Shexiang Baoxin Pill, a Traditional Chinese Herbal Formula, Rescues the Cognitive Impairments in APP/PS1 Transgenic Mice

Abstract

Background: Shexiang Baoxin Pill (SBP), a formulated traditional Chinese medicine (TCM), has been widely used to treat cardiovascular diseases for years. This herbal mixture has been shown to promote differentiation of cultured neuronal cells. Here, we aimed to investigate the effects of SBP in attenuating cognitive impairment in APP/PS1 transgenic mice.

Methods: Ethanol and water extracts of SBP, denoted as SBP_EtOH and SBP_water, were standardized and applied onto cultured rat pheochromocytoma PC12 cells. The potential effect of SBP_EtOH extract in attenuating the cognitive impairments in APP/PS1 transgenic mice was shown by following lines of evidence: (i) inhibition of Aβ fibril formation, (ii) suppression of secretions of cytokines, and (iii) improvement of behavioral tests by Morris water maze.

Results: SBP_water and SBP_EtOH inhibited the formation of β-amyloid fibrils and protected the Aβ-induced cytotoxicity in cultured PC12 cells. In APP/PS1 transgenic mice, the treatment of SBP_EtOH inhibited expressions of NO, NOS, AChE, as well as aggregation of Aβ. Besides, the levels of pro-inflammatory cytokines were suppressed by SBP treatment in the transgenic mice. Importantly, the behavioral tests by Morris Water maze indicated that SBP attenuated cognitive impairments in APP/PS1 transgenic mice.

Conclusion: The current result has supported the notion that SPB might ameliorate the cognitive impairment through multiple targets, suggesting that SBP could be considered as a promising anti-AD agent.

Keywords: Alzheimer’s disease; Chinese medicine; Shexiang Baoxin Pill; acetylcholinesterase; cognitive enhancing effect; neuroprotective.

Figure 1

SBP suppresses Aβ fibril formation and attenuated Aβ-induced toxicity in PC12 cells. (A) HFIP-reconstituted Aβ monomers (10 μM) were incubated at 37°C for 6 days with or without drugs (i.e. curcumin, SBP_EtOH or SBP_water, and other seven individual herbal materials). SBP_EtOH, ethanol extracts of various herbal materials, and Styrax solution were used at 100 μg/ml; and SBP_water, water extracts of various herbal materials, solutions of Ginseng Radix et Rhizoma extract and Borneolum Syntheticum were used at 500 μg/ml. Curcumin at 30 μM served as a positive control. The extent of Aβ aggregation was determined using ThT fluorescence assay. Data are expressed as Mean ± SEM of the percentage of control (no drug treatment), where n = 4; p < 0.05 (*); p < 0.01 (**); p < 0.001 (***) vs control group. p < 0.05 (^#) vs SBP_EtOH group. (B) Inhibition of cytotoxicity in PC12 cells by drug-modified Aβ_1-42 aggregates. Aβ_1-42 (15 μM) was incubated at 37°C for 6 days in the presence or absence of drugs (i.e. curcumin, SBP_EtOH or SBP_water, and other seven individual herbal materials), and then treated to PC12 cells for 48 h. The doses were in (A). MTT assay was performed to determine the cell viability. Data are expressed as Mean ± SEM of the percentage of control (untreated culture), where n = 4; p < 0.05 (*); p < 0.01 (**); p < 0.001 (***) vs Aβ_1-42-treated group.

Figure 2

The schedule of animal experiment. (A) The APP/PS1 transgenic mice and B6C3 wild type mice (all male at 5-month-old) were fed with standard food and water. The transgenic mice were randomly divided into five groups, i.e. model group, donepezil (2 mg/kg/day), SBP_EtOH (95% ethanol extract of SBP) low-dosage group (22.5 mg/kg/day), SBP_EtOH high-dosage group (45 mg/kg/day). Age-matched male wild-type (WT) B6C3 mice were used as control group (n = 6). In APP/PS1 mice model group and wild type mice group, the mice were injected with the corresponding volume of saline. Donepezil, dissolved in saline, and the SBP extract (2% DMSO, 6% cremophor EL, 92% NaCl) were administered intra-gastrically once per day starting on Day 1. Drug treatment lasted for 60 days. Behavioral test was started on Day 56. (B) The treatment of mice with herbal extracts were according to the protocol in Figure 2A. The latency to find a hidden platform during 4 consecutive days of training was determined in Morris water maze. (C) The total distance of swimming-tracking path during 4 consecutive days of training was determined. (D) The time near the wall spent in target quadrant (southeast, where the platform was placed during the training phase) during 4 consecutive days of training was determined. (E) The swim velocity during 4 consecutive days of training was determined. Values were expressed as mean ± SEM, where n = 6; p < 0.05 (^#); p < 0.01 (^##); p < 0.001 (^###) vs control group. *p < 0.05; **p < 0.01; ***p < 0.001 vs model group.

Figure 3

SBP rescues memory deficits in APP/PS1 transgenic mice. (A) The treatment of mice with herbal extracts were according to the protocol in Figure 2 . The typical swimming-tracking path on the fifth probe trial day was determined. The red square represents the place where the animal is being placed at the beginning of water maze test. The blue square represents the place where the animal is stopped at the end of water maze test. The blue circle represents the place where the platform is placed before water maze test. (B) the distance in target quadrant (southeast, where the platform had been placed during the training phase) in the probe trial (swimming 60 s without platform) was determined. (C) the time spent in the target quadrant was determined. (D) the number of target crossings in the target quadrant was determined. Values were expressed as mean ± SEM, where n = 6; p < 0.05 (^#) vs control group. *p < 0.05; **p < 0.01 vs model group.

Figure 4

SBP reduces Aβ expression in brain tissue. Brain tissues from drug-treated mice were collected from mice as described in Figure 2 . Brain tissues were immuno-stained with anti-Aβ antibody. The cerebral cortex was selected for analysis, as indicated Supplementary Figure 2 . Taupe area (star) represented the antibody staining. ImageJ software was used to do the quantification of Aβ plaque area. The control group was untreated wild type mice. Data are in Mean ± SEM of the Aβ density in terms of percentage of total area per view, where n = 6; p < 0.05 (*); p < 0.01 (**) vs model group. Bar = 100 μm.

Figure 5

SBP protects neurons from nuclear pyknosis in APP/PS1 transgenic mice. Brain tissues from drug-treated mice were collected from mice as described in Figure 2 . The hippocampus was selected for analysis, as indicated Supplementary Figure 2 . Brain tissues were collected and cut into sections at 4 μM thick. The sections were stained with hematoxylin and eosin stains. Typical pictures focusing the hippocampus were shown, in which the nucleus was blue, the cytoplasm was pink, and the red blood cells were bright red (left panel). The number of neurons having nuclear pyknosis (denoted as star for some but not all, for clarity) was determined under light microscope using Image proplus program (right panel). Results are expressed as the fold of change as compared to control (X Basal; untreated wild type mice), where the control was set as 1, Mean ± SEM, where n = 6; p < 0.01 (**) vs model group. Bar = 50 μm.

Figure 6

SBP promotes expressions of NO, NOS, AChE, and cytokine in APP/PS1 transgenic mice. Treatment of mice was as described in Figure 2 . For determination of levels of NO, NOS, and AChE, 0.1 g of brain tissue was homogenized using 0.9 ml of pre-cold saline. After reacted on ice for 15 min, the homogenate was centrifuged at 2,500 rpm/min for 10 min at 4°C, and the supernatants were used to measure the levels of NO, NOS, and AChE with application of corresponding kits according to the manufacturer’s instructions. Blood was collected for the measurement of the amounts of IL-6 and TNF-α. The blood was centrifuged at 3,500 rpm/min for 10 min, and the serum was obtained by discarding the precipitant. The levels of IL-6 and TNF-α in the serum was further determined using commercially available ELISA kits. Data are expressed as Mean ± SEM of the percentage of change as compared with control (untreated wild type mice), where n = 6; p < 0.05 (*); p < 0.01 (**); p < 0.001 (***) vs control group. p < 0.05 (#); p < 0.01 (##); p < 0.001 (###) vs model group.

Figure 7

SBP promotes neuroprotective effects via anti-apoptotic pathway. Treatment of mice was as described in Figure 2 . (A) Effects of SBP on the mRNA expressions of Bax and Bcl-2 in the brains in APP/PS1 transgenic mice. Data are expressed as Mean ± SEM of the percentage of change (up or down regulated) as compared with control (untreated wild type mice), where n = 6; p < 0.01 (^##) vs control group; p < 0.05 (*); p < 0.01 (**) model group. (B) Expression of β-Amyloid (~87 kDa), Bax (~21 kDa), and Bcl-2 (~26 kDa) in brain tissues of drug-treated mice (left panel). Expression of GAPDH (~36 kDa) served as a control. Quantitation of protein expression is shown (right panel). Results are expressed as the fold of change as compared to control (X Basal; untreated wild type mice), where the control was set as 1, Mean ± SEM, where p < 0.01 (^##) vs control group; n = 4; p < 0.05 (*); p < 0.01 (**) vs model group.