Qingxin Kaiqiao Recipe Improves Cognitive Performance, Inhibits Apoptosis, and Reduces Pathological Deposits in APP/PS1 Double Transgenic Mice via the PI3K/Akt Pathway

The traditional Chinese medicine of Qingxin Kaiqiao Recipe (QKR) is effective in the treatment of Alzheimer's disease (AD). This study aims to investigate whether QKR improves the cognitive ability and takes neuroprotective effect on APP/PS1 double transgenic mice via the PI3K/Akt pathway. APP/PS1 double transgenic mice were randomly divided into a model, donepezil-treated, or QKR-treated group (L-QKR: 4.75 mg/kg/d, M-QKR: 9.5 mg/kg/d, and H-QKR: 19 mg/kg/d, respectively). Wild-type C57/BL6J mice were used as the control group. Morris water maze (MWM) was used to test the ability of spatial navigation and memorization; terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay was applied to test the apoptosis; amyloid protein granule deposition was detected via Methenamine silver staining; Western blot (WB) analysis, immunohistochemistry, and RT-PCR were applied to measure the expression of Aβ and corresponding indicators of the PI3K/Akt pathway. Compared with the model group, QKR significantly relieved the cognitive impairment, reduced the deposition of senile plaques, decreased the expression of GSK-3α and Aβ, and increased the expression of p-PI3K, p-Akt, and IDE. In addition, the number of TUNEL-positive cells decreased after treatment using QKR. The current study proved that QKR, especially at the high dose tested, exerted a protective effect on improving learning and memory, inhibiting apoptosis, and reducing the process of pathological degeneration in the hippocampus of AD mice.


Introduction
Alzheimer's disease (AD), the most common cause of dementia, is characterized by the progressive development of cognitive dysfunction syndrome, including impairment of language, visual space, and memory function to varying degrees [1,2]. According to a report by the Alzheimer's Disease Association in 2019, with the extension of people's life expectancy, the risk of AD among people over 65 years of age has increased to 1 in 10, and that among people over 85 years old has increased to nearly 1 in 2 [3], which imposes a huge burden on both families and society. e main pathological features of AD currently known are neurofibrillary tangles (NFTs), neuronal death, and senile plaque (SP) [4]. e major component of SP is amyloidbeta (Aβ), whose abnormal aggregation and deposition in the brain can induce a series of neurotoxic effects leading to the occurrence of AD, such as neuronal cell death, synaptic loss, and cognition impairments [5,6]. Based on the existing studies, the metabolism of Aβ can be influenced by the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway [7], which is related to a variety of intracellular regulation and is one of the key pathways for cell cycle regulation and apoptosis [8]. At present, the main methods of treating AD at home and abroad are western medicine and rehabilitation [9]; the former causes undesirable side effects, and the latter is costly and unstable. Given the relative poverty status of the conventional therapy, more and more researchers turn their attention to the traditional Chinese medicine [10,11]. Qingxin Kaiqiao Recipe (QKR), based on Fumanjian, a notable traditional Chinese medicine compound from Jingyue Quanshu written by Zhang Jingyue in Ming Dynasty, has been widely used in clinic for many years and has produced remarkable effects. QKR is usually used to treat the combination of excess and deficiency syndrome, which can manifest as follows: (1) Qi stagnation (mild disorders of behavior and language, sentimentality, chest oppression, and taut pulse).

Experimental Schedule.
e 3-month-old APP/PS1 mice were randomly divided into 5 groups by the random number table method (model group, n � 20; donepezil group: 1.67 mg/kg/d, n � 20; high dose of QKR: 19 mg/kg/d, n � 20; medium dose of QKR: 9.5 mg/kg/d, n � 20; low dose of QKR: 4.75 mg/kg/d, n � 20), and then administered with abovementioned medicines via oral gavage. Wild-type C57/BL6J mice were used as a normal control (n � 20) throughout the study and were given the same volume of saline water (0.2 mL/10 g weight). Each group was treated once a day at 10:00 am for a period of 3 months (Figure 1).

Behavioral
Test. Spatial learning and memory of mice was assessed in Morris water maze (MWM) as published previously by two investigators completely blind to the treatment of the animals [13]. e 90 cm diameter pool was divided into four equal quadrants (South, East, North, and West), and it was filled with water maintained at 24°C with a hidden platform (10 cm diameter and 1.5 cm below the surface of water) located in the middle of the third quadrant. e test was performed for 6 days, once per day. In the first five days, each mouse was placed randomly into the water at one of the four quadrants, facing the wall of the pool. e route of navigation was recorded by the camera above the pool and analysed by the MWM system. When finding the platform within 60 s, the mouse was allowed to stand on the platform for 10 s. If not, it was placed on the platform by investigators for 10 s. e 6th day was for spatial probe, investigators removed the platform and placed the mice into the pool to swim freely for 60 s. e number of times they crossed the original platform within 60 s was recorded by the camera.

Hippocampus Collection.
In order to conduct different experiments, we divided mice in each group into two parts after the MWM test. Five mice selected at random in each group were anesthetized with an intraperitoneal injection of 10% chloral hydrate; the brains were removed quickly and placed in 4% paraformaldehyde, and the tissues were fixed in the 4% paraformaldehyde for 24 h, and then, dehydrated by passage through a gradient alcohol dehydration at room temperature. e tissues, transparentized with xylene, were then soaked in wax, embedded in paraffin, and finally, cut into sections of 5 μm thick. e rest of mice (n � 15/group) were killed after anaesthesia; the hippocampus was dissected under a dissecting microscope, placed in the EP tube, and stored at −80°C refrigerator.

TUNEL Staining.
TUNEL technique was performed according to the manufacturer's protocol supplied within the TUNEL pod kit (Roche, USA). e dewaxed slices were placed into 3% hydrogen peroxide in methanol for 15 min and were added 100 μl protease K (20 μg/ml) for 20 min. After washing with PBS, TUNEL reaction solution was added to the slices at 37°C for 1 h, and then, the POD was added to the slices. Finally, the slices were rinsed 3 times and stained with DAB and hematoxylin. e number of viable TUNEL-positive neurons in the hippocampus was observed by an Olympus fluorescence positive microscope.

Methenamine Silver Staining.
After the slices were conventionally dewaxed, 0.5% periodate and 8% chromic acid were added for 15 min and 60 min, respectively. en, the slices were washed with distilled water for 5 min, placed into preheated methenamine silver (Leagene, Beijing), and kept in an incubator at 60°C for 45 min. Finally, the slices were washed and added 1% gold chloride aqueous solution to tint for 2 min. e positive granules were calculated in Image-Pro Plus 6.0 (IPP 6.0).

Western Blot Analysis.
e frozen hippocampal tissues were taken out from −80°C refrigerator and lysed in RIPA lysis buffer containing a protease inhibitor cocktail. After calculated by the BCA protein assay kit, the total protein was separated by 6% or 8% SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. e membranes were blocked in a skim milk blocking buffer for 2 h,  e bands were detected by the chemiluminescence reagents ECL Western blotting detection reagents (USA) and finally analysed by AlphaEase FC gel image analysis software.

Quantitative Real-Time PCR.
Total RNA from hippocampus was isolated from TRIzol reagent according to the manufacturer's procedures. e absorbance values of RNA at OD 260 and OD 280 as well as the RNA content were detected by an M200 pro full-wave length multifunction microplate reader (Tecan, Switzerland). e cDNA was synthesized with reverse transcriptase ( ermo, USA). e quantitative polymerase chain reaction (qPCR), which includes 0.5 μl of each primer, 1 μl of the cDNA sample, 5 μl of the SYBR Green I Master, and 3 μl of the DEPC water, were performed in LightCycler ® 480II/96 (Roche, Switzerland). e reaction was set as following: 95°C for 4 min, followed by 35 cycles of 95°C for 10 s, 54°C for 10 s, and 72°C for 10 s, the signal was detected at 72°C, and each PCR reaction was performed in duplicate. Finally, the results were analysed by the 2 −△△Ct method (Table 1).

Statistical
Analysis. All data were processed using SPSS 18.0 statistical software and presented as the mean ± standard deviation. e data of each group were compared by the  Evidence-Based Complementary and Alternative Medicine 3 independent samples t-test and one-way ANOVA. Intergroup comparison was performed by the LSD method. In all analyses, P < 0.05 was taken to indicate statistical significance.

Behavioral Test Results.
In the MWM test, compared with the control group, the path tracking of APP/PS1 mice was disorganized. e QKR-treated group, especially the group of H-QKR, showed a selective search way for the platform and shorter path length than the model group did (Figure 2(a)). e average escape latency of 5 consecutive days of each group was displayed in curves and histogram (Figures 2(b) and 2(c)).
ere was no clear distinction among different groups in the first two days. However, from the third day onwards, the differences became more and more obvious. e model group maintained a longest escape route from start to finish, while the control group showed the shortest one on each day. e latency in the donepezil and H-QKR groups presented significant differences compared with that in the model group from the 3rd to 5th day (P < 0.01). e above results of the navigation tests with hidden platform were supported by a subsequent probe trial without the platform. e typical path tracking of each group within 60 s was shown in Figure 2(d). Within the similar total path length, the model group swam randomly throughout the tank. Compared with the model group, the H-QKR group and donepezil groups had more cross-platform locations and higher percentage of target quadrant searching time (P < 0.01) (Figures 2(e) and 2(f )). ese results provided evidence on the significant compensating effect of H-QKR on cognitive deficits. Furthermore, to exclude the possibility that the above results were due to visual or motor impairment, the swimming speed of mice was measured using a visible platform after the probe test. As shown in Figure 2(g), there were no statistically significant differences in average swimming speed among groups (P > 0.05).

TUNEL Staining Results.
e apoptotic cells stained by TUNEL staining were brown, the nucleus was blue, and the background was light blue. e results showed that the apoptosis in the model group was quite obvious, compared with other groups (P < 0.01), but the situation got better after treatment with donepezil or QKR, especially the donepezil group, which showed the most palpable effect in the treatment groups (P < 0.01). In addition, H-QKR produced the strongest effect among the three doses of QKR groups (P < 0.01) (Figure 3).

Methenamine Silver Granule Staining
Results. Senile plaques were black with a light brown background. Compared with other groups, the number of positive plaques in the hippocampus of mice in the model group increased significantly (P < 0.01). After treatment with donepezil or QKR, the number of positive plaques became fewer; however, the effect of the donepezil group was better than that of QKR groups (P < 0.01).
ere was also a significant difference in the number of senile plaques between the high dose of the QKR group and the medium and low dose of QKR groups (P < 0.01) (Figure 4).

Protein Levels of PI3K, p-PI3K, Akt, p-Akt, GSK-3α, IDE, and Aβ in the Hippocampus.
e expression levels of Aβ and GSK-3α protein in the model group were higher than that in other groups (P < 0.05, P < 0.01), while the levels of p-PI3K, p-Akt, and IDE were lower than that in the remaining groups (P < 0.01). e dose of 19 mg/kg/d QKR group produced the strongest effect among the three doses of QKR groups, which exhibited the highest expression levels of p-PI3K, p-AKT, and IDE protein (P < 0.01) and the lowest expression levels of GSK-3α and Aβ protein (P < 0.01); however, there were no evident differences in the expression of PI3K and Akt protein among QKR groups (P > 0.05) ( Figure 5).

Immunohistochemistry Results.
After immunohistochemical staining, the positive cells were stained brown with a light blue or brown-grey background. e results showed that the expression levels of Aβ in the model group were significantly higher than that in other groups, and the expression levels of PI3K, p-PI3K, AKT, and p-AKT in the model group was much lower than the remaining groups (P < 0.05, P < 0.01). Although the medication group did not achieve the effect of the normal group, the results were improved after treatment with donepezil or QKR. Among them, the donepezil group was the most effective one, followed by the H-QKR group ( Figure 6).

Gene Expression of PI3K, Akt, and Aβ in the Hippocampus.
e expression levels of Aβ in the model group was the highest among all groups (P < 0.05, P < 0.01); meanwhile, the model group showed the lowest expression levels of PI3K and Akt (P < 0.05, P < 0.01). After treatment with QKR or donepezil, the expression of Aβ decreased to varying degrees, and the expression of PI3K and Akt increased to different levels ( Figure 7).

Discussion
As a major component of senile plaques, Aβ peptide causes neurotoxicity and plays a key role in the pathogenesis of AD  Evidence-Based Complementary and Alternative Medicine [14,15]. e accumulation of Aβ can activate apoptosis and result in neuron cell death [16]. It was demonstrated that the level of Aβ is increased in AD brains and is related to the severity of the disease [17]. A classical AD model, APP/PS1 double transgenic mice were used in this research. e structure of the mice was based on Aβ pathology, with learning and memory impairment and detectable Aβ in the brain at 4 months old and significant amyloidosis at 6 months of age. And three-month-old mice usually have shown mild cognitive impairment accompanied by mild Aβ deposition [18,19]. From the age of 3 months, the mice were treated with diluted QKR extract for 3 months. It was exciting to see from the results of MWM and silver granule staining that after 3 months of QKR application, the cognitive performance of QKR-treated AD mice was improved compared with the model group, and the number of senile plaques was significantly reduced, indicating its role in improving cognition ability and reducing Aβ deposition.
QKR is a formula based on Fu Man Jian, a famous decoction composed of 10 traditional Chinese herbs. e major effective chemical compounds are summarized as follows: (1) Rhizoma Acori Tatarinowii (Shi chang pu):  asarone is one of the major ingredients of Rhizoma Acori Tatarinowii. Asarone and levodopa coadministration increase the striatal dopamine level in 6-hydroxydopamineinduced rats by modulating P-glycoprotein and tight junction proteins at the blood-brain barrier (BBB) and promoting levodopa into the brain [20]. (2) [24]. erefore, the effect of QKR may be related to its effective chemical components which can penetrate the bloodbrain barrier and finally enter the brain. Our previous in vivo experiments have proved that QKR could exert neuroprotection in AD mice by inhibiting apoptosis via multiple pathways. QKR could affect apoptosis-related protein expression, such as stimulation of antiapoptotic protein regulator Bcl-2 and inhibition of proapoptotic protein regulator Bax and caspase-3 [25]. Existing studies have reported that the PI3K/Akt pathway is involved in the activity of different kinds of cells, including the hippocampal neuronal cells [26,27]. Akt is a crucial downstream effector of phosphatidylinositol 3-kinase (PI3K), and its phosphorylation is essential in cell proliferation and cell death [28]. When the Akt is activated by the PI3K pathway, it can lead to phosphorylation of glycogen synthesis kinase-3α (GSK-3α), thus inhibiting the activity of GSK-3α and then suppressing the formation of Aβ by interfering with the activity of secretase [29,30]. Besides, the PI3K/Akt pathway may also be involved in the regulation of IDE activity, an important Aβ degrading enzyme, thereby affecting the degradation of extracellular Aβ [31,32]. It could be seen from the results of WB analysis that QKR-treated AD mice showed higher levels of p-PI3K, p-Akt, and IDE and lower levels of GSK-3α and Aβ than the model group. e results of immunohistochemistry and RT-PCR showed that the QKR-treated AD mice showed higher levels of p-PI3K and p-Akt and lower levels of Aβ than the model group, indicating the role of QKR in increasing the activity of the PI3K/Akt pathway. Furthermore, the TUNEL results revealed that after QKR treatment, there were fewer apoptotic cells in the AD mice, which unfolded an antiapoptotic role of QKR.
To sum up, QKR has a good effect on treating AD. e present experiment showed that the 19 mg/kg/d QKR (H-QKR) group was the most effective one among the three doses of QKR groups. However, whether there exists a higher dose of QKR that produce more effective role remains to be further explored. e effect of QKR may be related to its effective chemical components which can penetrate the blood-brain barrier and finally enter the brain. We will perform HPLC-fingerprint analysis in our next experiment to explore the mechanism of QKR. Moreover, an inhibitor of PI3K/Akt pathway needs to be applied in the future to better prove that QKR can indeed work through this pathway.

Conclusion
QKR may slow down the progress of AD by alleviating the level of Aβ, improving the cognitive function, and inhibiting apoptosis in the APP/PS1 double transgenic mice via the PI3K/Akt pathway, especially the high dose group of 19 mg/ kg/d, which showed almost similar effects with the positive control group of donepezil, an drug with clinical validity certification.
Data Availability e basic studying data used to support the findings of this study were supplied by Dr. Tianqi Wang under license and so cannot be made freely available. Requests for access to these data should be made to Dr. Tianqi Wang (tenkiou@ foxmail.com).

Conflicts of Interest
e authors declare that there are no conflicts of interest.