Korean traditional herbal formula Soshiho-tang attenuates memory impairment and neuronal damage in mice with amyloid-beta-induced Alzheimer's disease

Abstract

Background: Soshiho-tang (SST), also known as Xiaochaihu-tang in China and Sho-saiko-to in Japan, is an Oriental herbal formula traditionally used to treat febrile diseases. Recently, several in vitro and in vivo studies have reported the anti-cancer, anti-liver disease, and anti-inflammatory activities of SST. However, there is little evidence of its effects on neurological diseases. We previously reported the inhibitory effects of SST on in vitro acetylcholinesterase (AChE) activation and amyloid-β (Aβ) aggregation, which are crucial hallmarks of Alzheimer's disease (AD). In the present study, we report that SST has preventive effects on memory impairment and neuronal cell changes in an Aβ-induced AD-like mouse model.

Methods: Male mice underwent injection of Aβ aggregates and administered SST (500, 1,000, or 2,000 mg/kg/day) for 20 days. Behavioral tests (passive avoidance task [PAT] and Morris water maze [MWM] test) were conducted. Lastly, brain sections were obtained from sacrificed mice for quantitative analysis.

Results: Intracerebroventricular (ICV) injection of Aβ aggregates significantly decreased the latency time in the PAT and MWM test compared to normal control. In contrast, SST administration markedly reversed the latency caused by Aβ injection. Additionally, our data revealed that SST-mediated improvements in memory impairment are related to its neuroprotective and anti-neuroinflammatory effects. On histological analysis, SST treatment protected neuronal loss and damage as well as microglial activation, and ameliorated amount of Aβ in brain of mouse model of AD.

Conclusion: Our findings suggest that SST may be a promising candidate for the development of novel drugs for AD.

Keywords: Alzheimer's disease; Anti-neuroinflammation; Memory improvement; Neuroprotection; Soshiho-tang.

Fig. 1

HPLC chromatograms of the ethanol extract of Soshiho-tang (SST) and standard mixture. (A) The five standard compounds liquiritin (a), baicalin (b), baicalein (c), wogonin (d), and glycyrrhizin (e) were separated by a Gemini C₁₈ analytical column at 30°C at 275 or 254 nm. (B) Chemical structures of the standard compounds of SST. Liquiritin (a), baicalin (b), baicalein (c), wogonin (d), glycyrrhizin (e).

Fig. 2

Establishment of the Aβ-induced AD mouse model. (A) Experimental timeline of Aβ injection into the brain and SST administration. Aβ aggregates were prepared and injected in the intracerebroventricular (ICV) area of male C57BL6 mice with a stereotaxic apparatus. One day after Aβ injection, SST extracts were orally administered at 500, 1,000, or 2,000 mg/kg/day for 20 days. Passive avoidance task (PAT) and Morris water maze (MWM) tests were performed between the 8^th-10^th day and 15^th-21^st day, respectively. Morin was used as a positive control of Aβ inhibition. (B) Mean body weight changes of the experimental mice during the three weeks of schedule. NOR: normal group, Aβ: Aβ-injected group, SST-0.5, -1, or -2: 500, 1,000, or 2,000 mg/kg of SST-treated groups into Aβ mice, and M: morin (10 mg/kg)-treated group into Aβ mice.

Fig. 3

Effects of Soshiho-tang (SST) on memory impairment in an Aβ-induced AD mouse model using passive avoidance task. The passive avoidance task was performed on the 8^th to 10^th day after Aβ injection with or without SST administration. Transfer latency time was recorded as the time that mice remained in the lighted compartment within a 5 min period. The results are presented as the mean ± SD (n=9/group). ^⁎⁎p<0.01 vs NOR group and ^#p<0.05 vs Aβ group. NOR: normal group, Aβ: Aβ-injected group, SST-0.5, -1, or -2: 500, 1,000, or 2,000 mg/kg of SST-treated groups into Aβ mice, respectively, and M: morin (10 mg/kg)-treated group into Aβ mice.

Fig. 4

Effects of Soshiho-tang (SST) on memory impairment in an Aβ-induced AD mouse model using the Morris water maze (MWM) test. The MWM test was performed on the 15^th to 21^st day after the injection of Aβ. (A) The time to reach the platform during training trials (escape latency). (B) The swimming distance traveled to the platform in the training trials. (C) The number of crossings of the platform area in the spatial probe trial. The results are presented as the mean ± SD (n=9/group). *p<0.05 or ^⁎⁎p<0.01 vs NOR group, and ^#p<0.05 or ^##p<0.01 vs Aβ group. (D) Representative swimming traces of mice in the spatial probe trial test. NOR: normal group, Aβ: Aβ-injected group, SST-0.5, -1, or -2: 500, 1,000, or 2,000 mg/kg of SST-treated groups into Aβ mice, and M: morin (10 mg/kg)-treated group into Aβ mice.

Fig. 5

Effects of Soshiho-tang (SST) on neuronal loss in the brain tissues of Aβ-induced AD mice. (A) Multiple 5-μm paraffin sections of the hippocampus and cortical regions were prepared from the sacrificed mouse brains. Nissl staining was conducted using cresyl violet solution. Representative photomicrographs are shown at magnification of x400. (B and C) Graphs display the quantitative analysis of Nissl stained cell bodies in hippocampus (B) and cortex (C). The results are presented as the mean ± SD (n=3/group). ^⁎⁎p<0.01 vs NOR group and ^#p<0.05 vs Aβ group. NOR: normal group, Aβ: Aβ-injected group, SST-0.5, -1, or -2: 500, 1,000, or 2,000 mg/kg of SST-treated groups into Aβ mice, respectively, and M: morin (10 mg/kg)-treated group into Aβ mice.

Fig. 6

Effects of Soshiho-tang (SST) on NeuN in the brain tissues of Aβ-induced AD mice. (A) Multiple 5-μm paraffin sections of hippocampus and cortical regions were prepared from the sacrificed mouse brains. The expression of NeuN, a neuronal marker, was determined by immunohistochemistry at a magnification of x400. (B and C) Graphs display the quantitative analysis of NeuN positive cells in the hippocampus (B) and cortex (C). The staining intensities are reported in arbitrary units (AU). The results are presented as the mean ± SD (n=3/group). ^⁎⁎p<0.01 vs NOR group and ^#p<0.05 vs Aβ group. NOR: normal group, Aβ: Aβ-injected group, SST-0.5, -1, or -2: 500, 1,000, or 2,000 mg/kg of SST-treated groups into Aβ mice, respectively, and M: morin (10 mg/kg)-treated group into Aβ mice.

Fig. 7

Effects of Soshiho-tang (SST) on microglial activation in brain tissues of Aβ-induced AD mice. (A) Multiple 5-μm paraffin sections of hippocampus and cortical regions were prepared from the sacrificed mouse brains. The expression of Iba-1, a microglial activation marker, was determined by immunohistochemistry at a magnification of x400. (B and C) Graphs display the quantitative analysis of Iba-1 positive cells in the hippocampus (B) and cortex (C). The staining intensities are reported in arbitrary units (AU). The results are presented as the mean ± SD (n=3/group). ^⁎⁎p<0.01 vs NOR group and ^#p<0.05 vs Aβ group. NOR: normal group, Aβ: Aβ-injected group, SST-0.5, -1, or -2: 500, 1,000, or 2,000 mg/kg of SST-treated groups into Aβ mice, respectively, and M: morin (10 mg/kg)-treated group into Aβ mice.

Fig. 8

Inhibitory effects of Soshiho-tang (SST) on the Aβ deposition and of AChE activity in Aβ-induced AD mice. (A) AChE activity in mouse brain lysates were measured according to the commercial manufacture's protocol using an Acetylcholinesterase Activity Assay Kit (Biomax, Seoul, Korea). (B) Multiple 5-μm paraffin sections of hippocampus and cortical regions were prepared from the sacrificed mouse brains. The expression of Aβ was determined by immunohistochemistry at a magnification of x400. (C and D) Graphs display the quantitative analysis of Aβ-positive cells in the hippocampus (C) and cortex (D). The staining intensities are reported in arbitrary units (AU). The results are presented as the mean ± SD (n=3/group). ^⁎⁎p<0.01 vs NOR group and ^#p<0.05 vs Aβ group. NOR: normal group, Aβ: Aβ-injected group, SST-0.5, -1, or -2: 500, 1,000, or 2,000 mg/kg of SST-treated groups into Aβ mice, respectively, and M: morin (10 mg/kg)-treated group into Aβ mice.