Qingxin Kaiqiao Fang Inhibits Aβ25-35-Induced Apoptosis In

Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2020, Article ID 9058135, 14 pages https://doi.org/10.1155/2020/9058135

Research Article

Qingxin Kaiqiao Fang Inhibits Aβ25-35-Induced Apoptosis in Primary Cultured Rat Hippocampal Neuronal Cells via the p38 MAPK Pathway: An Experimental Validation and Network Pharmacology Study

Tian-Qi Wang,1,2 Xiao-Xiao Lai,1,2 Lu-Ting Xu,1,2 Yan Shen,1,2 Jian-Wei Lin,1,2 Shi-Yu Gao,1,2 and Hai-Yan Hu 1,2

1 e Second Aﬃliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, 109 Xue Yuan Xi Road, Lu Cheng District, Wenzhou 325000, China 2 e Second Clinical College, Wenzhou Medical University, Wenzhou 325003, China

Correspondence should be addressed to Hai-Yan Hu; [email protected]

Received 5 April 2020; Revised 7 July 2020; Accepted 13 July 2020; Published 4 August 2020

Academic Editor: Yoshiki Mukudai

Copyright © 2020 Tian-Qi Wang et al. 0is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Qingxin kaiqiao fang (QKF), a traditional Chinese medicine compound, has been applied to treat Alzheimer’s disease (AD) for many years and has exhibited remarkable eﬀects. However, the underlying mechanism is still not explicit. 0e current study aims to investigate whether QKF exerts an antiapoptotic role through the p38 MAPK pathway in the course of AD. Network pharmacology analysis was applied to study the eﬀective components, possible therapeutic targets, and AD-related pathway of QKF. Further, the AD cell model was established using amyloid-beta (Aβ)25-35 peptide and primary hippocampal neuronal cells extracted from newborn Sprague-Dawley rats. Microtubule-associated protein-2 (MAP-2) imaging was used to detect the morphology of hippocampal neurons. Western blot (WB) analysis was applied to detect the protein expression levels of p38 MAPK, p-p38 MAPK, Bcl-2, Bax, caspase-3, and cleaved caspase-3. Cell viability and apoptosis were determined using cell counting kit-8 (CCK-8) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays, respectively. SB203580 and U46619 were used to detect changes in cell morphology, cell viability, and apoptosis upon inhibiting or activating p38 MAPK. Our present work showed that QKF protects hippocampal neuronal morphology, enhances cell viability, and reduces the number of TUNEL-positive cells. In addition, our results showed that QKF increased the expression levels of antiapoptotic proteins and decreased the expression of proapoptotic proteins. QKF at 25 mg·mL−1 best inhibited neuronal apoptosis among the three doses of QKF by suppressing p38 MAPK activity. Collectively, QKF plays an antiapoptotic role via the p38 MAPK pathway.

1. Introduction aggregation and deposition of amyloid-beta (Aβ) fragments, the main component of SP. Among the Aβ fragments As a serious neurodegenerative disease in aged individuals, studied to date, Aβ 25-35 is the shortest, but it retains the Alzheimer’s disease (AD) is characterized by the formation toxicity of the full-length peptide and exhibits a significant of intracellular neurofibrillary tangles, extracellular deposits level of molecular aggregation [3, 4]. Evidence suggests that of senile plaques (SP), and the loss of neurons [1]. It is the excessive deposition of Aβ in the hippocampus leads to generally accepted that the apoptosis-induced death of a changes in nerve function and eventually cognitive dys- large number of neurons is a common feature in the brains function, as the hippocampus is one of the most delicate of patients with AD [2]. 0is programmed cell death can be areas in the brain and primarily responsible for learning and induced by multiple factors, one of which is the abnormal memory [5]. Moreover, some studies have shown that 2 Evidence-Based Complementary and Alternative Medicine hippocampal neuronal damage induced by Aβ can be me- which the network degrees and betweenness were greater diated by the regulation of p38 mitogen-activated protein than the mean values. kinase (p38 MAPK) [6, 7]. A member of the MAPK family, p38 MAPK, is closely related to the regulation of cell proliferation, differentiation, survival, and death. Recent studies 2.3. Gene Pathway and Functional Analysis. 0e core target have found that its phosphorylation is markedly increased in genes were identified using the DAVID database (http:// both the hippocampi of AD rats and in cultured neurons david.nifcrf.gov/). 0e signaling pathways and biological [8, 9]. In addition, p38 MAPK activation leads to pro- processes of QKF relevant to the treatment of AD were grammed neuronal cell death primarily through alterations analyzed by gene ontology (GO) enrichment analysis (http:// in the expression of proteins involved in apoptosis, including www.geneontology.org) and the Kyoto Encyclopedia of caspase-3, the antiapoptotic protein regulator Bcl2, and the Genes and Genomes (KEGG) pathway enrichment analysis proapoptotic protein regulator Bax [10]. In view of the role (http://www.kegg.jp/) (P ≤ 0.01). of p38 MAPK in apoptosis, SB203580, a direct inhibitor of p38 MAPK, and U46619, an agonist of p38 MAPK, are 2.4. Main Reagents. Poly-L-lysine (PLL) (10x) was pur- currently applied by many researchers to assess whether the chased from Beijing Solarbio Science and Technology Co., antiapoptotic role of p38 MAPK is mediated by its sup- Ltd. (Beijing, China). DMEM/F12 (1 :1), Neurobasal Plus pression [11, 12]. Qingxin kaiqiao fang (QKF), which is Medium, B27 Plus Supplement (50x), HBSS (1x), FBS, and based on Fumanjian, a notable traditional Chinese medicine 0.25% trypsin were obtained from Gibco (Grand Island, New compound from Jingyue Quanshu that was first described by York, USA). 0e Aβ25-35 fragment and dimethyl sulfoxide Zhang Jingyue during the Ming Dynasty, is made mainly (DMSO) were purchased from Sigma (St. Louis, MO, USA). from Radix Rehmanniae (Sheng Di Huang), Radix Paeoniae Donepezil hydrochloride, SB203580, and U46619 were ob- Alba (Bai Shao), Radix Ophiopogonis (Mai dong), Cortex tained from Santa Cruz Biotechnology Inc. (Delaware, Moutan Radicis (Mu dan pi), Poria cocos (Fu Ling), Herba USA). L-Glutamine was obtained from 0ermo Fisher Dendrobii Rhizoma (Shi Hu), Rhizoma Acori Tatarinowii (Massachusetts, USA). 0e rabbit polyclonal antibodies (Shi Chang Pu), Rhizoma Anemarrhenae (Zhi mu), Soph- against MAP2 were purchased from Proteintech Group, Inc. orae Flavescentis (Ku Shen), and Pericarpium Citri Retic- (Chicago, USA). Rabbit monoclonal antibodies against p38 ulatae (Chenpi). A network pharmacology analysis, which MAPK, p-p38 MAPK, caspase-3, cleaved caspase-3, and Bax integrates high-throughput data integration, database re- were purchased from Cell Signaling Technology (Beverly, trieval, data mining, target prediction, laboratory simula- MA, USA). 0e primary antibody against Bcl-2 was pur- tions, and other research methods [13], was applied in this chased from Abcam (Cambridge, UK). Horseradish per- study to reveal the complex mechanism of QKF in the oxidase- (HRP-) conjugated IgG secondary antibody was treatment of AD. purchased from Beijing Bioss Biotechnology Co., Ltd. (Beijing, China), and the BCA protein assay kit was obtained 2. Materials and Methods from Beyotime Biotechnological Technology Co., Ltd. (Shanghai, China). 2.1. Active Ingredients Screening and Targets Identification. 0e BATMAN-TCM database (a bioinformatics analysis tool for determining the molecular mechanism of TCM) 2.5. Preparation of QKF and Aβ25-35. QKF is composed of 10 (http://bionet.ncpsb.org/batman-tcm/) was used to search herbs: Radix Rehmanniae (Sheng Di Huang), Radix Paeo- for the compounds contained in QKF. Further, we predicted niae Alba (Bai Shao), Radix Ophiopogonis (Mai dong), the potential targets of the above drugs based on the Cortex Moutan Radicis (Mu dan pi), Poria cocos (Fu Ling), BATMAN-TCM database and set a target score≥20 as the Herba Dendrobii Rhizoma (Shi Hu), Rhizoma Acori threshold for targets. Afterward, a “QKF-compound-target” Tatarinowii (Shi Chang Pu), Rhizoma Anemarrhenae (Zhi network was constructed and analyzed using Cytoscape 3.7.2 mu), Sophorae Flavescentis (Ku Shen), and Pericarpium software. AD-related targets were retrieved from the fol- Citri Reticulatae (Chenpi), all of which are recorded in the lowing five databases, namely, Online Mendelian Inheri- Chinese Pharmacopoeia, as being present in QKF at a ratio tance in Man (OMIM; https://www.omim.org/), PharmGKB of 2 : 2: 2 : 2: 2 : 2: 2 :1.5 :1.5 :1 on a dry-weight basis, re- (http://www.pharmgkc.org/), GeneCards (http://www. spectively. All herbs were provided by the Second Affiliated genecards.org/), GAD (https://geneticassociationdb.nih. Hospital of Wenzhou Medical University and verified by the gov/), and KEGG (http://www.kegg.jp/) databases. Department of Chinese Materia Medical of Wenzhou Medical University. To make the 1 g·mL−1 stock solution, the raw herbs were decocted with 10 times the volume of dis- 2.2. Network Establishment. 0e drug-related targets and tilled water, extracted twice, filtered, and concentrated, and disease targets were standardized by using the UniProt the drug stocks were then sterilized in an autoclave database. 0e String database (https://string-db.rog/) was (Panasonic Co., Ltd. Tokyo, Japan; MLS-3751L) and stored ° used to analyze protein-protein interactions (PPIs). After- at 4 C until use. 0e Aβ25-35 peptide was dissolved in DMSO ward, the network analyzer function of Cytoscape 3.7.2 at a concentration of 100 μmol L−1 and then placed in a 37°C software was used to analyze the network parameters. 0e thermostat water bath three days prior to use. Prior to use, it topological analysis was used to identify the key genes for was diluted to 10 μmol L−1 with a maintenance medium. Evidence-Based Complementary and Alternative Medicine 3

2.6. Primary Hippocampal Neuron Cultures. One-day-old culture, the neurons were washed with PBS three times Sprague-Dawley (5-6 g) rats were purchased from Beijing (5 min each time), fixed with 4% paraformaldehyde for Vital River Laboratory Animal Technology Co., Ltd. (cer- 20 min at room temperature, washed with PBS another three tification number SCXK 2012-0001; Beijing). 0e rats were times, permeated with 0.2% Triton X-100 at room tem- sterilized using 75% ethanol and then euthanized. 0e skin perature, washed with PBS a further three times, blocked and skull were cut with a pair of sterile scissors. 0e brain with 5% BSA, and finally washed with PBS three more times. was removed from the skull with a pair of tweezers and 0e neurons were cultured with anti-MAP2 polyclonal placed in a small Petri dish with a small amount of HBSS. antibody (1 : 200) in a wet box at 4˚C for 24 h, washed with 0e cerebral hemispheres were peeled back, and the hip- PBS three times, incubated with anti-rabbit IgG (H + L) at pocampus was removed and placed in a Petri dish with the 37°C for 1 h in the dark, and washed with PBS three times. proper amount of HBSS. After all hippocampi were isolated, 0e neurons were incubated with DAPI for 10 min at room they were cut into 1 mm sections with a pair of ophthalmic temperature, treated with antifluorescence quenching and scissors and washed with PBS, and then 3 ml of 0.125% sealing tablets, and washed with PBS three times (5 min each trypsin was added to the sections which were incubated for time). Images of neurons and nuclei were captured under a 15 min at 37°C. Fifteen minutes later, the same amount of high-power microscope. DMEM/F12, containing 10% FBS and 1% penicillin-streptomycin solution, was added to terminate cell digestion, and the solution was gently triturated into a cell suspension with 2.9. Western Blot Analysis. 0e total protein was extracted a sterile pipette. 0e cell suspension was then filtered with a with lysis buffer, and the total protein concentration was 200-mesh copper sieve, and the filtered cell suspension determined using the bicinchoninic acid assay. Proteins underwent centrifugation (4°C; 500 r) for 2 min, after which were separated by 10% PAGE and subsequently transferred the supernatant was retained. 0e retained suspension again onto polyvinylidene difluoride (PVDF) membranes (Roche underwent centrifugation (4°C; 1000 r) for 5 min, and the Applied Science, Indianapolis, IN, USA). 0e membranes supernatant was discarded, following which fresh culture were blocked for 2 h in skim milk blocking buffer and medium was added. Finally, the cells were placed in an washed 3 times with TBS/0.1% Tween 20 before incubation ° with primary antibodies (1 :1000) against p38 MAPK, p-p38 incubator with 5% CO2 and incubated at 37 C for 1 h to induce differential adhesion. Following cell counting using a MAPK, Bcl2, Bax, caspase-3, cleaved caspase-3, and β-tu- ° blood cell counting chamber, the cells were seeded in a 6- bulin overnight at 4 C. 0e membranes were then incubated well plate at a density of 2 ×106/well. 0e culture medium with HRP-conjugated goat anti-rabbit secondary antibody was replaced with maintenance medium composed of as appropriate for 2 h at room temperature. After being Neurobasal Plus Medium, B27 Plus, L-glutamine, and washed three times with TBST, the immunoreactive proteins penicillin-streptomycin solution four hours later. 0ereafter, were visualized using an ECL Plus reagent kit. Finally, the half of the maintenance medium was replaced with fresh density of each band was quantified using AlphaEaseFC, and medium every two days. 0e abovementioned animal ex- β-tubulin served as the internal control. periments were conducted in accordance with the ethical requirements approved by the Chinese Association of Ac- 2.10. Cell Viability Assay. Cell viability was assessed by cell creditation of Laboratory Animal Care. counting kit-8 (CCK-8) assay (Dojindo Laboratories, Tokyo, Japan) according to the manufacturer’s instructions. In 2.7. Cell Grouping. After 7 days of culture, the neurons were short, hippocampal neurons were plated directly in 96-well 4 randomly grouped as follows: control group; model group, plates at a density of 1 × 10 cells/well and grouped into five −1 which was then treated with 10 μmol·L Aβ25-35 alone on wells for each group. After treatment with the corresponding th −1 −1 the 8 day of culture; 25 mg mL QKF group; 12.5 mg mL drugs for 24 h, the cells were exposed to Aβ25-35 −1 QKF group; 6.25 mg mL−1 QKF group; donepezil, which was (10 μmol L ) for an additional 24 h and then cultured in used as a positive control group and treated with 10 μmol·L−1 maintenance medium (100 μL) and CCK-8 reagent (10 μL) donepezil hydrochloride; 25 mg mL−1 QKF + SB203580 for 4 h at 37°C. Finally, cell viability was determined by group, which was treated with 10 μmol L−1 SB203580 and reading the optical density (OD) at a wavelength of 450 nm 25 mg mL−1 QKF; and 25 mg mL−1 QKF + U46619 group, using an enzyme-labeled instrument (Infinite 200Pro, Tecan, which was treated with 10 μmol·L−1 U46619 and 25 mg mL−1 Switzerland). QKF. After 24 h of drug treatment, Aββ25-35 (at a final concentration of 10 μmol L−1) was added to all groups 2.11. TUNEL Staining. TUNEL staining was performed with (except for the control group) for 24 h to establish the AD an In Situ Cell Death Detection Kit (Roche, Mannheim, cell model. Meanwhile, the control group was incubated Germany), according to the manufacturer’s instructions. with the same volume of culture medium for the same length Neurons at day 9 were washed with PBS three times (5 min of time. each time), fixed with 4% paraformaldehyde for 20 min at room temperature, washed with PBS another three times, 2.8. Cell Observation. Cell morphology was observed under permeated with 0.1% Triton X-100 in 0.1% sodium citrate for a Nikon inverted fluorescent microscope (Nikon Corp., 2 min on ice, and washed with PBS a further three times. 0e Tokyo, Japan) at 4 h and 1, 3, 5, and 7 days. After 9 days of cells were then incubated with 50 μL of TUNEL reaction 4 Evidence-Based Complementary and Alternative Medicine mixture in a humidified atmosphere for 1 h at 37°C in the (Figures 2(b)–2(d)). KEGG enrichment analysis identified dark, rinsed three times with PBS, subjected to fluorescent 22 pathways (P < 0.01) (Supplementary Table 9) counterstaining with DAPI for 10 min at room temperature, (Figure 2(e)). 0e results indicated that apoptosis may be the washed with PBS three times, and finally treated with an main way where QKF exerts its effects during the treatment antifluorescence quenching sealing tablet. 0e ratio of of AD. 0erefore, we designed an in vitro experiment to TUNEL-positive cells to the total number of cells was de- verify our hypothesis. termined under a light microscope (Leica DM 3000) and used to calculate the proportion of apoptotic. 3.3. QKF Protected Hippocampal Neuron Morphology. 0e morphology and growth of rat hippocampal neurons 2.12. Statistical Analysis. SPSS 22.0 statistical software (IBM were observed under Nikon inverted fluorescence micro- Corporation, Armonk, NY, USA) was used to analyze the scope at 4 h and 1, 3, 5, and 7 days (Figure 3(a)). Hippo- data. 0e measurement data are expressed as the mean- campal neurons were resuspended just after their ± standard deviation. 0e data from different groups were inoculation. Four hours later, most of the cells were adherent compared using the independent-samples t-test and one- and presented a rounded shape that was surrounded by a way ANOVA. Pairwise comparisons of homogeneous data halo; a few cells also showed protrusions. At day 1, the were performed using the least significant difference (LSD) neurons were completely adherent; grew well with protru- test. P < 0.05 indicated statistical significance for all analyses. sions of different lengths; and showed a spindle-like, tri- angular, or irregular morphology; however, no clear 3. Results connections between neurons were established. After 3 days of culture, the number of cell protrusions was increased, the 3.1. Identification of Active Ingredients and Target Selection. cells were obviously thickened and elongated in shape, and A total of 295 active compounds were identified in the ten connections between neurons began to form. After 5 days of herbs contained in QKF. 0e 9 active compounds in Radix culture, the neurons were plump with a surrounding halo Rehmanniae were identified. 0irty-five active compounds and had become more connected. After 7 days of culture, the were identified in Radix Paeoniae Alba. Twenty-two active cell body was enlarged and plump, there was an obvious halo compounds were identified from Radix Ophiopogonis. around the cells, and the synapse had become longer and Eighteen active compounds were identified in Cortex thicker. 0e neurons had moved closer to each other and Moutan Radicis. Twenty-one active compounds were began to form cell populations, and the cell protrusions had identified in Poria cocos. Twelve active compounds were formed a dense nerve fiber network. We next examined the identified in Herba Dendrobii Rhizoma. Fourteen active neuronal cell morphology in each group following different compounds were identified in Rhizoma Acori Tatarinowii. treatments using an immunofluorescence microscope to 0irty-two active compounds were identified in Rhizoma image microtubule-associated protein-2 (MAP-2), which Anemarrhenae. A total of 116 active compounds were was predominantly localized within cell bodies and den- identified from Sophorae Flavescentis. 0irty-five active drites. 0e neurons in the control group were plump and compounds were identified in Pericarpium Citri Reticulatae dense, and their cell protrusions formed a dense and (Supplementary Tables 1 and 2). A total of 7433 target elaborate nerve fiber network. 0e neurons in the group proteins were identified in QKF. Topological analysis of the treated with Aβ25-35 alone showed a smaller cell body with protein interaction network nodes revealed a total of 1368 thinner and more damaged dendrites than those in the target proteins when the duplicates were removed. We control group and sometimes presented a fragmented or constructed an ingredient-target network (Supplementary beaded appearance, and the number of connections between Table 3) (Figure 1). For disease target identification, we neurons was decreased. However, after treatment with identified the AD-related targets in the OMIM, GeneCards, donepezil or QKF, the cell body became plumper, the PharmGKB, GAD, and KEGG databases. We retrieved 1689 dendrites became thicker, and the junctions between cells target genes corresponding to disease targets and identified were tighter. 0e 25 mg mL−1 QKF group showed an im- 1218 target genes by screening (Supplementary Table 4). proved effect compared to that in the QKF groups at the other tested doses (Figure 3(b)). 3.2. PPI Network Construction and Functional Enrichment Analysis of QKF. 0ere is a certain degree of signaling 3.4. QKF Alleviated Neuronal Apoptosis Induced by Aβ25-35. transduction between different signal pathways and targets. A TUNEL assay was performed to examine apoptosis in 0erefore, the mechanism of action between targets can be hippocampal neurons. TUNEL-positive cells in the selected analyzed by determining the interactions between proteins. visual field were counted by the positive cell counting We used String 11.0 to determine the PPIs among the 1368 method, and the final values are presented as the ratio of the targets of QKF and the 1218 targets related to AD, which number of TUNEL-positive cells to the number of total cells. include 284 nodes and 4183 edges. According to the As shown in Figures 4(a) and 4(b), the apoptotic rate of the established screening rules, 66 targets were identified as key hippocampal neurons in the model group was visibly in- targets related to the QKF treatment of AD (Supplementary creased compared with that in the control group (P < 0.01). Table 5) (Figure 2(a)). GO enrichment analysis revealed 58 In comparison with that in the model group, treatment with entries related to AD (Supplementary Tables 6, 7, and 8) QKF and donepezil reduced the apoptotic rate (P < 0.05, Evidence-Based Complementary and Alternative Medicine 5

Cortex Pericarpium Radix Rhizoma Herba moutan Radix citri Sophorae paeoniae Radix acori Poria dendrobii Rhizoma radicis rehmanniae reticulatae ﬂavescentis alba ophiopogonis tatarinowii cocos rhizoma anemarrhenae

Soyasaponin Rehmaglutin 1-allyl-2,4,5-trimethoxy-benzene P-cymene I Uridine C

Xilingsaponin Kosamol O-xylene Neral Beta-myrcene B A

Isomatrine Lactiﬂorin D-catechin 1-methyl-4-ethylbenzene Dendrobine

Decanoic Campesterol M-xylene Degalactotigonin Chrysanthemaxanthin acid

Gamma-aminobutyric Leachianone Alpha-terpineol Alpha-pinene Baptifoline acid A methyl

Sophoraisoﬂavanone Lauric Ophiopogonanone Neoligustilide N-methylcytisine A acid A

Ophiopogonanone Citronellal 2,4-decadienal Ergosterol Limonene A

FOXA1 PPARG CTNNB1 AARS MTRR STAR RYR3 PIK3CG OXCT2 ARHGEF2 SCN1A CD47 SEC14L4 CHRM3CACNA2D1MGP DGKA GOT2 GABRA4 NARS IL1 F3 HDAC9 BCHE GABRG1 POLA1 COMT CDC42 NOTCH2 SOAT1 EPHX2 MC5R SCN8A SNW1 FGF2 GLYR1 SQRDL

LARS NRXN2 TTPA P4HA3 ADH7 RIPK2 GPD1L ATP1A1 SLC22A4 KCNB2 ACE PHB ADRA2C PDE3A SOCS1 CRYM RPL3 GPRC5A SREBF1 ADRA1A SOD3 ALAS2 RDH8 MAPT PHGDH CAT HCN4 BDH1 ASPA KCNMA1 NRB1 AKR1D1 THNSL1 BMP6 LEP NUDT9 ZFP42

PLCB1 SCN4B POLD1 ALOX15 GPR27 TNFSF11 FNTA RAB3B MAS1 ERO1B SNAI1 KLKB1 FURIN KCNA5 GPT2 TPO CTNNBIP1NLGN3 RDH11 SDHAF2 IHH FOLR1 TRPM8 RGS2 ADORA2AATP1A3 PRKDC AGT SLC25A29HOMER1 KDM1A CYGB HTR3C FLNA BCL11B TARS FCER1G

PROC GNAS GRIK5 KIT GLDC PARK2 OTC TRPA1 NCOR1 AGXT GABRB1 PPP2CA CFTR POLE4 F12 GCSH LILRB1 FOXL2 NFS1 CCL5 CHAT PIK3R3 MATN1 DRD1 PTGER1 EPHB1 SRI ACE2 WNT2 FCER2 HCN2 CRYZ CHUK ADRB1 SERPINE2 HMOX1 XRCC4

CROT SELP FBP1 NT5C2 ZFPM1 ADORA2BACTN3 DLL1 SLC1A6 PML FAS PAXBP1 MAOB CASQ2 CYP4F2 RARA CACNA1GPDE2A GABRA6 CAMK2GTRDMT1 DPP4 NPPA NT5M RXRG DPPA3 KIF14 SRD5A3 NFIB HRH3 PDE8B CARTPT CYP2R1 KCNJ12 TH P2RX3 KCNC2

ALDH5A1 SUMO1 QDPR HSD17B11 BAAT JUP SIX1 NEUROD2NCBP2 P3H3 PDGFB ALDH8A1 GHRH RGCC COLGALT1 CHRNB2 TRPV1 GSK3A CNTNAP4 HTN1 COX7C TYR ZP4 SLC7A11 ITPR2 PPIAL4CHSD17B2 F1 CHRNA5 MMP28 PRKCG OXCT1 SLC7A4 OXT ESR1 LTF TIRAP

SLC38A3 LGALS3 RAB8B FECH HELB PTGIS ARG2 RDH14 NR1H4 PLA2R1 HTR6 SLC8A1 PLOD1 GRIA3 AKR1C1 VDAC3 MC1R KCNC3 ITFG2 CHRFAM7ASRD5A1 STAT5A HSD17B1 TFAP2C SLC13A4 CEND1 DNMT3B APRT TAS1R3 PPARD GCDH CAMK2ASERPINB7 RFK CA7 SRD5A2 POLE3

RYR2 NRP1 FLOT1 ACADSB GNPAT SLC9A3R1 DMTN PIK3CB SULF2 GFI1 HNF1B VCAM1 ACAD8 PIK3R1 DARS ARRB1 HAP1 CYP2E1 RXRA NOS2 PRKCB CRP COX3 HEG1 PNLIPRP2HIBADH P3H2 PPAT PCYT1A ADSS NEDD4L STX1A DPYS PCSK6 CDH11 DLX5 DKK3

LTK CA2 MTTP SCN3B SMO EGR1 AQP8 CETN2 MAPK1 PIPOX S1G GABRB2 HTR7 GJA5 PNPO AMELX CPLX2 ENPP1 CD36 PCCB DHRS9 UROS FBXO45 OCA2 SUCLG2 AK9 CPT1A CYP11A1 TNF TICAM2 COLQ PF4 TAC1 NAPRT ACPP LRRC4B COX8A

NKX3-1 CLDN5 COX5A CHRNA3 NAMPT CCS SLCO1B1 PDE9A VCP GRIA4 LRAT LPL TNR TPM1 KANK2 HPRT1 ASCL1 NPY5R DNAJC15 FOLR2 SLC6A3 NT5C1A NAV2 DDC DRD5 OCLN MC2R ACAT1 ASL P2RX1 CX3CR1 ADAM8 RDH12 KARS TNFSF13 PLIN5 KCNA3

CCR7 EGLN2 ADRB2 ANAPC2 SLC44A4 DRD3 PYGL CREB1 SLC26A6 HPX SLC28A3 CACNG1 PLD2 VKORC1L1RETSAT RARB GLYATL1 PLN YBX3 GLRA4 LCMT1 ARRDC3 P4HA2 ATP2A1 DGUOK ACSL4 TPMT PPP1R15A HTR1B GRIN2B PRKAA1 AMN SDC4 ALDH3B2 IVD CACNA1I DHRS3

LRRK2 RNASE4 KCNA7 ADCY6 TNFSF13B KYNU PRKAB1 GPLD1 FKBP1A DTNBP1 OPRL1 OAT ETHE1 FGA WNT4 GPR18 OGFOD1 KISS1 DARS2 CACNA2D2ATIC P4HB PGR AOC3 SHANK1 DHFRL1 LPCAT2 P2RX2 F2RL1 ADIRF AVPR2 NF2 ABCG1 COX6A2 ADRBK1 UGT3A2 CYR61

BCKDK CACNG2 SLC7A2 ANGPT1 KCNJ6 LHCGR UGT8 CACNA1BMTNR1ARAP1GAP ACSL3 C2CD5 SLC6A9 GAS6 LAMP2 SCN7A LRRC8A EDN2 CECR1 GABRQ ACIN1 AQP2 ABCB4 POU1F1 SLC9A6 PPIF GNA15 SIX4 HSP9AB1 MMAA ALDOA TALDO1 PDE6A KCNJ1 PDE7A LGALS9 F7

CSF1 FCER1A SLC25A2 PAICS DLG4 HTR3A CYP3A4 AQP1 ALDH2 KDM6B SMAD7 C5 RTN2 TLR3 TGFB1 PIK3CA CSF2 DCT KCNJ11 EOMES YWHAE AMD1 CRH RAG2 CETP EPO NOD2 ACO2 TIGAR METRNL KMT2A RNASE8 GPR143 NCKAP1LSLC22A5 G6PD ZPR1

ADCY3 ADH1A PPP3R2 APLN PLOD3 ADRA1B TAL1 SLC6A5 MAP2K1 MYO5A VARS TBC1D32 FDXR HSD17B3 TPI1 SIX3 ALDH1A3MAT1A SAMHD1 ARPIN SLC22A6 PDXP PIK3R5 FFAR1 HTR1A NOX1 FABP6 ABCG2 ATP2B4 HTR1F SLC32A1 C1QTNF1 CNTN2 KCND3 FGF4 BCAT2 AKT1

SLC38A7 GABRA5 GRIA1 NMUR2 IMPDH1 CNR2 PLD1 PPP1R9B PTK2B GGCX PDE11A CD74 TGFB2 P4HA1 BICD1 PRDM8 ROCK2 LCMT2 EPM2A EZR AVP CXCL13 HSD17B8 OAZ1 RNASE1 ANXA1 STUB1 MGLL SLC7A3 SLC25A13PHYKPL GPER1 PCK1 PPIAL4E IMPA1 NCK2 RNF27

PAH ENPP3 CACNA1F RERE TARS2 ACADS ALDH1A2 C3 SPARC NQO1 MED1 8-Mar PLD4 ALDOC PLA2G2DATP13A2 BCL1 STIM2 ARID1A ABHD6 CACNA1CNFKB1 NUDT15 SI TBXAS1XRCC6BP1SCN11A PDE1B SLC36A1 APOC2 AKR1C3 RHOA PLA2G1B AZIN2 ADK SLITRK6 ITPA

SHANK3 GDF5 SOAT2 ADORA3SLCO1B3 PPP2CB ALDOB MTAP PHOSPHO1COX6C PPARGC1BSCGB1A1 PODXL GUK1 PTPN2 ZNF536 POLB CHRM2 WLS MTNR1B KCNB1 RBP1 FADS2 EPHA4 AGTRAP MIP BGLAP DHRS2 PTGER3 PRPS1 ELOVL4 GPT NOS1AP SULF1 SOX15 ACY3 HCAR2

GLRA3 WDR77 NRG1 MAP2 GPBAR1 CRACR2AFKBP1B FEZF2 SCN4A GRIN2D EIF2AK1 KLF5 S1A9 VDR WNT11 SERPINE1CAMK2D DAB2 KCNQ1 OGDHL FASN GABRP HMGCR ADCY1 SCN2B CALCA FNDC5 TERT PDE4C PPIA MAOA TFPI PDE3B ALDH1A1ALOX15BPPIAL4F ACSL1

GSS ALAS1 KCNE5 CCL2 CHRNA1 CTPS1 GRIN2C NR1I3 HTR1D SHMT2 HTR4 RINT1 ITGAL BCKDHB CDH5 CHRM1 GRIN2A IRX5 PRKCA DCK NEFH RDH1 ITGB2 SLC18A3 PPP3CB PTGER4 UHRF1 NUDT16 OAZ3 CHRNA2 PRPH OPRK1 PFKL AMICA1 EGLN3 P2RY1 CNR1

RRM2 GLI3 TOP1 GLYAT ACSS1 CDH8 TMEM11 SPR NR1I2 POLE2 HTR3D AOC1 HCRT BCL2 BDKRB2 HIF1AN WNT5A ADRA1D TAAR1 STAP1 GNMT SCN3A SUCLA2 IARS2 SPRY1 SUGCT GABBR1 KCNA4 PDGFA HPS4 RLBP1 PLCG2 SULT2A1 UTS2R RPS6KA1 PCSK9 CD28

RXFP4 PDE1C CPT2 KDM3A WNT2B CMPK2 HDAC2 TMBIM6 DHRS4 GABRG2 GNB5 HTR2C CRHR1 CDH3 VKORC1 CALHM1 HELLS SDHC KCNJ9 CRHBP TAC4 COX6B1 BCKDHAGABRA3 SEC14L2 CLN3 CD4 MCM3 ARV1 ARCN1 GFER GLRA2 HYAL2 ARX OPRM1 AVPR1A GABRB3

HMGA2 LONP1 ADH1C RAG1 SLCO2B1 SLC18A2 DRD4 CRLF1 OXTR SLC29A1 GRIK4 LARS2 SLIT2 SOD2 IL13 SULT1A2 USP7 HTR5A ALDH3A1PDE5A SORCS3 SEC14L3 TCF3 CALB1 TOP2A DLST TUBB1 MLXIPL NRXN3 TUBA4A MUT PLA2G2ETNFAIP3 SDHA IKBKB CBS KCNJ8

ASNS MTR BMP5 CYP27B1 HIPK2 ADA CACNB1COLGALT2WNT1B GHRL ATP1A2 PIK3R2 PRKCD IMPDH2 AGTR2 BAX IL6 GNAT1 KCNJ14 CCL3 JMJD6 GABRA2 COL27A1 GRIN3B KCNIP2 TOX3 DNM3 SIRT1 ALDH9A1KHDRBS1CHRNA6 PAX2 PPP2R1A GLUL IGF1 SULT1A1 PIK3R6

NT5E COX1 PAX5 F2 NCOA1 CYP1A1 DRD2 HIF1A VDAC1 GABRD CXCR4 CCM2L OPN5 AMPD3 SNAI2 ABAT CACNA1H EEA1 APOE FOXP3 PLD3 PHKG2 IL17A F9 FGFR1 KCNK4 ATP11C PPP3CA ASRGL1 GRIN1 SALL1 NTSR1 HDAC1 TNFRSF13CADAM17 APOA2 ARFGEF2

TNNC1 ALKBH7 IARS KCND1 SLC7A1 CDKN1A MSN MGEA5 SERPINF2 DHFR AOC2 GPR55 SEC14L6 GABRG3 EDN1 HSP9AA1 PDE4D SCN2A NUDT12 ATP8B1 NDRG2 AP3D1 ADRB3 MMAB SLC1A3 IL18R1 REST SLC5A7 SLC1A1 TWIST1 GRIK1 NCOA3 RRM2B ACTN2 PCYT1B TET1 SPX

SLC1A4 KLF4 CHKA CHRM5 NQO2 ADH1B LPCAT1 LTA KL NOS3 NKX2-1 THRA DAGLA F11 OGDH COX7B AVPR1B KCND2 NARS2 PNP CHRNB4 DHODH BMP4 PPIAL4DGAL3ST1 CBR3 GPI OAZ2 AGXT2 ADRA2B IL1RN APEX1 RS1 CYP27A1 ADSSL1 AHCYL1 CAMLG

CAV3 RPS6KA2 HTT PSAT1 TRIM24 SULT1E1 PAWR PKD2 RARG ARG1 ADAP2 PDE7B DTYMKCACNA1ABNIP3 SLC13A2 APOA1 CES1 LEF1 MTOR PIM1 ITPR1 XCL1 AURKA SLC25A1 ATM UCN2 MPO GC PROS1 GATA3 IL4I1 BCL2L11 RHO RDH5 CD34 FUT7

ABCC2 CYP1A2 ANG SIPA1 POU4F3 PIK3CD RARRES1 MECOM CASQ1 HACD1 SLC6A4 PPARA AGER HTR1E XDH NEDD4 CBR1 CACNA1DGABRE NFX1 CHRNA4 ACHE DBT PRSS12 IDO1 CYP4F11 IDNK ANK2 MAPK8IP2 ALDH1B1 SUCNR1 TBR1 MIF HTR2B GAMT NR3C1 AARS2

MRPS36 INSR SLC18A1 RBP3 ADAP1 KCNH2 COX4I1 SDHB CDK5R1 DAO APOH SLC2A1 MEX3C BBOX1 PGD GALC HSD17B7 TKT POR SHMT1 CYP4B1 REN SFXN5 IL1B NLGN1 DOCK5 ETFDH CHDH ALDH7A1NRXN1 POLA2 RYR1 MAP4 ALDH3B1 ENPP6 B4GALT1 HPN

SCN5A DCSTAMPACOT4 VTI1A ADCY5 ZNF219 IL2 MAT2A AOX1 GNAT3 AGRN CTCF AREG ADRA2A ILVBL EGFR IL17RA CETN1 DPYD MGMT DAB2IP TYMS CBR4 PTGFR FER TRPV3 CREBRF GRIK2 PTGS2 SYT2 INS PLAT PRDM16 BRCA1 SCN1B AHCY SNCA

PTGER2 TP53 KCNC1 UCHL1 CHGA TACR2 SUCLG1 CRTAP GPX7 GLRB BMP2 CTSH APLP1 ANK3 DHTKD1 PDE6C IL34 ACVR2A CRAT TSPO NEFL ADIPOQCYP24A1 PTGS1 MC3R PDXK SERPINH1 ANKH GRIA2 PALM UTS2 MMACHCCACNA1SUGT1A1 RXRB PARP1 UHRF2

CHRNB3 DNMT1 DLD SLC13A5 ESRRG EDA STIM1 ABCC4 HTR2A UROD TRIM28 ITPR3 UBE2B IFNG SLC13A1 ARRB2 GATM CD63 P3H1 KCNA1 GRIN3A TAS1R2 UBR5 KCNA2 GCAT HINT1 TAS1R1 CKB NR3C2 ADORA1 RIPK1 AFM GRIK3 CRHR2 ABCB1 SPRY2 COL5A1

NOTCH1GLYATL2 CYB5R3 NPPC HTR3E SLC6A2 TCN1 HRH1 AK5 ADAL HRH2 SNTG2 LHX6 FBLN1 CABP1 SOX9 IYD LPCAT4 LANCL2 RNASE2 PDE6B IL4 RAB3A BAD LYZ CTR9 ALAD FSCN1 MAP2K5 OSBPL8 POLE CHRNA9 DNMT3A CYB5R1 COX7A1 GLRA1 PPP1R1B

PDE4A PTEN EPCAM RET NCK1 DUT MAD2L2SLC25A12 CUBN TREM1 PARK7 POLG GSR KCNJ5 MYD88 ACADM DOCK4 DMGDHSLC29A2 PPP3R1 GRM7 KCNA6 SEMA4D ADH4 HRH4 HRC SLC17A7 CHRM4 JUN UBIAD1 PKP3 FGFR2 GABRA1 PHOX2B ALOX5 FADS1 FGF23

NGFR SLC25A15 PDE8A FASLG AIFM1 RAPGEF2CYP17A1 HACL1 ACY1 ZP3 COX2 GARS PDE4B NUDT1 SLC13A3 ASPH DGKI AGTR1 TYRP1 SHBG CAD HSD17B6 ACSS2 RDH13 ASS1 PRKAB2 S1PR2 ROCK1 PROZ PAX7 TST KCNJ15 AKAP13 SLC1A5 CAV2 NAPEPLDSULT2B1

GOT1 MAPK9 CYP19A1 FOS PKP2 TBX21 SERPINB3 SDHD PDE1A CYP39A1 PLG HOXA5 MAGI2 RRM1 SHH CBSL HMBS GUCY1B3 SOD1 ESR2 SIRT2 SRC OPN4 CHRNA7 PLOD2 VDAC2 SLC22A1 KCNJ3 PINK1 EGLN1 TXNRD1 STATH ICAM1 CBFA2T3 COX5B BCAT1 DERA

SLC7A8 MC4R PGLS WT1 SPI1 TMLHE FADD PPIAL4A IGF2 TBPL1 DNAJA3 AR CDC2 DBH CDK5R2 CD3A SCN9A SUOX LIG4 HTR3BFGF1 AKR1C2 THRB AIF1 ACOX1 NOS1 Figure 1: Compound-target network diagram. 0e red triangle represents herbs in Qingxin kaiqiao fang, yellow diamond indicates candidate compounds, and the green circle indicates predicted targets.

P < 0.01). 0e 25 mg mL−1 QKF group showed a greater Bcl-2 to the greatest extent among the three doses of QKF decrease than the other two QKF groups that were tested examined (P < 0.05, P < 0.01). 0e expression levels of p-p38 (P < 0.01). MAPK, cleaved caspase-3, and Bax were increased in the 0e expression levels of apoptosis-related proteins in rat model group compared with those in the control, donepezil, hippocampal neurons were measured by Western blot (WB) and 25 mg mL−1 QKF groups (P < 0.01). Compared with analysis (Figures 4(c), 4(d)). 0ere were no evident differ- those in the model group, the expression levels of Bax were ences in the total p38 MAPK or caspase-3 protein expression decreased in the 6.25 mg mL−1 QKF and 12.5 mg mL−1 QKF levels among the six groups (P > 0.05). WB analysis showed groups (P < 0.01), and the expression levels of p-p38 MAPK significantly lower Bcl-2 protein levels in the model group were decreased in the 6.25 mg mL−1 QKF (P < 0.05) and compared with those in the control group (P < 0.01). 0e 12.5 mg·mL−1 groups (P < 0.01). 0e 12.5 mg mL−1 QKF Bcl-2 protein expression level in the QKF and donepezil group exhibited downregulated expression levels of cleaved groups was increased compared to that in the model group caspase-3 (P < 0.05), while the 6.25 mg mL−1 QKF group did (P < 0.01). QKF at 25 mg mL−1 increased the expression of not show an evident difference in the expression of cleaved 6 Evidence-Based Complementary and Alternative Medicine

Biological process ESR1 EDN1 SIRT1 ADRB2 Positive regulation of nitric oxide biosynthetic process CYP3A4 IL4 Response to ethanol IL6 ACE HMGCR Response to drug PIK3CA AVP TP53 NOS1 Aging AR Positive regulation of transcription from INS NOS2 PTEN RNA polymerase II promoter CAT CREB1 PPARG Positive regulation of smooth muscle cell proliferation AKT1 PIK3R1 MAPT RXRA Response to hypoxia –log (P value) SNCA MTOR 10 DNMT1 SOD1 Regulation of blood pressure NFKB1 ADIPOQ COMT AGTR1 Response to nutrient 15.0 HMOX1 TNF MAPK1 Positive regulation of transcription, DNA-templated GRIN2B APOA1 AGT IL1B IGF1 CALB1 Response to nicotine 12.5 NOTCH1 CHAT Response to lipopolysaccharide SLC6A4 NOS3 PTGS2 VDR 10.0 CCL2 CNR1 Positive regulation of apoptotic process APOE FOS Negative regulation of gene expression FGF2 VCAM1 GRIN1 DRD1 SOD2 Protein kinase B signaling TH F2 DLG4 Positive regulation of gene expression REN IFNG CACNA1C GRIA2 Positive regulation of sequence-specific CHRNA4 DNA binding transcription factor activity DRD2 ACHE Cellular response to drug Response to activity Social behavior

0 10203040 Gene ratio

Gene counts 10 20

15 25

(a) (b)

Cellular component Molecular function

Extracellular space Enzyme binding Extracellular region Identical protein binding Plasma membrane Tetrahydrobiopterin binding Transcription regulatory region DNA binding Dendrite Protein binding Cytosol Cytokine activity Axon –log (P value) NADP binding 10 Lysosome –log (P value) 10 Transcription factor binding 12.5 10 Neuron projection Heme binding 10.0 Dendritic spine Protein homodimerization activity 8 Caveola Insulin−like growth factor receptor binding 7.5 Postsynaptic membrane Nitric−oxide synthase activity 6 Receptor binding 5.0 External side of plasma membrane Steroid hormone receptor activity Synapse 4 Arginine binding Perikaryon RNA polymerase II transcription factor activity, ligand−activated sequence−specific DNA binding Nuclear chromatin Growth factor activity Endoplasmic reticulum Hormone activity Phosphatidylinositol 3−kinase complex Core promoter sequence−speciﬁc DNA binding Terminal bouton Dopamine binding Nucleus 0 25 50 75 100 Dendrite cytoplasm Gene ratio 0204060 Gene counts Gene ratio 10 40

Gene counts 20 50 10 30

30 (c) (d)

KEGG pathway

HIF−1 signaling pathway TNF signaling pathway Chagas disease (American trypanosomiasis) Prostate cancer Cocaine addiction CAMP signaling pathway Insulin resistance –log (P value) 10 MTOR signaling pathway 11 Pathways in cancer 10 Amphetamine addiction AMPK signaling pathway 9 Type II diabetes mellitus 8 Leishmaniasis 7 Prolactin signaling pathway Osteoclast diﬀerentiation Amyotrophic lateral sclerosis (ALS) FoxO signaling pathway PI3K−Akt signaling pathway Cholinergic synapse Hepatitis B

0102030 Gene ratio

Gene counts 8 14

10 16

12 18 (e)

Figure 2: Network construction and enrichment analysis. (a) 0e blue circle represents the key target of Qingxin kaiqiao fang in the treatment of Alzheimer’s disease; the edge represents the interaction between the targets predicted by String database, and the thicker the line is, the higher the conﬁdence level is. (b) Biological process in GO enrichment analysis. (c) Cellular component in GO enrichment analysis. (d) Molecular function in GO enrichment analysis. (e) KEGG pathway enrichment analysis. GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes. Evidence-Based Complementary and Alternative Medicine 7

4h 1d 3d 5d 7d

(40X)

(a) MAP2 DAPI Merge Control Model Donepezil 6.25mg/mL QKF 12.5mg/mL QKF 25mg/mL QKF (20X) (b) Figure 3: Qingxin kaiqiao fang reduces the damage of Aββ25-35 to cell morphology. (a) Morphology characteristic of hippocampal neuronal cells at different stages (4 h, 1 day, 3 days, 5 days, and 7 days, respectively) (original magnification, ×400). (b) Immunofluorescence images of microtubule-associated protein-2 (green) and nucleus (blue) (original magnification, ×200). 8 Evidence-Based Complementary and Alternative Medicine

TUNEL DAPI Merge ∗∗ 80 ∗ ∗∗ ∗∗ ∗∗ 60 Control

40 Model

Apoptotic ratio (%) ratio Apoptotic 20

0 Model L-QKF H-QKF Donepezil Control M-QKF Donepezil QKF 6.25mg/mL QKF 12.5mg/mL QKF

25mg/mL (20X) (a) (b)

Control Model Donepezil 6.25mg/mL 12.5mg/mL 25mg/mL (QKF) 4 ∗∗ ∗∗

∗∗ ∗∗∗∗ ∗ ∗∗ p38MAPK ∗ ∗∗ 3 ∗∗ ∗∗ ∗ ∗∗ ∗∗ ∗ p-p38MAPK 2

∗∗ ∗∗ ∗∗ Fold over control over Fold 1 ∗ Caspase-3

0 Cleaved caspase-3 Bax Bcl-2 Caspase-3 p38 MAPK

Bax p-p38 MAPK Cleaved caspase-3 Cleaved

Control 6.25mg/mL QKF Bcl-2 Model 12.5mg/mL QKF Donepezil 25mg/mL QKF

β-tubulin

Figure 4: Qingxin kaiqiao fang reduces hippocampal neuronal apoptosis. (a) TUNEL staining of hippocampal neurons in each group (original magnification, ×200). (b) Statistical analysis results of TUNEL staining. (c) 0e gray value of p38 MAPK, p-p38 MAPK, caspase-3, cleaved caspase-3, Bax, and Bcl-2 in each group. (d) ODs indicative of p38 MAPK, p-p38 MAPK, caspase-3, cleaved caspase-3, Bax, and Bcl- 2 protein expression. Data are expressed as mean ± SD (n � 5), significant differences between the two groups are indicated as ∗P < 0.05 and ∗∗P < 0.01. QKF, Qingxin kaiqiao fang. caspase-3 compared with the model group (P > 0.05). 3.5. QKF Increased Cell Viability. A CCK-8 assay was used to Moreover, the altered protein expression levels in both the test hippocampal neuron viability. As shown in Figure 5, 25 mg mL−1 QKF and donepezil groups showed a consistent there was a sharp decline in hippocampal neuron viability trend, and there were no clear differences in the effects after 24 h of treatment with Aβ25-35 alone compared with between these two groups (P > 0.05). that in the control group (P < 0.01). In comparison with the Evidence-Based Complementary and Alternative Medicine 9

1.5 ∗∗

∗∗ ∗∗ 1.0

CCK8 OD 0.5

0.0 Model Control Donepezil 25mg/mL QKF 6.25mg/mL QKF 12.5mg/mL QKF Figure 5: CCK8 assay demonstrates that Qingxin kaiqiao fang promotes the cell viability of injured hippocampal neurons. Data are expressed as mean ± SD (n � 5); significant differences between the two groups are indicated as ∗∗P < 0.01. QKF, Qingxin kaiqiao fang. model group, the QKF and donepezil groups showed sig- (P < 0.01) and lower expression levels of p-p38 MAPK, nificantly elevated cellular viability (P < 0.01). 0e cleaved caspase-3, and Bax (P < 0.05, P < 0.01). 0e ex- 25 mg mL−1 QKF group showed an obviously improved pression levels of p-p38 MAPK, Bax, and cleaved caspase-3 effect compared to the QKF groups treated with the other in the U46619-treated group were significantly higher than tested doses (P < 0.01), indicating the concentration-de- those in the 25 mg mL−1 QKF group (P < 0.01), and the level pendent neuroprotective effect of QKF. of Bcl-2 was markedly lower than that in the 25 mg mL−1 QKF group (P < 0.01). 0ere was no evident difference in the levels of p38 MAPK and caspase-3 (P > 0.05) in the 3.6. Inhibiting or Activating p38 MAPK Affected the Hippo- 25 mg mL−1 QKF + SB203580 or 25 mg. mL−1 QKF + U46619 campal Neuron Morphology. 0e abovementioned results − groups compared with the H-QKF group. 0ese results demonstrated that 25 mg mL 1 QKF showed the best effect demonstrated that inhibiting p38 MAPK blocked apoptosis, in terms of preserving the morphology and inhibiting the while p38 MAPK activation produced an inverse effect apoptosis of hippocampal neuronal cells among the three − compared to that of p38 MAPK pathway inhibition. doses of QKF assessed. 0erefore, 25 mg mL 1 QKF was used for the subsequent experiments. In addition, p38 MAPK activity was inhibited by SB203580 and activated by U46619 3.8. Inhibiting or Activating p38 MAPK Affected Cell Viability. to determine whether the antiapoptotic effect of QKF on 0e cell survival rate was calculated as (OD experimental neurons was mediated by p38 MAPK silencing. Neurons in −1 group−OD blank group)/(OD 25 mg/mL QKF group - OD blank group) the 25 mg mL QKF + SB203580 group were plumper, their × 100%. In comparison with that in the 25 mg mL−1 QKF synapses were longer and thicker, and the nerve fiber net- group, hippocampal neuron viability in the 25 mg mL−1 work was much more elaborate and dense compared to that QKF + SB203580 group was obviously increased (P < 0.01), in the other two groups (Figure 6). and cell viability in the 25 mg mL−1 QKF + U46619 group was notably decreased (P < 0.01) (Figure 8). 0ese findings 3.7. Inhibiting or Activating p38 MAPK Affected Hippocampal indicated that inhibiting p38 MAPK activity elevates hip- Neuronal Apoptosis. As shown in Figures 7(a) and 7(b), the pocampal neuron viability and that activating the p38 25 mg mL−1 QKF + SB203580 group showed a lower apo- MAPK pathway inhibits hippocampal neuron viability. ptotic rate than the 25 mg mL−1 QKF group (P < 0.01), and the rate of hippocampal neuron apoptosis was higher in the 4. Discussion 25 mg mL−1 QKF + U46619 group than that in the −1 25 mg mL QKF group (P < 0.05). 0e apoptotic rate in the Aβ25-35, a short fragment of the Aβ peptide that is related to AD, SB203580-treated group was notably lower than that in the has been found in experimental studies to have amyloidogenic, −1 25 mg mL QKF + U46619 group. aggregative, and neurotoxic features. Aβ25-35 sedimented faster, WB analysis was employed to measure the extent of p38 indicating an increased tendency toward aggregation compared MAPK phosphorylation and the protein expression levels of with Aβ1-42, which is another peptide widely used to develop AD p38 MAPK, caspase-3, cleaved caspase-3, Bax, and Bcl-2 models in vivo and in vitro and to generate apoptotic signals among the three groups. As shown in Figures 7(c) and 7(d), leading to cell death [14, 15]. Neuronal apoptosis, which is an compared with the 25 mg mL−1 QKF group, the SB203580- important cell process and the main mechanism of neuronal treated group showed higher expression levels of Bcl-2 death, plays an essential role in neurodegeneration in AD. 0e 10 Evidence-Based Complementary and Alternative Medicine

MAP2 DAPI Merge 25mg/mL QKF 25mg/mL + SB203580 QKF

25mg/mL + U46619 QKF (20X)

Figure 6: Immunofluorescence images of microtubule-associated protein-2 (green) and nucleus (blue) in the QKF group, QKF + SB203580 group, and QKF + U46619 group (original magnification, ×200). QKF, Qingxin kaiqiao fang. significance of apoptosis in the pathogenesis of AD has attracted apoptosis in APP/PS1 double transgenic mice through its increasing attention [16]. In a normal physiological environ- effects on the p38 MAPK pathway [19]. Mammalian p38 ment, apoptosis and the natural antiapoptotic defense system MAPK, a member of the MAPK family, is mainly involved in exist in a dynamic balance and the excessive accumulation and promoting apoptosis. Aβ aggregated as extracellular plaques deposition of Aβ disrupt this balance [17]. 0e present study phosphorylates p38 MAPK, which then induces tau revealed that Aβ25-35 at 10 μmol·L-1 destroyed cell morphol- hyperphosphorylation, reduces synaptic plasticity, and ogy, disrupted the connections between neurons, decreased cell eventually induces neuronal apoptosis [20]. 0e results of viability, and increased the number of TUNEL-positive cells. the WB analysis in our present in vitro study showed that the However, these effects were improved after treatment with QKF, phosphorylation of p38 MAPK was significantly increased in which has been used to treat AD for many years and has shown the model group compared with the control group. In the remarkable effects on early symptoms such as cognitive dys- QKF-treated group, a significant decrease in the protein function and behavioral symptoms [18]. expression of p-p38 MAPK was observed compared with the Network pharmacology prediction was applied to deeply model group. In addition to the role of p38 MAPK in ap- explore the potential mechanisms underlying the effects of optosis, a family of intracellular cysteine proteases known as QKF on AD. We first screened the active compounds of QKF caspases are known to exhibit apoptotic effects and to be and identified the target proteins of QKF. 0en, the QKF- involved in the intrinsic mitochondrial apoptosis pathway compound-target-AD network was constructed, and en- [21]. Caspase-3, the final executor of apoptosis and the best- richment analysis was performed. Our analysis indicated characterized apoptosis executor within the caspase family, that QKF was mainly targeted in the cell cycle, cell prolif- can be cleaved by an activated upstream caspase [22]. eration, apoptosis, and inflammation in the treatment of AD. Following the exposure of cells to apoptotic stimuli, caspase It can be seen from the above enrichment analysis that both activation has been demonstrated to rely on the presence of the MAPK pathway and the apoptotic process are very cytochrome c released from mitochondria; however, apo- important. In our previous in vivo study, QKF was found to ptosis can be prevented by the presence of Bcl-2 in these successfully ameliorate memory impairment and inhibit organelles [23]. Bcl-2 is an integral membrane protein Evidence-Based Complementary and Alternative Medicine 11

Tunel DAPI Merge ∗ ∗∗ ∗∗ 30

20 25mg/mL QKF

10 Apoptotic ratio (%) ratio Apoptotic

0 SB203580 25mg/mL + QKF 25mg/mL QKF U46619 25mg/mL + U46619 QKF 25mg/mL + SB203580 QKF 25mg/mL + QKF

(a) (b) 25mg/mL QKF + 25mg/mL QKF + ∗∗ ∗∗ ∗∗ 25mg/mL QKF SB203580 U46619 2.0 ∗∗ ∗∗ ∗∗ ∗∗ p38MAPK ∗∗ 1.5 ∗ p-p38MAPK 1.0

0.5 Caspase-3 H-QKF over Fold

0.0 Cleaved caspase-3 Bax Bcl-2 Bax Caspase-3 p38 MAPK p-p38 MAPK

Bcl-2 caspase-3Cleaved 25mg/mL QKF 25mg/mL QKF + SB203580 β-tubulin 25mg/mL QKF + U46619

Figure 7: 25 mg mL−1 QKF alleviates neuronal apoptosis by p38 MAPK silencing. (a) TUNEL staining in each group of hippocampal neurons (original magnification, ×200). (b) Statistical analysis results of TUNEL staining. (c) 0e gray value of associated proteins in each group. (d) Relative protein expression in each group, as detected by WB analysis. Data are expressed as mean ± SD (n � 5); significant differences between the two groups are indicated as ∗P < 0.05 and ∗∗P < 0.01. QKF, Qingxin kaiqiao fang. located mainly on the outer membrane of mitochondria that decrease p38 MAPK activation by many researchers [25, 26]. are known for improving cell survival and preventing cells In this study, to verify that the inhibition of p38 MAPK from undergoing apoptosis [24]. In contrast, the related activity has an antiapoptotic effect, U46619, an agonist of protein Bax facilitates apoptosis and counters the anti- p38 MAPK, was applied [27, 28]. Our results demonstrated apoptotic role of Bcl-29 (the potential mechanism is shown that the 25 mg mL−1 QKF + SB203580 group, but not the in Figure 9). 0e results of the WB analysis in the present U46619-treated group, exhibited increased preservation of study showed that QKF, especially at a dose of 25 mg mL−1, neuronal morphology, enhanced neuron viability, a de- rescued hippocampal neurons from Aβ-induced apoptosis creased number of TUNEL-positive cells, upregulated ex- by reducing the expression of Bax and cleaved caspase-3 and pression of an antiapoptotic protein, and downregulated by enhancing the expression of Bcl-2. expression of proapoptotic proteins. 0ese findings suggest Because p38 MAPK plays a role in the process of apo- that QKF prevents apoptosis in AD by silencing the p38 ptosis, the p38 MAPK inhibitor SB203580 has been used to MAPK pathway. 12 Evidence-Based Complementary and Alternative Medicine

2.0 ∗∗

∗∗ 1.5

1.0 CCK8 OD

0.5

0.0 25mg/mL QKF 25mg/mL + U46619 QKF 25mg/mL + SB203580 QKF Figure 8: 25 mg mL−1 QKF promotes cell viability by p38 MAPK silencing. Data are expressed as mean ± SD (n � 5); signiﬁcant diﬀerences between the two groups are indicated as ∗P < 0.05 and ∗∗P < 0.01. QKF, Qingxin kaiqiao fang.

Aβ

Bcl Bax 2 Bax Bax Bax Bax p38MAPK – p-p38MAPK SB203580 QKF Cytoc

p38MAPK U46619 Apoptosis

Caspase cascade (including caspase-3)

Promotion Inhibition Figure 9: Aβ treatment significantly overexpressed Bax protein and has a little effect on reducing the expression of Bcl2 protein. 0ese changes released cytochrome c from mitochondria, which resulted in activation of the caspase cascade, including caspase-3, the downstream protein of the caspase family. 0e activation of caspase cascade indirectly stimulated p38 MAPK, resulting in p38 MAPK phosphorylation. Finally, p-p38 MAPK led to apoptosis. QKF enhanced the expression of Bcl-2 and reduced the expression of Bax and cleaved caspase-3, thus affecting subsequent steps.

5. Conclusion Data Availability QKF, especially the high-dose group of 25 mg mL−1, may inhibit 0e basic studying data used to support the findings of this Aβ25-35-induced hippocampal neuron apoptosis by blocking the study were supplied by Dr. Tian-Qi Wang under license and p38 MAPK pathway. Moreover, our finding also demonstrated so cannot be made freely available. Requests for access to that network pharmacology was a reliable way to find targets and these data should be made to Dr. Tian-Qi Wang, tenkiou@ possible mechanisms of traditional Chinese medicine. foxmail.com. Evidence-Based Complementary and Alternative Medicine 13

Conflicts of Interest [8] L. Jong and K. Nam, “Recent advances in the inhibition of p38 MAPK as a potential strategy for the treatment of Alzheimer’s 0e authors declare that there are no conflicts of interest. disease,” Molecules (Basel, Switzerland), vol. 22, no. 8, 2017. [9] G. Xiao, C. Yu, and Z. Li, “p38 mitogen-activated protein kinase gene silencing rescues rat hippocampal neurons from Authors’ Contributions ketamine-induced apoptosis: an in vitro study,” International Tian-Qi Wang wrote the manuscript text. Tian-Qi Wang, Journal of Molecular Medicine, vol. 42, no. 3, pp. 1401–1410, 2018. Xiao-Xiao Lai, Lu-Ting Xu, and Yan Shen prepared figures [10] N. Hironori, M. Ryuichi, Y. Kei et al., “Phosphorylation of p38 and collected samples. Jian-Wei Lin and Shi-Yu Gao ana- mitogen-activated protein kinase downstream of bax-caspase- lyzed data. Hai-Yan Hu and Tian-Qi Wang designed the 3 pathway leads to cell death induced by high D-glucose in experiment. All authors contributed toward data analysis human endothelial cells,” Diabetes, vol. 50, no. 6, pp. 1472– and drafting and critically revising the paper; gave final 1481, 2001. approval of the version to be published; and agree to be [11] Z. Wang, X. Bao, L. Song, Y. Tian, and P. Sun, “Role of miR- accountable for all aspects of the work. 106-mediated mitogen-activated protein kinase signaling pathway in oxidative stress injury and inflammatory infil- Acknowledgments tration in the liver of the mouse with gestational hyperten- sion,” Journal of Cellular Biochemistry, 2019. 0is work was supported by the Second Affiliated Hospital [12] R. Haitao, M. Qinghe, and Y. Natesh, “Protective effects of and Yuying Children’s Hospital of Wenzhou Medical glutathione on oxidative injury induced by hydrogen peroxide University Scientific Research Center, the National Natural in intestinal epithelial cells,” e Journal of Surgical Research, vol. 222, pp. 39–47, 2018. Science Foundation of China (grant no. 81573896), and the [13] B. Diaodiao, S. Zhuo, Z. Junbo, M. Mei, and W. Changli, Natural Science Foundation of Zhejiang Province (grant no. “Study of the mechanism underlying therapeutic effect of LY15H270016). Compound Longmaining on myocardial infarction using a network pharmacology-based approach,” Biomedicine & Supplementary Materials Pharmacotherapy, vol. 118, Article ID 109234, 2019. [14] M. Elisabetta, S. Marini, M. Coletta, F. Orsini, B. Giardina, 0e data of active ingredients and targets are listed in and F. Misiti, “Abeta(31-35) and Abeta(25-35) fragments of Supplementary Tables 1, 2, 3, and 4. 0e data of PPI Network amyloid beta-protein induce cellular death through apoptotic and Functional Enrichment Analysis can be seen in Sup- signals: role of the redox state of methionine-35,” FEBS plementary Tables 5, 6, 7, 8, and 9. (Supplementary Letters, vol. 579, no. 13, pp. 2913–2918, 2005. Materials) [15] C. Pike, D. Burdick, A. Walencewicz, C. Glabe, and C. Cotman, “Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state,” e References Journal of Neuroscience : e Official Journal of the Society for Neuroscience, vol. 13, no. 4, pp. 1676–1687, 1993. [1] F. Xiao, S. Gao, Q. Yi, and P. Yu, “TGF-β1 protection against [16] D. Telegina, K. Suvorov, S. Kozhevnikova, and G. Kolosova, Aβ1-42-induced hippocampal neuronal inflammation and “Mechanisms of neuronal death in the cerebral Cortex during apoptosis by TβR-I,” Neuroreport, vol. 29, no. 2, pp. 141–146, aging and development of Alzheimer’s disease-like pathology 2018. in rats,” International Journal of Molecular Sciences, vol. 20, [2] S. Shimohama, “Apoptosis in Alzheimer’s disease--an up- no. 22, 2019. date,” Apoptosis : An International Journal on Programmed [17] H. Sui, L. Zhang, Z. Liu, and Y. Jin, “Atorvastatin prevents Aβ Cell Death, vol. 5, no. 1, pp. 9–16, 2000. oligomer-induced neurotoxicity in cultured rat hippocampal [3] S. Ami and K. Dimitri, “Binding of cytotoxic a β 25-35 peptide neurons by inhibiting Tau cleavage,” Acta Pharmacologica to the dimyristoylphosphatidylcholine lipid bilayer,” Journal of Chemical Information and Modeling, vol. 58, no. 5, Sinica, vol. 36, no. 5, pp. 553–564, 2015. pp. 1053–1065, 2018. [18] H. Hai, C. Zhi, L. Hui et al., “Fumanjian, a classic chinese [4] F. Sengnou, Z. Bei, L. Ping et al., “PI3K/AKT/mTOR/p70S6K herbal formula, can ameliorate the impairment of spatial pathway is involved in aβ25-35-induced autophagy,” BioMed learning and memory through apoptotic signaling pathway in Research International, vol. 2015, Article ID 161020, 19 pages, the hippocampus of rats with Aβ 1-40 -induced alzheimer’s 2015. disease,” Evidence-Based Complementary And Alternative [5] L. Stephanie, L. Susan, B. Rachal, and J. William, “Hippo- Medicine, vol. 2014, Article ID 942917, 9 pages, 2014. campal activation is associated with longitudinal amyloid [19] L. Xu, X. Lai, Y. Shen, J. Lin, S. Gao, H. Hu et al., “Qingxin accumulation and cognitive decline,” eLife, vol. 6, 2017. kaiqiao fang ameliorates memory impairment and inhibits [6] X. Yan, C. Da, W. Gui, and H. Xio, “Involvement of apoptosis in APP/PS1 double transgenic mice through the P38MAPK activation by NMDA receptors and non-NMDA MAPK pathway,” Drug Design, Development and erapy, receptors in amyloid-β peptide-induced neuronal loss in rat vol. 13, pp. 459–475, 2019. hippocampal CA1 and CA3 subfields,” Neuroscience Research, [20] G. Kheiri, M. Dolatshahi, F. Rahmani, and N. Rezaei, “Role of vol. 85, pp. 51–57, 2014. p38/MAPKs in Alzheimer’s disease: implications for amyloid [7] T. Wipawan, P. Nicha, C. Vorapin et al., “N-benzylcinnamide beta toxicity targeted therapy,” Reviews in the Neurosciences, protects rat cultured cortical neurons from β-amyloid pep- vol. 30, no. 1, pp. 9–30, 2018. tide-induced neurotoxicity,” Neuroscience Letters, vol. 556, [21] O. Brustugun, K. Fladmark, S. Døskeland, S. Orrenius, and pp. 20–25, 2013. B. Zhivotovsky, “Apoptosis induced by microinjection of 14 Evidence-Based Complementary and Alternative Medicine

cytochrome c is caspase-dependent and is inhibited by Bcl-2,” Cell Death and Differentiation, vol. 5, no. 8, pp. 660–668, 1998. [22] W. Shu, N. Lean, and L. Chun, “Effects of antioxidants and caspase-3 inhibitor on the phenylethyl isothiocyanate-induced apoptotic signaling pathways in human PLC/PRF/5 cells,” European Journal of Pharmacology, vol. 518, pp. 96–106, 2005. [23] J. Yang, “Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked,” Science, vol. 275, no. 5303, pp. 1129–1132, 1997. [24] K. Kuan, L. Shing, L. Wei et al., “0e deubiquitinating enzyme inhibitor PR-619 enhances the cytotoxicity of cisplatin via the suppression of anti-apoptotic bcl-2 protein: in vitro and in vivo study,” Cells, vol. 8, no. 10, 2019. [25] S. Jantira, P. Eakkapote, A. Punyanuch et al., “Combination of metformin and p38 MAPK inhibitor, SB203580, reduced myocardial ischemia/reperfusion injury in non-obese type 2 diabetic Goto-Kakizaki rats,” Experimental and erapeutic Medicine, vol. 18, no. 3, pp. 1701–1714, 2019. [26] W. Ya, Z. Tianming, D. You et al., “Genipin ameliorates carbon tetrachloride-induced liver injury in mice via the concomitant inhibition of inflammation and induction of autophagy,” Oxidative medicine and cellular longevity, vol. 2019, Article ID 3729051, 12 pages, 2019. [27] S. Liping, K. Xiaocen, L. Szeyun et al., “0e prostaglandin H2 analog U-46619 improves the differentiation efficiency of human induced pluripotent stem cells into endothelial cells by activating both p38MAPK and ERK1/2 signaling pathways,” Stem Cell Research & erapy, vol. 9, no. 1, p. 313, 2018. [28] R. Zhou, X. Yang, X. Li et al., “Recombinant CC16 inhibits NLRP3/caspase-1-induced pyroptosis through p38 MAPK and ERK signaling pathways in the brain of a neonatal rat model with sepsis,” Journal of Neuroinflammation, vol. 16, no. 1, p. 239, 2019.