Research ArticleClassic Prescription, Kai-Xin-San, AmelioratesAlzheimer’s Disease as an Effective Multitarget Treatment:From Neurotransmitter to Protein Signaling Pathway

Sirui Guo,1 Jiahong Wang ,2 Huarong Xu,1 Weiwei Rong,1 Cheng Gao ,2 Ziyue Yuan,1

Fucheng Xie,1 Kaishun Bi,1 Zhou Zhang ,2 and Qing Li 1

1School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, 110016 Liaoning, China2School of Life Sciences and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, 110016 Liaoning, China

Correspondence should be addressed to Zhou Zhang; zzhouzhang@163.com and Qing Li; lqyxm@hotmail.com

Received 20 February 2019; Accepted 7 May 2019; Published 1 July 2019

Academic Editor: Ryuichi Morishita

Copyright © 2019 Sirui Guo et al. This is an open access article distributed under the Creative Commons Attribution License, whichpermits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Alzheimer’s disease (AD) is a widespread neurodegenerative disease caused by complicated disease-causing factors.Unsatisfactorily, curative effects of approved anti-AD drugs were not good enough due to their actions on single-target, whichled to desperate requirements for more effective drug therapies involved in multiple pathomechanisms of AD. The anti-ADeffect with multiple action targets of Kai-Xin-San (KXS), a classic prescription initially recorded in Bei Ji Qian Jin Yao Fang andapplied in the treatment of dementia for thousands of years, was deciphered with modern biological methods in our study.Aβ25-35 and D-gal-induced AD rats and Aβ25-35-induced PC12 cells were applied to establish AD models. KXS couldsignificantly improve cognition impairment by decreasing neurotransmitter loss and enhancing the expression of PI3K/Akt. Forthe first time, KXS was confirmed to improve the expression of PI3K/Akt by neurotransmitter 5-HT. Thereinto, PI3K/Akt couldfurther inhibit Tau hyperphosphorylation as well as the apoptosis induced by oxidative stress and neuroinflammation.Moreover, all above-mentioned effects were verified and blocked by PI3K inhibitor, LY294002, in Aβ25-35-induced PC12 cells,suggesting the precise regulative role of KXS in the PI3K/Akt pathway. The utilization and mechanism elaboration of KXS havebeen proposed and dissected in the combination of animal, molecular, and protein strategies. Our results demonstrated thatKXS could ameliorate AD by regulating neurotransmitter and PI3K/Akt signal pathway as an effective multitarget treatment sothat the potential value of this classic prescription could be explored from a novel perspective.

1. Introduction

Alzheimer’s disease (AD) is the widespread neurodegenera-tive disease that accounted for 60-70% cases of dementiacharacterized by amnesia, cognition impairment, and mentalbehavioral changes [1]. It was reported that there are morethan 50 million cases of dementia worldwide in 2018, signify-ing one new case every 3 seconds [2]. Currently, all FDA-approved drugs were developed for specific single-target,such as cholinesterase inhibitors (donepezil, rivastigmine,and galantamine) and N-methyl-D-aspartate receptor ago-nist (memantine) [3], and the symptoms of AD could be alle-viated to some extent. However, their therapeutic effects werefar from the expectation by reason of their actions on single-

target, which could not match multiple pathogenetic factorsmainly including abnormal β-amyloid and Tau [4, 5], thusproviding imperious demands for more effective multitargetdrug therapies.

Clinical curative effects of traditional Chinese medicinehave been continuously verified in thousands of years, andseveral classic formulas specially aiming at various diseaseswere gradually developed. As a common formula for thetreatment of “amnesia,” the main symptom of AD, KXSwas initially recorded in Bei Ji Qian Jin Yao Fang in the Tangdynasty (Year 652), consisting of Polygala tenuifolia Willd(PR), Panax ginseng C.A. Mey (GR), Poria cocos (Schw.)Wolf (PO), and Acorus tatarinowii Schott (AT) [6]. Basedon literatures to date, researches of KXS were mainly focused

HindawiOxidative Medicine and Cellular LongevityVolume 2019, Article ID 9096409, 14 pageshttps://doi.org/10.1155/2019/9096409

on component identification or mechanism dissection, andmost pathological studies were related to neurotransmitterregulation that involved a single pathway, which could notcomprehensively elaborate therapeutic effects of KXS andthe scientificity of AD treatment [7, 8]. Therefore, there isan urgent need for explaining multidimensional beneficialeffects of KXS with modern biological methods.

Aβ25-35 and D-gal-induced rats were usually selected asthe most pertinent model to simulate characteristic patholog-ical changes of AD [9]. Meanwhile, PC12 cells were com-monly used for in vitro research to verify the underlyingcellular mechanism against AD, which was important toevaluate biological function. Hence, we explained the phar-macological effects of KXS at animal, molecular, and cellularlevels, multidimensionally clarifying the biological mecha-nism and scientificity for AD treatment, which could provideresearch reference for studies on the mechanism of Chineseclassical prescriptions.

2. Materials and Methods

2.1. Chemicals and Reagents. The purity of the following ref-erence standards was all over 98%. Isoproterenol hydrochlo-ride was purchased from the National Institutes for Food andDrug Control (Beijing, China). Arachidonic acid (AA), leu-kotriene-B4 (LTB4), thromboxane-B2 (TXB2), 5-hydrox-yeicosatetraenoic acid (5-HETE), 8-hydroxyeicosatetraenoicacid (8-HETE), 12-hydroxyeicosatetraenoic acid (12-HETE),15-hydroxyeicosatetraenoic acid (15-HETE), and 15-hydroxyeicosatetraenoic acid-d8 (15-HETE-d8) were obtainedfromCayman.Dopamine (DA), γ-aminobutyric acid (GABA),acetylcholine (ACh), tryptophan (Try), tyrosine (Tyr), 5-hydroxytryptophan (5-HTP), 5-hydroxyindoleacetic acid(5-HIAA), 5-hydroxytryptamine (5-HT), metanephrine(ME), 3-methoxytyramine (3-MT), Aβ25-35, and D-gal wereobtained from Sigma-Aldrich Corporation (USA).

2.2. Preparation of Herbal Extracts. The herbal pieces of GR,PR, PO, and AT were purchased from Beijing TongrentangCo. Ltd., China, and identified by Prof. Ying Jia, ShenyangPharmaceutical University. The decoction pieces were mixedin a weight ratio of 3 : 2 : 3 : 2, then boiled in 10 volumes ofdouble distilled water for 1 h and extracted twice. The com-bined extracts were concentrated to 1mg/mL by a rotaryevaporator and stored at -20°C.

2.3. Animals.Male SD rats weighing 200-250 g were providedby the Experimental Animal Center of Shenyang Pharma-ceutical University. SD rats were maintained in a controlledenvironment (humidity 50 ± 10%, 23 ± 2°C) under a 12 hlight/dark cycle and allowed to water and standard diet adlibitum. All experiments were approved by the Guideline ofAnimal Experimentation of Shenyang Pharmaceutical Uni-versity. Rats were divided into four groups, namely, the con-trol group, model group, KXS group, and huperzine A (HupA) group. Briefly, AD rats were injected with D-gal(50mg/kg/d) intraperitoneally for 6 weeks, and control ratswere given the same volume of saline. KXS (10 g/kg/d) andHup A (30 μg/kg/d) were oral administrated from the third

week until the end of the experiment once a day [10]. The ste-reotaxic injection of 4 μg Aβ25-35 (1μg/μL) was operated asdescribed previously [11]. At the seventh week, the Morriswater maze performance was tested as described previously[12]. Drug administration and Morris water maze test werescheduled according to the experimental timelines (Figure 1).

2.4. Measurement of Endogenous Substances. The plasmasamples were collected after Morris water maze behavioraltesting. Aliquots of 200 μL plasma samples were spiked with20μL of IS (isoproterenol hydrochloride and 15-HETE-d8)and 20 μL of methanol by vortexing for 30 s. The mixtureswere extracted with 600μL acetonitrile (containing 1% for-mic acid) by vortex-mixing for 3min. After centrifugationat 12000 rpm, 4°C for 5min, the supernatants were trans-ferred and evaporated to dryness at 35°C. Then, the residuewas reconstituted in 50 μL 0.05% formic acid in methanol-water (15 : 85, v/v) for UFLC-MS/MS analysis.

Liquid chromatographic runs were performed on an XRLC-20 AD Prominence™ UFLC system (Shimadzu, Japan).Chromatographic separation was achieved with an InertsilODS-EP column (150mm × 4 6mm, 5 μm particles), (Shi-madzu, Japan) at 30°C. Mobile phase A was 0.05% formicacid in water, and mobile phase B was 0.05% formic acid inmethanol. A mobile phase was achieved with gradient elutionusing a complex gradient, as the following: 85%A→ 85%A at0–0.01min, 85%A→ 55%A at 0.01–0.5min, 55%A→ 0%Aat 0.5–5min, 0%A→ 0%A at 5–10min, 0%A→ 85%A at10–10.01min, and 85%A→ 85%A at 10.01–12min. Sym-metrical and efficient peaks were obtained at a flow rate of0.5mL/min with a sample injection volume of 5 μL.

Mass spectrometric detection was performed by a 4000QTrap™ MS/MS system from AB Sciex (Foster City, CA,USA) in the MRM modes. The ion spray voltage was main-tained at 5500V for positive and -4500V for negative moni-toring. The source temperature was set at 500°C with high-purity nitrogen as Gas 1, Gas 2, and curtain gas set at 50,50, and 20 psi, respectively. The acquisition, instrument con-trol, and analysis of data were dealt with Analyst software(version 1.5.2, AB Sciex, USA).

2.5. Drug Treatment and Cell Viability Detection. Highlydifferentiated rat pheochromocytoma cells, PC12, werepurchased from the Boster Biological Technology Co. Ltd.(Wuhan, China) cultured in H-DMEM supplemented with10% FBS, 1% L-glutamine, and 1% penicillin/streptomycin.Cells were incubated at 37°C in moist air containing 5%CO2. PC12 cells were seeded in culture plates (1 × 105cells/mL) for 24h and then treated with series of concentra-tions of KXS (0, 0.05, 0.1, 0.5, 1, 5, 10, and 20mg/mL) for24 h. Subsequently, Aβ25-35 was added to the medium andcocultured with KXS for an additional 24 h. LY294002which was the inhibitor of the phosphoinositide 3-kinase(PI3K)/Akt signaling pathway was employed 1h prior toKXS treatment. PC12 cells were divided into four groups,namely, the control group, Aβ group, KXS group, andpretreatment of LY294002+KXS group. Meanwhile, PC12cells were pretreated with LY294002 for 1 h, incubated with1 μmol/L 5-HT and 20μmol/L Aβ25-35 for 24h, and divided

2 Oxidative Medicine and Cellular Longevity

into the control group, Aβ group, 5-HT group, and pre-treatment of LY294002+5-HT group [13].

Cell viability was measured by the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assayand LDH assay kit. After the KXS treatment, the mediumwas removed and replaced with 20 μL MTT (5mg/mL,Sigma) in phosphate buffer solution. The plates were incu-bated for 4 h at 37°C, followed by the addition of 150μLdimethyl sulfoxide (DMSO). The absorbance was detectedat 490nm wavelength by a microplate reader. The concen-trations of LDH assessed as a cytotoxicity assay indicatorin cell culture supernatants were evaluated by the LDH assaykit (Nanjing Jiancheng, China) according to the manufac-turer’s instructions. The absorbance was detected at 450 nmwavelength. The LDH release activity was calculated asthe following:

ODsample −ODcontrolODstandard −ODblank

∗ concentrationstandard 1

2.6. Measurement of Intracellular Reactive Oxygen Species(ROS) and Mitochondrial Membrane Potential (MMP).Intracellular ROS was measured using the ROS assay kit(Nanjing Jiancheng, China). 2,7-Dichlorofluorescin diace-tate was a sensitive intracellular reactive oxygen detectionprobe which was widely used. According to the descriptionof the previous study [14], the fluorescence in fresh hippo-campal tissue and PC12 cells was evaluated with the exci-tation at 500 ± 15 nm and the emission at 525 ± 15 nm.Rhodamine 123 (Beyotime, China) was a fluorochromewhich could penetrate cytomembrane and selectively stainmitochondria in alive cells. Rhodamine 123 was dissolved inDMSO and diluted to the working concentration (2 μmol/L)with cell culture medium. The fluorescence was evaluatedwith a flow cytometry (BD, USA) at 507nm excitation and529nm emission.

2.7. Assessment of Annexin V-FITC/PI Staining andProinflammatory Factor (IL-1β and TNF-α). The rate of apo-ptosis was quantified with an Annexin V-FITC/PI apoptosisdetection kit (KeyGen BioTECH, China). The washed cellswere resuspended in binding buffer and incubated withannexin V-FITC and propidium iodide. The treated cellswere measured with a flow cytometry within 1 h. Theannexin V-FITC and PI were tested through FL1 and FL3and quantified with FlowJo 10 software, respectively. IL-1β

and TNF-α ELISA assays (Boster, China) were conductedto determine the level of IL-1β and TNF-α in the hippocam-pal tissue samples and PC12 cell culture medium obtainedfrom the treated PC12 cells in order to test the anti-inflammatory effect of KXS. Concentrations of IL-1β andTNF-α were measured according to the standard protocolfrom the manufacturer. The absorbance was evaluated withthe microplate reader at 450nm.

2.8. Western Blotting. The protocol of western blotting wasproceeded as described previously [15]. In brief, the hippo-campal tissue samples and PC12 cells were harvested andlysed with lysis buffer (Beyotime, China) containing 1%phosphatase inhibitors and protease inhibitor. The quan-titative proteins (40 μg) were separated on 10% SDS-polyacrylamide gels. The primary antibodies were PI3Kp110а (#4249, CST, 1 : 1000), PI3K (ab40776, Abcam,1 : 1000), Tau (ab32057, Abcam, 1 : 1000), Tau (phosphoS396) (ab109390, Abcam, 1 : 5000), Tau (phospho S199)(ab81268, Abcam, 1 : 5000), Bax (ab32503, Abcam, 1 : 1000),bcl-2 (WL01556, Wanleibio, 1 : 800), GSK-3β (#12456, CST,1 : 1000), phospho-GSK-3β (Ser9) (#5585, CST, 1 : 1000),Akt (#4691, CST, 1 : 1000), Akt (phospho S473) (ab81283,Abcam, 1 : 5000), and cleaved-caspase-3 (25546-1-AP, Pro-teintech, 1 : 1000). The bands were covered with ECL andphotographed with the Chemiluminescent Imaging System(Tanon, China). β-Actin was calculated as a control for pro-tein expression in every membrane.

2.9. Immunohistochemistry and Immunofluorescence. Inorder to detect the phosphorylation of Tau, immunohisto-chemical analysis was performed on hippocampus sections(4 μm thickness) using a monoclonal anti-Tau (phosphoS396) antibody (1 : 1000). A Rabbit SP detection kit (ZSBIO)was adopted to visualize immunolabeling according to themanufacturer’s instructions. Images of the immunohisto-chemical sections were acquired using a CIC microscope(XSP-C204). Histologic sections were rehydrated with gradi-ent alcohol, antigen-repaired with citric acid buffer (pH 6.0),and then quenched with 3% H2O2 for 25min. After blockingwith goat serum, sections were incubated with anti-Tau(phospho S396) antibody at 4°C overnight, followed withstreptavidin-horseradish peroxidase (Servicebio, G1211) for50min at room temperature. The sections were colored withperoxidase-3,3′-diaminobenzidine (DAB) substrate (Servi-cebio, G1211) and scanned at 400x.

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3Oxidative Medicine and Cellular Longevity

Immunofluorescence for neuronal nuclei (NeuN) expres-sion was performed. Brain tissue sections were rehydratedwith gradient alcohol, antigen-repaired with citric acid buffer(pH 6.0), and then blocked with BSA for 30min. The sectionswere incubated with NeuN antibody (1 : 200) at 4°C over-night, followed with fluorescence secondary antibodies (Ser-vicebio, 1 : 300) for 50min at room temperature. NeuN (redfluorescence) was visualized by excitation at 520nm anddetection of emission at 590nm. DAPI (blue fluorescence)was visualized by excitation at 380 nm and detection of emis-sion at 420nm. Images of the immunofluorescence sliceswere acquired with Nikon Eclipse C1. The percentage ofthe integrated optical density (IOD) occupied by positiveexpression products above background staining was mea-sured in the hippocampal CA1 region (3 sections per rat)using Image-Pro Plus software.

2.10. Statistical Analysis. All statistical data were analyzedusing Student’s t-test and one-way ANOVA with SPSS 19.0software. The principal component analysis (PCA) and par-tial least squares discrimination analysis (PLS-DA) wereproceeded with the SIMCA-P software package (UmetricsAB, Umea, Sweden). The statistical results were expressedas the mean ± SD, and statistical significances were classifiedas ∗P < 0 05, ∗∗P < 0 01, and ∗∗∗P < 0 001.

3. Results

3.1. KXS Improved Learning and Memory Impaired in ADRats Induced by D-Gal and Aβ25-35. The AD animal modelinduced by D-gal and Aβ25-35 was applied in order to simu-late human pathological processes. The quality control ofKXS was achieved by HPLC fingerprint (Figure S1). Thewater maze test was adopted to detect the effect of KXS onlearning and memory in AD rats. Hup A (AChEIs), analkaloid isolated from the Chinese herbal medicine Huperziaserrata, was licensed as a nutraceutical in the United Statesand widely used for AD treatment in China [16]. From thesecond day of training trials, the rats in the model groupshowed longer escape latency and swimming distance,meaning obvious learning and memory impairmentcompared to the control (Con) group (Figures 2(a) and2(b)). On the fifth day of the Morris water maze, the spatialprobe test was tested. The rats treated with Hup A or KXSshowed significantly less escape latency and swimmingdistance to find the removal platform in the target quadrant(Figures 2(c)–2(e)). In addition, Hup A and KXS group ratsshowed more platform crossings than the model group(Figure 2(f)). Neuronal nuclei (NeuN), a neuronal specialnuclear marker, was detected by immunohistochemistrystaining to evaluate the neuroprotection of KXS on neuronaldensity in hippocampal CA1 regions [17]. The fluorescenceintensity of NeuN in the model group was significantlyreduced, which was a hallmark pathology of neuronaldamage in AD and indicated a successful AD model.KXS treatment observably increased fluorescence intensityof NeuN compared with the model group (Figures 2(g)and 2(h)). The results of the Morris water maze andimmunohistochemistry staining of NeuN suggested that

KXS could be potentially used for the prevention and/ortreatment of AD.

3.2. KXS Treatment Increased the Concentration of CentralNeurotransmitters in AD Rats. In order to study the influenceof KXS on central neurotransmitter loss, a typical pathologicmanifestation, the UFLC-MS/MS method was used to detectthe concentrations of related endogenous neurotransmitters.The method was validated according to the European Medi-cines Agency guidance for bioanalytical method validation(Tables S1-S2) [18]. Compared with the model group,concentrations of four types of neurotransmitters, namely,ACh, GABA, dopamine (Tyr, dopamine, ME, and 3-MT),and 5-HT (5-HTP, 5-HT, and 5-HIAA), were increased inrat plasmas after drug treatment (Table 1). Therefore, KXScould protect an impaired central nervous system byameliorating neurotransmitter loss. Neurotransmitter datawere handled with PCA. In the light of unsupervised PCAscore plots, plasma samples in KXS and Hup A groups werelocated between the AD model group and the control group.Nevertheless, there was no significant difference between theKXS group and the Hup A group (Figure 3(a)). The PCAresult also proved the efficacy of KXS on regulating the levelsof central neurotransmitters. After PLS-DA processing, 5-HIAA, 5-HT, ME, ACh, 5-HTP, and DA were successivelyscreened from detected neurotransmitters based on thevalues of variable importance in the projection (VIP > 1)(Figures 3(b) and 3(c)). These all demonstrated that the 5-HT pathway played an important role in inhibiting effect ofKXS on neurotransmitter loss.

3.3. KXS Played a Role in Neuroprotective Effect against ADvia the PI3K/Akt Signaling Pathway. Previous studies haveshown that neurotransmitter 5-HT could regulate PI3K/Aktand glycogen synthase kinase-3 (GSK-3) signaling pathways[19]. Hence, we examined the effect of KXS on thePI3K/Akt/GSK-3β protein signaling pathway by immuno-blotting. Our data showed that AD model rats had dramat-ically impaired PI3K/Akt/GSK-3β signaling caused by Aβ25-35 and D-gal, including reduced p-PI3K, p-Akt, and p-GSK-3β (Ser9), while the rats treated with KXS or Hup A resultedin sufficient restoration of PI3K/Akt GSK-3β signaling(Figures 4(a) and 4(b)). NFTs, another typical pathologicalchange in AD, are mainly caused by the Tau hyperpho-sphorylation activated by GSK-3β [20]. To address this, weevaluated the p-Tau level with immunoblot and immuno-histochemistry. The results of western blot (Figures 4(a)and 4(b)) and immunohistochemistry (Figures 4(c) and4(d)) showed that KXS could prominently reduce theexpression of p-Tau (S199 and S396). In brief, the aboveresults demonstrated that the neuroprotection of KXS wasachieved with modulating Tau hyperphosphorylation viathe PI3K/Akt/GSK-3β signaling pathway.

Inhibition of inflammatory factor and ROS-induced neu-ronal apoptosis via PI3K/Akt and GSK-3β pathways has beenapproved as an available therapeutic strategy for AD [21].Various studies have indicated that there was a vicious circlebetween oxidative stress and neuroinflammation which wererepresentative pathomechanisms of AD [22]. With the

4 Oxidative Medicine and Cellular Longevity

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Figure 2: KXS treatment ameliorated memory and learning impairment. The mean escape latency (a) and swimming distances (b) wereobtained from different test days, and the KXS group spent less time and distances to reach the fourth quadrant platform.Representative rat search paths from different groups (c) were shown on the fifth day. The rats treated with Hup A or KXS showedsignificantly less escape latency (d), swimming distance (e), and platform crossings (f) to find the removal platform in the fourthquadrant. Impact of KXS on modulating hippocampal NeuN levels was assessed by immunohistochemistry. The fluorescence intensitywas quantified using Image-Pro Plus software (g and h). The treatment of KXS showed improvement in spatial memory. Values werethe mean ± SD (n = 6). ∗P < 0 05, ∗∗P < 0 01, and ∗∗∗P < 0 001vs. the control group; #P < 0 05, ##P < 0 01, and ###P < 0 001vs. the modelgroup; $$P < 0 01vs. the Hup A group. Scale bar = 20 μm.

5Oxidative Medicine and Cellular Longevity

Table 1: The concentrations of central neurotransmitters were determined by UFLC-MS/MS.

Analytes Control (ng/mL) Model (ng/mL) KXS (ng/mL) Hup A (ng/mL)

ACh 166 7 ± 21 8 99 0±10 0∗∗∗ 133 4 ± 10 6### 125 7 ± 7 6##

Tyr 7170 ± 897 4918±716∗∗∗ 5886 ± 608# 6407 ± 637##

DA 6 785 ± 1 678 2 673±0 773∗∗∗ 5 601 ± 1 383## 5 218 ± 0 965##

ME 10 76 ± 1 45 4 507±0 6441∗∗∗ 8 96 ± 1 10### 9 00 ± 0 72###

3-MT 1 821 ± 0 434 0 6645±0 3005∗∗∗ 1 505 ± 0 405## 1 425 ± 0 272##

Try 1 637 × 104 ± 0 332 × 104 1 580 × 104 ± 0 435 × 104 1 848 × 104 ± 0 465 × 104 1 763 × 104 ± 0 219 × 104

5-HTP 15 56 ± 3 96 4 144±0 985∗∗∗ 10 10 ± 3 38### 11 12 ± 1 38###

5-HT 959 ± 30 509 4±110 7∗∗∗ 787 9 ± 27 5### 719 1 ± 46 0###

5-HIAA 32 28 ± 2 80 5 864±0 847∗∗∗ 29 00 ± 1 43###$$$ 24 52 ± 2 33###

GABA 53 17 ± 7 05 37 02 ± 10 26∗ 49 05 ± 9 19# 48 07 ± 7 34#

Data were expressed as themean ± SD (n = 6). ∗P < 0 05 and ∗∗∗P < 0 001vs. the control group; #P < 0 05, ##P < 0 01, and ###P < 0 001vs. the model group; and$$$P < 0 001vs. the Hup A group.

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6 Oxidative Medicine and Cellular Longevity

treatment of KXS, the ROS level measured with dichlor-ofluorescein (DCF) fluorescence and concentrations ofinflammatory factors such as IL-1β and TNF-α were signif-icantly reduced in the rat hippocampus (Figures 5(a)–5(c)).Moreover, the arachidonic acid pathway, leading to theincrease of proinflammatory mediators, was evaluated bythe UFLC-MS/MS to examine the anti-inflammatory effectsof KXS [23]. We found that KXS could significantlydecrease the concentration of AA as well as its metaboliteslike 5-HETE, LTB4, and TXB2 (Table 2). In the light of

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Figure 4: KXS inhibited hyperphosphorylation of Tau by activating the PI3K/Akt/GSK-3β signaling pathway. KXS upregulated the PI3K/Aktsignaling pathway, restored the phosphorylation activity of GSK-3β (Ser9), and decreased the degree of Tau phosphorylation caused by Aβ25-35 and D-gal. Representative western images (a) and quantification (b) of protein related to the Tau signaling pathway in the rat hippocampuswere represented. The area fraction of the hippocampus occupied by p-Tau (S396) was calculated using Image-Pro Plus software (c and d).Values were the mean ± SD (n = 6). ∗∗∗P < 0 001 vs. the control group; #P < 0 05, ##P < 0 01, and ###P < 0 001 vs. the model group; $P < 0 05and $$P < 0 01 vs. the Hup A group. Scale bar = 25μm.

7Oxidative Medicine and Cellular Longevity

3.4. KXS Treatment Decreased the Activity of PC12 Cells andthe Expression of Tau Signaling Pathway In Vitro. To investi-gate whether KXS treatment could protect PC12 cells againstAβ25-35-induced damage, we measured the cell viability with

MTT and LDH release assays [24]. According to MTTresults, alive PC12 cells at 24 h coculture with Aβ25-35 at 10,20, and 40 μmol/L were 76 96 ± 2 85%, 57 23 ± 3 67%, and16 26 ± 1 54%, respectively (Figure 6(a)). 20μmol/L Aβ25-35

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Figure 5: KXS inhibited oxidative stress, neuroinflammation, and apoptosis in AD rat hippocampus. The levels of ROS (a), IL-1β (b), andTNF-α (c) in the rat hippocampus were measured using commercial kits. The representative western images (d) and quantification (e) ofBax/bcl-2 and cleaved-caspase-3 in the rat hippocampus were showed. The score plot of arachidonic acid and its metabolites in plasmawas showed in unsupervised PCA (f). Values were the mean ± SD (n = 6). ∗∗∗P < 0 001vs. the control group; ###P < 0 001vs. the modelgroup; and $P < 0 05 and $$$P < 0 001vs. the Hup A group.

Table 2: The concentrations of arachidonic acid and its metabolites in rat plasma determined by UFLC-MS/MS.

Analytes Control (ng/mL) Model (ng/mL) KXS (ng/mL) Hup A (ng/mL)

5-HETE 0 807 ± 0 276 1 748±0 248∗∗∗ 0 934 ± 0 192###$$ 1 344 ± 0 0695##

8-HETE 39 19 ± 6 00 55 78±10 24∗∗∗ 34 68 ± 7 88### 38 21 ± 4 67###

12-HETE 35 19 ± 8 97 69 06±8 73∗∗∗ 45 14 ± 7 57### 52 82 ± 3 67###

15-HETE 28 33 ± 2 92 47 90±6 83∗∗∗ 31 33 ± 2 04###$$$ 48 22 ± 5 87AA 1541 ± 228 3571±242∗∗∗ 1684 ± 136###$$$ 3076 ± 391##

LTB4 20 10 ± 6 68 34 92±6 78∗∗∗ 27 63 ± 4 37# 31 67 ± 4 58TXB2 474 1 ± 83 7 975 2±106 7∗∗∗ 676 9 ± 64 4###$ 850 ± 172Values were themean ± SD (n = 6). ∗∗∗P < 0 001vs. the control group; #P < 0 05, ##P < 0 01, and ###P < 0 001vs. the model group; and $P < 0 05, $$P < 0 01, and$$$P < 0 001vs. the Hup A group.

8 Oxidative Medicine and Cellular Longevity

was chosen in the later experiments which would result in 50-60% cell death. In the presence of an increased dose of KXS(0.05 to 20mg/mL), we found that KXS at 1mg/mL couldinhibit cell death induced by Aβ25-35; however, with thehigher concentration of KXS (20mg/mL), the cell viability

was significantly reduced (Figure 6(b)). Meanwhile, KXScould also effectively meliorate Aβ25-35-caused LDH releasein PC12 cells (Figure 6(c)). LY294002 (20μmol/L), a classicPI3K inhibitor, was applied to further verify the effect ofKXS on the neuronal activity via the PI3K signaling pathway

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Figure 6: KXS treatment regulated the activity of neurons and the expression of the Tau signaling pathway in vitro. The treatment of PC12cells with Aβ25-35 (10, 20, and 40 μmol/L) alone for 24 h (a) or pretreatment with different concentrations of KXS for 24 h (b) was representedby the MTT assay. The concentration of LDH was assessed as the cytotoxicity assay indicator to assess the effect of different concentrations ofKXS (c). The inhibiting effects of LY294002 (5, 10, 20, and 40 μmol/L) were investigated through pretreatment for 1 h (d). The expression ofp-Akt was significantly increased by 5-HT (e). KXS had neuroprotection by modulating the hyperphosphorylation of Tau through thePI3K/Akt signaling pathway. Representative western images (f) and quantification (g) of proteins related to the Tau signaling pathway inPC12 cells were represented. Values were the mean ± SD (n = 6). ∗P < 0 05, ∗∗P < 0 01, and ∗∗∗P < 0 001vs. the control group; #P < 0 05,##P < 0 01, and ###P < 0 001vs. the Aβ group; &P < 0 05, &&P < 0 01, and &&&P < 0 001vs. the LY+KXS group; and $$P < 0 01vs. the LY+5-HT group.

9Oxidative Medicine and Cellular Longevity

(Figure 6(d)). Then, the effect of 5-HT on the expression ofp-Akt was explored. After the treatment of 5-HT, the expres-sion of p-Akt was significantly increased, while the regulationof 5-HT was inhibited by LY294002. It was verified that KXScould improve the expression of PI3K/Akt by neurotransmit-ter 5-HT (Figure 6(e)). What is more, the treatment withKXS upregulated the PI3K/Akt signaling pathway, restoredthe phosphorylation activity of GSK-3β (Ser9) affected byAβ25-35, and promoted the downregulation of phosphory-lated Tau (S199 and S396) (Figures 6(f) and 6(g)). The useof inhibitor affected the efficacy of KXS, further demonstrat-ing that KXS exerted its efficacy through the PI3K signalingpathway, which was in consistent with the results of KXSin vivo.

3.5. KXS Modulated Oxidation and Neuroinflammation-Induced Apoptosis In Vitro. There were abundant evidencessuggesting that excess ROS production and neuroinflamma-tion were involved in Aβ-induced neurotoxicity and harmfulto mitochondrial function [25]. Coculture of KXS at1mg/mL could dramatically reverse Aβ25-35-induced ROSaccumulation (Figure 7(a)) and reduced the MMP level(Figure 7(b)). Interestingly, pretreating the cells withLY294002 resulted in the blockage of ROS reduction andMMP increase, suggesting that PI3K signaling played a rolein oxidative stress. We also found that the treatment ofAβ25-35 alone significantly increased the levels of IL-1β andTNF-α compared to the control (Figures 7(c) and 7(d)).However, the increase was significantly reduced in the pres-ence of KXS at 1mg/mL. The neuroinflammation and ROSproduction resulting in mitochondrial dysfunction could alsoactivate the following caspase cascade up to apoptosis [26].KXS treatment could significantly reduce the expressions ofBax/bcl-2 and cleaved-caspase-3 (Figures 7(e) and 7(f)) andthe percentage of apoptotic cells (Figures 7(g) and 7(h)) com-pared to the control, which was evidenced by immunoblot-ting and flow cytometry. Furthermore, the pretreatment ofLY294002 influenced the effect of KXS on inhibiting oxida-tion and neuroinflammation-induced apoptosis, indicatingthe PI3K signaling pathway played a vital role in neuropro-tection of KXS.

4. Discussion

Nowadays, FDA-approved drugs for the treatment of ADwere single-target, which did not have satisfactory therapeu-tic effects aiming at various pathologic changes includingabnormal β-amyloid and Tau [27, 28]. For these reasons, aperfect therapeutic strategy against AD should be compre-hensive pharmacological improvements with multiple targets[29]. In the last years, the pharmaceutical development hasturned to search for Chinese herbs with multiple targetsand pathways rather than just pursuit medicines that specif-ically target a single disease-causing factor [30]. The classicalprescription, KXS, was used for dementia treatment for thou-sands of years by means of restoring the body dynamic bal-ances [31]. The development of KXS was restrained due tothe lack of a definite mechanism. Thus, KXS was investigatedby previously optimizing the pharmacokinetic study on AD

rats [10], and the underlying mechanisms of KXS againstAD were researched in vivo and in vitro.

In China, Hup A, a natural alkaloid, was widely applied toimprove cognition impairments of dementia in the clinicalapplication via multiple pathways [32]. Therefore, Hup Awas used as the positive control in all animal studies andproved the improvements on cognition impairment, neuro-transmitter imbalance, oxidative stress, etc. The behavioralexperiments indicated the improving effect of KXS on learn-ing and memory abilities. To explore the underlying mecha-nism, the effects of KXS on the classic pathologic pathways ofAD were further studied. Based on our finding, a model forthe neuroprotective effect of KXS against AD pathogenesiswas proposed (Figure 8). Firstly, the mechanism researchwas carried out with quantitative determination of neuro-transmitters which could be potential biomarkers and pro-vide the evidences for early diagnosis and treatment of AD.The pathogenesis of AD was closely related to the changesof neurotransmitter homeostasis [33]. Aβ could impairpatient’s memory and executive function by reducing theconcentration of neurotransmitters in the cerebral cortexand hippocampus such as dopamine and 5-HT [34]. Thedetected neurotransmitters mainly include GABA, ACh,dopamine pathway, and 5-HT pathway in target metabolo-mics, and it was confirmed that KXS played a crucial rolein treating AD by increasing those neurotransmitters(Table 1). In PCA result, there was no significant separationbetween the KXS group and the Hup A group which alsoproved that KXS and Hup A have the same effect onimproving neurotransmitters. According to PLS-DA result,the 5-HT pathway made great contribution to the samplegrouping. Previously, it was reported that 5-HT receptorscould modulate the release of ACh [35]. These all demon-strated that 5-HT played an important role in inhibitingeffect of KXS on neurotransmitter loss. Moreover, thePI3K/Akt/GSK-3 signal pathway was affected by the centralneurotransmitter signaling pathways such as 5-HT [19].We also confirmed that it was reasonable to amelioratethe expression of PI3K/Akt by improving the concentrationof 5-HT.

GSK-3, a serine/threonine protein kinase, which wasfound closely related to Aβ and Tau, belonged to theproline-directed kinase and had two subtypes: GSK-3α andGSK-3β [36]. The GSK-3α was involved in regulating theproduction of Aβ peptide, while the latter was involved inthe phosphorylation of Tau and the formation of NFTs[37]. Deposition of Aβ could further cause synaptic changes,abnormal phosphorylation of Tau proteins, transmitter loss,and memory dysfunction [38, 39]. The abnormal hyperpho-sphorylated Tau was present in the neurofibrillary tangles,disrupted microtubule system, and constituted a threat tothe stability of neurons. Therefore, it was involved in thepathogenesis of AD as the main pathological feature of AD[40]. It was demonstrated that KXS could upregulatePI3K/Akt, along with the inhibition of GSK-3β activationas well as the progressive reduction of the phosphorylationlevel of Tau protein (S199 and S396) in the hippocampus,and these protein signaling pathways were mediated by cen-tral neurotransmitters.

10 Oxidative Medicine and Cellular Longevity

There were often metabolic disorders and increased oxi-dative stress in the brain of AD patients [41]. Deposition ofAβ could produce a large number of free radicals and further

cause apoptosis [42]. Available evidences showed thatoveraccumulation of ROS would cause mitochondrial mem-brane damage [43]. Our experiments found that KXS could

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11Oxidative Medicine and Cellular Longevity

significantly improve mitochondrial function by reducingROS levels and increasing mitochondrial membrane poten-tial. Several studies suggest that a vicious circle betweenoxidative stress and neuroinflammation was a significantpathogenic feature in AD where the circle might aggravatethe Aβ-induced pathology and the hyperphosphorylationof Tau [44]. In vitro studies have demonstrated that theseproinflammatory cytokines such as IL-1β and TNF-α wereneurotoxic and could induce neuronal death [45]. AA is anessential fatty acid that plays an important role in signaltransduction of neuronal cells and glial cells, which canbe converted into prostaglandin, thromboxane, leukotriene,etc. In AD, the increased ROS and proinflammatory factorsinduced by Aβ can cause excessive activation of phospholi-pase A2 (PLA2) and release of excessive AA, which ulti-mately leads to nerve cell apoptosis [46]. We found thatKXS had significantly reduced inflammatory factors (IL-1β and TNF-α) and AA as well as its metabolites inplasma, indicating that KXS has the anti-inflammatoryeffect. Thus, we thought that the antiapoptosis effect ofKXS was achieved by inhibiting mitochondrial dysfunctionand inflammation.

In summary, our studies indicated that the treatmentof KXS significantly improved cognition impairment bydecreasing neurotransmitter loss and further causing theintensified expression of PI3K/Akt, which could furtherinhibit the hyperphosphorylation of Tau as well as theapoptosis induced by oxidative stress and neuroinflamma-tion. We dissected the KXS as a new therapy for the treat-ment of AD at the animal, molecular, and cellular levels; itmay be considered as a valuable modality to explore thevalue of Chinese classical prescription.

Data Availability

The data used to support the findings of this study areincluded within the article and the supplementary informa-tion files.

Conflicts of Interest

The authors announce no conflict of interest to currentresearch.

Authors’ Contributions

Sirui Guo and JiahongWang contributed equally to the work.

Acknowledgments

This study was supported by the National Natural ScienceFoundation (No. 81473324). This study was also supportedby the Liaoning Innovative Research Team in University(No. LT2013022), Liaoning Distinguished Professor Projectfor Qing Li (2017), and Shenyang Pharmaceutical UniversityInnovative Research Team.

Supplementary Materials

Figure S1: UPLC fingerprints of eleven batches of KXS.Similarity analysis was performed with “Similarity Evalua-tion System for Chromatographic Fingerprint of Tradi-tional Chinese Medicine (version 2009).” The similarityvalues of all imported chromatograms with respect to thereference fingerprint were greater than 0.96. Table S1: listof selected ESI mode, MRM parameters, declustential (DP),entrance potential (EP), collision energy (CE), and cell exitpotential (CXP) for each analyte. Table S2: the linearitydata for analytes in plasma using the proposed method.(Supplementary Materials)

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