and underlying mechanisms of a triterpenoid saponin (polygalasaponin XXXII, PGS32) isolated from the roots of Polygala tenuifolia Willd.

Methods: The Morris water maze was used to evaluate the spatial learning and memory of mice. To detect the basic properties of synaptic transmission and long-term potentiation (LTP) in the dentate gyrus of rats, electrophysiological recordings were made of evoked potentials. Western blotting analysis and immunofluorescence assays were used to determine the phosphorylation of extracellular signal-regulated kinase (ERK), cAMP response element-binding protein (CREB), synapsin I and the expression of brain derived neurotrophic factor (BDNF).

Results: When administered at 0.125, 0.5, or 2 mg/kg, PGS32 could significantly prevent scopolamine-induced cognitive impairments in mice. Intracerebroventricular (icv) administration of PGS32 greatly enhanced basic synaptic transmission in the dentate gyrus of rats and induced LTP. In primary hippocampal neurons, as well as in the hippocampus of maze-trained mice, PGS32 activated the mitogen-activated protein (MAP) kinase cascade by promoting phosphorylation of ERK, CREB and synapsin I. The expression of BDNF was also greatly enhanced in the hippocampus.

Conclusion: Our findings suggest that PGS32 can improve hippocampus-dependent learning and memory, possibly through improvement of synaptic transmission, activation of the MAP kinase cascade and enhancement of the level of BDNF. Therefore, PGS32 shows promise as a potential cognition-enhancing therapeutic drug.

Figure 1

Effects of PGS32 on performance in the training trials (A) and in the probe trial (B) of the Morris water maze task in mice with scopolamine-induced cognitive deficits. Data represent means±SEM (n=12). Mice were orally treated with PGS32 (0.125, 0.5, and 2 mg/kg), the same volume of saline, or huperzine-A (Hup A, 0.05 mg/kg) for 16 days for a period ranging from 10 d prior to beginning the behavioral experiments to the end of the water maze task. Scopolamine (1 mg/kg) or the same volume of saline was administered ip to mice 30 min before the training trial and the probe trial. (A) Mean escape latency to find the hidden platform over 5 consecutive days of the training trial. The data were analyzed using two-way ANOVA (group×days, 6×5) with repeated measures. (B) Swimming time in the target quadrant in 90 s probe trial (no platform). Each mouse was subjected to 90 s observation on the 6 th day of the water maze tasks.^cP<0.01 vs control group. ^eP<0.05, ^fP<0.01 vs model group.

Figure 2

Effects of PGS32 on basic synaptic transmission in the DG of anesthetized rats. (A) Anatomical diagram of a rat skull. The points R and S represent the approximate location of the recording electrode and the stimulating electrode, respectively. (B) Measurement of PS amplitude. a: stimulus artifact; b: initial point of excitatory postsynaptic potential (EPSP); c: middle point of bd; d, e, f: end points of the peaks; length of ge: amplitude of PS. (C) Original figure of PS in control and PGS32 group at baseline, 10 min, 30 min, and 60 min after icv. (D) Time-course plots of perforant path-evoked population spikes (PS) of the control group (white triangle) and PGS32 group (black rhombus). The stimulating electrode and the recording electrode were placed within the perforant path (PP) and the granular cell layer of the dentate gyrus, respectively. An evoked response was generated in the dentate gyrus granular cell layer by stimulating the PP at low frequency (0.033 Hz); PS of the granular cell layer in DG was recorded throughout the test. The baseline was recorded from 30 min before administration. At 0 min, 5 μL 400 μmol/L PGS32 (icv, final concentration was 1 μmol/L) was injected in the test subject, whereas 0.4% DMSO-PBS was injected in a control animal.

Figure 3

Phosphorylation of ERK, CREB, and synapsin I and the expression of BDNF of hippocampal lysates of mice following the Morris water maze task (n=6). (A) Representative immunoblots of hippocampal lysates of the mice. The mice were administered and trained for the water maze tasks, and then hippocampus samples from six mice of each group were homogenated for immunoblotting. (B) The relative density of the bands was analyzed using one-way ANOVA. The six columns from the left to the right in each protein represent the control group, model group, PGS32 0.125, 0.5, 2 mg/kg group and HupA group, respectively. ^bP<0.05, ^cP<0.01 vs control group. ^eP<0.05, ^fP<0.01 vs model group.

Figure 4

Effects of PGS32 on the phosphorylation of ERK1/2, CREB, and synapsin I in primary hippocampal neurons. The 5 DIV neurons were incubated with either 1 μmol/L PGS32 (PGS32 group) or vehicle (0.001% DMSO-PBS, Control group) at 37°C for 4 min; cells were then fixed and phosphorylation of ERK, CREB, and synapsin I was detected (n=3). The green fluorescence was used to label the levels of phospho-ERK, phospho-CREB, and phospho-synapsin I in the control and PGS32 groups (arrow pointed). Rhodamine-labeled phalloidin was used to detect the F-actin of the neurons, so that their outline was displayed.