Kai Xin San ameliorates scopolamine-induced cognitive dysfunction

Abstract

Kai Xin San (KXS, containing ginseng, hoelen, polygala, and acorus), a traditional Chinese herbal compound, has been found to regulate cognitive dysfunction; however, its mechanism of action is still unclear. In this study, 72 specific-pathogen-free male Kunming mice aged 8 weeks were randomly divided into a vehicle control group, scopolamine group, low-dose KXS group, moderate-dose KXS group, high-dose KXS group, and positive control group. Except for the vehicle control group and scopolamine groups (which received physiological saline), the doses of KXS (0.7, 1.4 and 2.8 g/kg per day) and donepezil (3 mg/kg per day) were gastrointestinally administered once daily for 2 weeks. On day 8 after intragastric treatment, the behavioral tests were carried out. Scopolamine group and intervention groups received scopolamine 3 mg/kg per day through intraperitoneal injection. The effects of KXS on spatial learning and memory, pathological changes of brain tissue, expression of apoptosis factors, oxidative stress injury factors, synapse-associated protein, and cholinergic neurotransmitter were measured. The results confirmed the following. (1) KXS shortened the escape latency and increased residence time in the target quadrant and the number of platform crossings in the Morris water maze. (2) KXS increased the percentage of alternations between the labyrinth arms in the mice of KXS groups in the Y-maze. (3) Nissl and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining revealed that KXS promoted the production of Nissl bodies and inhibited the formation of apoptotic bodies. (4) Western blot assay showed that KXS up-regulated the expression of anti-apoptotic protein Bcl-2 and inhibited the expression of pro-apoptotic protein Bax. KXS up-regulated the expression of postsynaptic density 95, synaptophysin, and brain-derived neurotrophic factor in the cerebral cortex and hippocampus. (5) KXS increased the level and activity of choline acetyltransferase, acetylcholine, superoxide dismutase, and glutathione peroxidase, and reduced the level and activity of acetyl cholinesterase, reactive oxygen species, and malondialdehyde through acting on the cholinergic system and reducing oxidative stress damage. These results indicate that KXS plays a neuroprotective role and improves cognitive function through reducing apoptosis and oxidative stress, and regulating synapse-associated protein and cholinergic neurotransmitters.

Keywords: Kai Xin San; apoptosis; cholinergic system dysfunction; cognitive dysfunction; donepezil; neural regeneration; neuroprotection; oxidative stress; scopolamine hydrobromide; synaptic dysfunction.

Figure 1

Extraction procedure of Kai Xin San. d.w.: Delivered weight; h: hours.

Figure 2

Experimental procedure: drug administration and behavioral tests. Mice received drug administration after the 1-week adaptive feeding period. Behavioral tests (the Morris water maze and Y-maze spontaneous alternation test) were implemented after the 1-week drug administration period. Behavioral tests lasted 1 week, and during which drug treatment was continued. The total duration of the experiment was 2 weeks.

Figure 3

KXS ameliorates spatial learning and memory impairments. Escape latency (A), swimming trajectories (B), time spent in target quadrant (C), crossing times of the platform (D), and swimming speed (E) measured in the Morris water maze test. (F) Y-maze spontaneous alternation. Data are shown as the mean ± SEM (n = 12; one-way analysis of variance followed by Dunnett’s post hoc test). The experiment was performed in triplicate. #P < 0.05, ##P < 0.01, vs. CON group; *P < 0.05, **P < 0.01, vs. SCOP group. KXS: Kai Xin San; CON: vehicle control; SCOP: scopolamine; KXS-L: scopolamine + Kai Xin San (0.7 g/kg per day); KXS-M: scopolamine + Kai Xin San (1.4 g/kg per day); KXS-H: scopolamine + Kai Xin San (2.8 g/kg per day); DON: scopolamine + donepezil (3 mg/kg per day); d: days.

Figure 4

KXS reduces neuronal apoptosis. (A) Nissl staining in the hippocampus: neurons were disordered and Nissl bodies in the cells were reduced or even vacuolate (red arrow; × 200). (B) TUNEL staining in the cortex and apoptotic bodes (red arrow; × 200). (C, D) Protein expression of Bcl-2 and Bax was detected in the hippocampus and cortex, respectively. Data are shown as the mean ± SEM (n = 8; one-way analysis of variance followed by Dunnett’s post hoc test). The experiment was performed in triplicate. #P < 0.05, ##P < 0.01, vs. CON group; *P < 0.05, **P < 0.01, vs. SCOP group. KXS: Kai Xin San; CON: vehicle control; SCOP: scopolamine; KXS-L: scopolamine + Kai Xin San (0.7 g/kg per day); KXS-M: scopolamine + Kai Xin San (1.4 g/kg per day); KXS-H: scopolamine + Kai Xin San (2.8 g/kg per day); DON: scopolamine + donepezil (3 mg/kg per day). d: Day.

Figure 5

KXS reverses neuronal degeneration. (A, B) Western blot assay results showed that KXS administration increased the expression of PSD95, SYN, BDNF in the hippocampus and cortex compared with the model (SCOP) group, respectively. Data are shown as the mean ± SEM (n = 8; one-way analysis of variance followed by Dunnett’s post hoc test). The experiment was performed in triplicate. #P < 0.05, ##P < 0.01, vs. CON group; *P < 0.05, **P < 0.01, vs. SCOP group. KXS: Kai Xin San; CON: vehicle control; SCOP: scopolamine; KXS-L: scopolamine + Kai Xin San (0.7 g/kg per day); KXS-M: scopolamine + Kai Xin San (1.4 g/kg per day); KXS-H: scopolamine + Kai Xin San (2.8 g/kg per day); DON: scopolamine + donepezil (3 mg/kg per day). PSD95: Postsynaptic density-95; SYN: synaptophysin; BDNF: brain-derived neurotrophic factor; d: days.

Figure 6

Effect of KXS on cholinergic neurotransmitters. Assay detection showed that KXS administration increased the expression of ChAT and ACh, decreased the expression of AChE in the hippocampus and cortex compared with the SCOP group. AChE activities (A, B), ChAT activities (C, D), and ACh levels (E, F). Data are shown as the mean ± SEM (n = 8; one-way analysis of variance followed by Dunnett’s post hoc test). The experiment was performed in triplicate. #P < 0.05, ##P < 0.01, vs. CON group. *P < 0.05, **P < 0.01, vs. SCOP group. KXS: Kai Xin San; CON: vehicle control; SCOP: scopolamine; KXS-L: scopolamine + Kai Xin San (0.7 g/kg per day); KXS-M: scopolamine + Kai Xin San (1.4 g/kg per day); KXS-H: scopolamine + Kai Xin San (2.8 g/kg per day); DON: scopolamine + donepezil (3 mg/kg per day). ChAT: Choline acetyltransferase; ACh: acetylcholine; d: days.

Figure 7

KXS decreases oxidative stress injury. Assay detection showed that KXS administration increased the expression of SOD and GSH-Px, decreased the expression of ROS and MDA in the hippocampus and cortex compared with the SCOP group. SOD (A, B), GSH-Px (C, D), ROS (E, F), and MDA (G, H). Data are shown as the mean ± SEM (n = 8; one-way analysis of variance followed by Dunnett’s post hoc test). The experiment was performed in triplicate. #P < 0.05, ##P < 0.01, vs. CON group. *P < 0.05, **P < 0.01, vs. SCOP group. KXS: Kai Xin San; CON: vehicle control; SCOP: scopolamine; KXS-L: scopolamine + Kai Xin San (0.7 g/kg/per day); KXS-M: scopolamine + Kai Xin San (1.4 g/kg per day); KXS-H: scopolamine +Kai Xin San (2.8 g/kg per day); DON: scopolamine + donepezil (3 mg/kg per day). SOD: Superoxide dismutase; GSH-Px: glutathione peroxidase; ROS: reactive oxygen species; MDA: malondialdehyde; d: day.