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, re- spectively. 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 pro- liferation, 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.