Yangxue Qingnao is a widely applied Chinese traditional patent medicine, consisting of 11 active components, including Angelica sinensis, Ligusticum chuanxiong hort, White peony root, Prepared radix rehmanniae, Uncaria, Spatholobus suberectus, Prunella vulgaris, Cassia seed, Nacre, Rhizoma corydalis, and Asarum, effects of which are shown in Table 1. YXQN extract was provided by the TIANJIN TASLY Pharmaceutical co., LTD, the processing of the product followed strict quality control, and the ingredients were subjected to standardization. YXQN extract was dissolved in distilled water at 0.069 g, 0.208 g, and 0.624 g per mL for use. Positive control Aricept donepezil (HCl salt) tablets (commonly referred to as donepezil; Eisai (China) Pharmaceutical co., LTD) was prepared with a concentration of 0.103 mg/mL for use. The diluted YXQN and donepezil were used for oral administration by 0.1 mL/10 g of weight in mice. In all, the drug dosages are YXQN low-dose at 0.69 g/kg, YXQN middle-dose at 2.08 g/kg, YXQN high-dose at 6.24 g/kg, and donepezil at 1.03 mg/kg. Besides, the dosages of YXQN middle-dose and donepezil are equal with clinical application doses for patients in Pharmacology.

Amyloid precursor protein/presenilin 1 mice (B6C3-Tg (APPswe, PSEN1dE9) 85Dbo/J) were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). This study was carried out in accordance with the recommendations of the Chinese Council on Animal Care Guidelines, the Model Animal Research Center of Nanjing University. The protocol was approved by the Model Animal Research Center of Nanjing University.

In the AD model of APP/PS1 mice, degenerate cognitive function and Aβ deposits could be observed at 6–7 months (Jankowsky et al., 2004). Thus, we used equal numbers of female and male APP/PS1 mice at age of 8 months to explore the effects of YXQN and assessed AD pathology at the age of 10 months after 2 months administration. After 1 week of acclimatization to the cages, the 8-month APP/PS1 mice were randomly divided into five groups (vehicle group, n = 16; YXQN low-dose group, n = 17; YXQN middle-dose group, n = 18; YXQN high-dose group, n = 18; donepezil group, n = 16) and then orally administered with attenuated donepezil, YXQN (low-, middle-, and high-dose), or water (0.1 mL/10 g weight) for 2 months. Littermates were used as WT vehicle control (n = 16) throughout the study and were given distilled water for 2 months as well. Mice were housed in standard laboratory cages with a 12 h light and dark cycle along with free access to food and water.

The experimental design of behavior and biochemical analysis is shown in Figure 1. During the 2-month administration period, cognitive function of the five groups of APP/PS1 mice was measured by Y-maze at 30th and 60th day, and measured by Morris Water Maze (MWM) at the termination of drug supplementation. After that, a set of the biochemical index in the brain was investigated. Therefore, all AD pathological indices in this research were determined in 10-month-old APP/PS1 mice.

The short-term spatial memory ability for five groups of APP/PS1 mice was tested by Y-maze spontaneous alternation. In brief, we placed mice separately into a radially symmetric Y-maze with three arms (arms: 40 cm long, 4 cm wide; walls: 30 cm tall). The number and sequence of arm entries were scored over 8 min. Alternations were calculated when a mouse consecutively traveled to all three arms in any order without re-entering the previous arms. Percent of alternation was formulated as the ratio of the number of alternations to the number of total arm entries minus two (Lalonde, 2002; Town et al., 2008).

The spatial learning-memory ability was assessed by the MWM tests, which consists of the orientation navigation tests and the spatial probe tests. Mainly, the MWM contains a circular tank (diameter: 120 cm) filled with water at 24°C and a hidden platform (diameter: 15 cm) positioned 1–2 cm below the opaque water in the middle of the northeast quadrant. Before the measurement, mice were trained to find the platform for 3 days, orienting by cues on the wall of the tank as spatial references (Vorhees and Williams, 2006). For orientation navigation tests, mice were allowed to search for the platform for 120 s and to stay on the submerged platform for 30 s, before they were placed back in the cage under a heater to dry. Mice were tested four times a day for six consecutive days. The escape latency and the swim path tracking until the mice landed on the platform were recorded on videotape. For the probe trials, which were performed to determine memory retention on the next day (day 7), the platform was removed, and mice were placed into the pool from the opposite quadrant where the platform had been located. They were allowed to swim for 120 s, and the number of platform crossings, the percent of time spent in each quadrant, and the swim path tracking were recorded on videotape.

After behavioral analysis, mice were euthanized with pentobarbital sodium and fixed in 4% paraformaldehyde after myocardial perfusion. Brains were dissected and embedded in paraffin for preparing sagittal sections and further staining analysis. The sections from each mouse were separately stained according to the following procedures:

For thioflavin-S staining, brain sections were stained with 0.01% thioflavin-S in 50% ethanol, and following differentiation in 50% ethanol (Heneka et al., 2013). Then, stained sections were analyzed with the Hg-Lamp for fluorescence excitation.

Congophilic amyloid staining by congo-red conformed to standard protocols (Vom Berg et al., 2012). Briefly, sections were incubated with 0.5% congo-red in 80% methanol and 20% glycerol for 20 min, and following differentiation with 0.2% KOH in 80% ethanol. Subsequently, nuclei were stained blue with hematoxylin.

In addition, brain sections were subjected to EnVision system immunohistochemistry to detect Aβ, ADAM10, BACE1, and PS1. The primary antibody 6E10 (Covance) was used for assessing Aβ deposition at 1:500 dilution. Moreover, the specific antibodies ADAM10, BACE1, and PS1 (Bioss) antibodies were used for staining and analyzing the expression of ADAM10, BACE1, and PS1 at 1:200 dilution.

Finally, all stained sections were analyzed using a BX41 or IX71 microscope (Olympus) and collected using DP Controller software. We performed the quantitative assessment of three defined regions per mouse brain. Plaque number and staining area were calculated by Image-Pro Plus 6.0 software. The staining area fraction was determined by dividing total plaque area by the area of the microscopic field.

The homogenized brain tissues were eluted by boiling in SDS-sample buffer. Then, we performed SDS-PAGE to assess brain proteins using the Bio-Rad mini gel system (Vom Berg et al., 2012). In particular, Aβ and APPct levels were measured with urea-based electrophoresis, and then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were then probed with antibodies at the appropriate dilutions, including Aβ and full-length APP (6E10, Covance) and APPct (A8717, Sigma–Aldrich) at 1:1000 (Sarajarvi et al., 2009; Heneka et al., 2013), ADAM10 (Bioss), BACE1 (Bioss), and PS1 (Bioss) at 1:500. We used β-actin (Abcam) as internal controls and Image-Pro Plus software for densitometric analysis.

For ELISA test of Aβ40 and Aβ42 peptides from soluble and insoluble fractions, we homogenized frozen cerebral hemispheres (150 mg/mL wet weight) in PBS containing 1% SDS with protease inhibitors (Roche), followed by centrifugation at 100,000 g for 60 min at 4°C (Kawarabayashi et al., 2001; Vom Berg et al., 2012). The supernatant was removed as the soluble fraction, and the pellets (insoluble fraction) were dissolved in 70% formic acid. Then the insoluble fraction was neutralized by 1 M Tris buffer (pH 11). We used the Human Amyloid-β (aa1-40) or (aa1-42) Quantikine ELISA Kit to analyze Aβ40 and Aβ42 according to the manufacturer’s instructions (R&D Systems).

Data presented as means ± standard errors of the mean (SEM). All quantitative results including histology staining, Western blot, ELISA, and behavioral tests were analyzed by ANOVA with Dunnet’s post hoc. All analyses were carried out using SPSS 17.0 statistics software (Chicago, IL, United States).

Utilizing 8 month old APP/PS1 mice and their littermates (WT), we evaluated the possible effects of YXQN on cognitive function of mice after 2 months of drug administration by two kinds of behavioral test, including MWM tests and Y-maze tests. Firstly, the APP/PS1 mice were administrated with vehicle, YXQN low-dose 0.69 g/kg, middle-dose 2.08 g/kg, and high-dose 6.24 g/kg (equivalent to 33, 100, and 300% clinical application dose), or donepezil 1.03 mg/kg (equivalent to 100% clinical application dose) per day from 8 to 10 months of age. At the time of termination of drug supplementation, mice were subjected to the navigation tests in the MWM to assess their spatial learning-memory formation. The search time to find the platform (escape latency) and path tracking were recorded. As shown in Figure 2A, compared with WT mice, the typical path tracking of the vehicle APP/PS1 mice was disorganized, indicating the mice searched for the hidden platform by a random trajectory. YXQN administration APP/PS1 mice showed shorter path lengths and selective search tracking, similar to WT mice. These data suggested an improvement in the spatial memory of AD mice from YXQN groups. Moreover, the average escape latencies of 6 consecutive days of each group were displayed in curves (Figure 2B). By statistics, escape latencies were demonstrated tend to decrease over time, and overall, latencies were significant different among groups (F = 23.475 day: p < 0.0001 group: p = 0.002; RM-ANOVA). The vehicle APP/PS1 showed the longest latencies, and the vehicle WT mice showed the shortest latencies on each day. Compared with vehicle AD mice, YXQN low-, middle-, and high-dose APP/PS1 mice and donepezil APP/PS1 mice showed different degrees of shortened latencies, and YXQN high-dose group most closely approached the latencies of the WT group, and presented significant differences with vehicle group on day 3 and 4 (F = 2.948 and 2.572; p = 0.036 and 0.036). Especially, on day 6, the latencies of YXQN low-, middle-, and high-dose APP/PS1 mice were remarkably shorter by 32.10, 36.06, and 40.32%, respectively, relative to vehicle APP/PS1mice. Next, for quantificational evaluation, the area under the escape latency curve were calculated, which represented the general cognitive level over six consecutive days. As shown in Figure 2C, compared to WT mice, the vehicle APP/PS1 mice presented a substantial increase in AuC-latency (F = 4.015; p < 0.0001), suggesting the declining spatial memory. The reduction in the escape latency was observed in the different dosages of YXQN and donepezil groups compared with the vehicle group. Particularly, the AuC-latency of the high-dose YXQN group showed the most significant decrease (F = 4.015; p = 0.011), approximating the AuC-latency of the WT group. Thus, YXQN, especially in high-dose, could substantially ameliorate the severe deficit in spatial and long-term memory formation in aged APP/PS1 mice.

The above results of the navigation tests with hidden platform were supported by a subsequent probe trial without the platform. Again, the typical path tracking of each group in 120 s was shown in Figure 2D. Within the similar total path length, the vehicle APP/PS1 mice swam randomly throughout the tank, indicating their poor memory retention of the location of the platform. However, the WT mice and also the YXQN administrated APP/PS1 mice used a spatially biased search strategy to locate the platform, indicating their good memory retention. Further, we calculated the platform location crossing times and the percent of target quadrant search time, both of which evinced the memory retention of the location where the hidden platform had been placed. The data suggested that there were more platform location crossing times and a higher percent of target quadrant search time in the YXQN high-dose group than the vehicle group in APP/PS1 mice (F = 2.300; p = 0.012; F = 2.779; p = 0.023). The platform location crossing times and percent of target quadrant search time of YXQN high-dose group were both approximate to the outcomes of WT group (F = 2.300; p = 0.001; F = 2.779; p = 0.010) (Figures 2E,F). These results provided evidence on the significant compensating effect of YXQN high-dose on cognitive deficits.

To further confirm the above results, the Y-maze alternation tests were performed for the detection of short-term spatial memory ability. As shown in Figure 3A, compared with the vehicle APP/PS1 group, the percentage alternation of YXQN high-dose showed a notable increase in the memory test after 1 month of drug treatment (F = 1.490; p = 0.024). When 2 months of the drug treatment finished, again the YXQN high-dose group showed the most significant effect on increasing alternation (F = 2.183; p = 0.015), implying the enhancement of short-term memory by YXQN high-dose treatment. Further, in Y-maze alternation tests, the amelioration of cognitive ability was independent of motor ability (Reiserer et al., 2007), as manifested by the unchanged numbers of arms entered in each group (Figure 3B).

Above all, our results indicated that YXQN significantly improved cognitive deficits of APP/PS1 mice in a dose-dependent manner. Remarkably, the effect of decreased cognitive impairment was greater in the YXQN middle- and high-dose groups than the donepezil group. Intriguingly, with YXQN high-dose supplemented for 2 months, the spatial short- and long-term memory formation and retention of the APP/PS1 mice was similar to that of the littermates WT mice, suggesting that YXQN counteracts cognitive decline in APP/PS1 mice.

Based on the fact that YXQN extract supplementation may ameliorate cognitive decline in the AD mouse model, we next investigated the core pathology of the AD-amyloid burden in APP/PS1 mice of each group. Firstly, using specific antibody 6E10, the Aβ plaques were stained in the sagittal brain sections of each group. As a result, substantial cerebral amyloidosis could be observed by Aβ-immunoreactive in vehicle APP/PS1 mice at 10 months of age (Figure 4A). However, YXQN low-, middle-, and high-dose groups all presented a forceful reduction in the Aβ deposition when compared with the vehicle group. The Aβ covered areas were reduced by approximately 50% in the APP/PS1 mice treated with the three dose levels of YXQN extract (F = 9.212; p < 0.0001 and p < 0.0001) (Figure 4C). Next, to corroborate the finding of the reduction of amyloid burden in YXQN groups, congo-red staining was performed, which specifically stains the amyloid plaque. Again, YXQN extract significantly decreased the Aβ burden in the brain of APP/PS1 mice (Figure 4B). Particularly, compared with the vehicle group, the YXQN high-dose group showed a 72% decrease in plaque covered area (F = 12.080; p = 0.002, p = 0.001, and p < 0.0001) (Figure 4D).

The hippocampus (HC), entorhinal cortex (EC), and cingulate cortex (CC) form the CC-EC-hippocampus, which is the most important element in a brain for organizing spatial memory and transforming short-term memory to long-term memory. This region is the first area to suffer damage in the brain of AD patients and is closely associated with neurodegeneration (Khan et al., 2014; Lopez et al., 2014; Chang et al., 2016). We therefore performed thioflavin-S staining on the brain sections to investigate whether YXQN administration alters Aβ deposition in the hippocampus and cortical areas, including the EC and the CC. Thioflavin-S is a kind of fluorochrome specifically binding to amyloid deposits, and can be excited to produce green fluorescence. As shown in Figure 5A, the YXQN middle- and high-dose groups both significantly decreased the amount of thioflavin-S positive plaques in the hippocampus, EC, and CC, compared with the vehicle group. By quantifying, YXQN middle- and high-dose groups, respectively, reduced the Aβ plaques by 32 and 44% in hippocampus areas (F = 5.938; p = 0.028 and p = 0.002) (Figure 5B), and also by 39 and 57% in the cortex areas, relative to the vehicle group (F = 15.051; p = 0.003 and p < 0.0001) (Figure 5C). Taken together, YXQN treatment groups had substantially reduced Aβ deposition in the hippocampus and cortical regions in a dose-dependent manner, suggesting that YXQN extract ameliorated cognitive impairment in MWM and Y-maze probably through reducing the Aβ deposition in the hippocampus and cortical areas of APP/PS1 mice.

Accumulating evidence indicates that soluble Aβ oligomers and Aβ fibrils participate in the pathological and cognitive symptoms of AD through different processes (Larson and Lesne, 2012; Zahs and Ashe, 2013). To define which kind of Aβ assemblies YXQN is involved in, we examined the amount of Aβ40 and Aβ42 in the soluble (SDS-soluble) or in the insoluble (formic acid-soluble) fractions in cerebral homogenate by ELISA. The majority of extracellular aggregations of Aβ40 and Aβ42 were detected in the formic acid-soluble fractions. Of the two, Aβ42 is the more amyloidogenic form of the peptide, due to its more hydrophobic nature (Kayed et al., 2003; Sadigh-Eteghad et al., 2015). As shown in Figure 6A, compared with the vehicle group, there was a significant decrease in the levels of soluble Aβ40 (F = 9.069; p = 0.008, p = 0.001, and p = 0.001) and Aβ42 (F = 15.569; p < 0.0001 and p < 0.0001) in the YXQN middle- and high-dose groups. Moreover, compared with the vehicle group, the APP/PS1 mice in YXQN groups showed a 50–70% reduction in highly aggregated forms (insoluble, formic acid extract) both of Aβ40 (F = 43.774; p = 0.001, p < 0.0001, and p < 0.0001) and Aβ42 (F = 19.671; p < 0.0001, p < 0.0001, and p < 0.0001).

To further confirm the above results of ELISA that YXQN reduced the levels of Aβ, the amount of total cerebral Aβ were tested by Western blot. As shown in Figure 6B, the low-, middle-, and high-dose YXQN dramatically reduced the amount of Aβ in the brain, by 42, 62, and 77%, respectively, relative to the vehicle group (F = 6.339; p = 0.011, p = 0.003). To investigate APP processing involved in the reduction of Aβ levels, we further analyzed the expression of full-length APP and APP-derived C-terminal fragments (CTFα and CTFβ) in the brain of APP/PS1 mice in each group by immunoblot with the specific antibody. CTFα, one of the α-secretase-derived fragments from the non-amyloidogenic processing of APP, is a physiological product. Meanwhile, β-secretase-derived CTFβ from the amyloidogenic processing of APP is the pathologic product that further generates Aβ. As shown in Figure 6C, full-length APP was unchanged among different treatment groups. However, in YXQN treated APP/PS1 mice, the levels of CTFβ were remarkably decreased, along with an obvious increase of CTFα. Notably, the ratio of CTFα to CTFβ in the YXQN middle- and high-dose groups showed a 1.6∼1.8-fold elevation above that of the vehicle group by quantification (F = 3.593; p = 0.039 and p = 0.017). Thus, the results indicate a modulation effect of YXQN on suppressing amyloidogenic and promoting non-amyloidogenic processing of APP.

Subsequently, to corroborate the effect of YXQN extract on non-amyloidogenic processing, we examined the expression of N-terminal α-secretase-derived sAPPα in cerebral hemispheres of each group, which has been reported as antagonizing amyloidogenic processing of APP and playing a neurotrophic role. Our results showed that the expression of sAPPα in YXQN middle- and high-dose groups presented a robust augmentation compared with the vehicle group (Figure 7A), which was consistent with the increase of CTFα. Given that ADAM10 as a common α-secretase cleaves APP to generate sAPPα and CTFα, the expression of ADAM10 in each group was detected by immunoblot. As shown in Figure 7A, a similar notable augmentation of ADAM10 was observed in YXQN middle- and high-dose groups, compared with the vehicle group. Then, we immunostained brain slices of the five groups with ADAM10 antibody and analyzed the total staining area. As shown in Figure 7C, compared with the vehicle group, the degree of ADAM10 staining in the cortex of YXQN middle- and high-dose groups strikingly increased. There was a meaningful difference between middle- or high-dose YXQN and vehicle APP/PS1 mice in the total positive stain cover area of ADAM10 (F = 16.433; p = 0.006 and p < 0.0001) (Figure 7D), which is in accordance with the results from the Western blot (Figure 7A). These data provided evidence that YXQN improved non-amyloidogenic processing of APP by promoting ADAM10 expression.

BACE1 and PS1 as β- and γ-secretase play a core role in the pathological processing of APP (amyloidogenic processing) (Oddo et al., 2004; Willem et al., 2015). Based on the effects of YXQN in reducing the production of CTFβ and Aβ, the expression of BACE1 and PS1 were further detected in the brain of each group by both Western blot and immunohistochemistry, to confirm the roles of YXQN in the inhibition of APP pathological processing. As shown in Figure 7B, compared with the vehicle group, the expressions of BACE1 and PS1 in the brain tissues of YXQN groups were greatly reduced in a dose-dependent manner. A reduction of the BACE1 level was observed in the donepezil group as well. Moreover, in the analysis of the staining area of BACE1 and PS1 by immunohistochemistry, we found the BACE1 and PS1 levels markedly reduced in all three YXQN dosage level groups (Figure 7C). The total staining areas of BACE1 in low-, middle-, and high-dose groups were substantially decreased by 45, 57, and 79%, relative to the vehicle group (F = 15.754; p = 0.005, p = 0.001, and p < 0.0001); and that in the donepezil group also showed a slight reduction (F = 15.754; p = 0.032) (Figure 7E). Likewise, the three groups of YXQN, respectively, showed a 31, 45, and 54% reduction of PS1 expression in APP/PS1 mice, compared with vehicle APP/PS1 mice (F = 7.506; p = 0.005) (Figure 7F), suggesting the diminished production of Aβ peptide. Taken together, these data might indicate that YXQN extract inhibited the expression of BACE1 and PS1, and leaded to the reduction of Aβ in the brain of APP/PS1 mice. These results therefore addressed the suppression of amyloidogenic pathological processing of APP by YXQN.

All in all, our results confirm a significant role of YXQN against cognitive decline and Aβ aggregation in AD, through up-regulating the level of α-secretase ADAM10 in the physiological processing of APP, and down-regulating β-secretase BACE1 and γ-secretase PS1 in the pathological processing of APP.