A Network Pharmacology Approach for Uncovering the Osteogenic Mechanisms of Psoralea Corylifolia Linn

Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2019, Article ID 2160175, 10 pages https://doi.org/10.1155/2019/2160175 Research Article A Network Pharmacology Approach for Uncovering the Osteogenic Mechanisms of Psoralea corylifolia Linn Luna Ge,1 Kai Cheng,2 and Jinxiang Han 1 1Shandong Medicinal Biotechnology Center, Key Laboratory for Biotech-Drugs of National Health Commission, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan 250062, China 2Shandong Cancer Hospital and Institute, Shandong Academy of Medical Sciences, Shandong Cancer Hospital Affiliated to Shandong University, Jinan, Shandong, China Correspondence should be addressed to Jinxiang Han; [email protected] Received 12 June 2019; Revised 6 September 2019; Accepted 11 October 2019; Published 7 November 2019 Academic Editor: Raffaele Pezzani Copyright © 2019 Luna Ge et al. .is 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. Background and Aim. Psoralea corylifolia Linn (PCL) is an herb that is commonly used for alleviating osteoporosis and vitiligo. Although accumulating evidence has demonstrated the antiosteoporotic effect of PCL, the identities of the osteogenic compounds in PCL and their functional targets remain elusive. To investigate the osteogenic ingredients in PCL and their functional mechanisms, network pharmacology analysis was performed on the targets of PCL and osteogenesis. Methods. .e active components of PCL were screened by literature review. .e potential protein targets of the active PCL components were predicted with the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), Search Tool for Interactions of Chemicals (STITCH), SwissTargetPrediction, and PubChem. .e target networks related to PCL and osteogenic differentiation were constructed by using Cytoscape. MC3T3-E1 cells were used to verify the targets. Results. Twenty-three active components of PCL and 162 potential target proteins were identified. Further analysis reduced the number of potential target proteins to 71. Of the 23 components, bavachalcone, psoralen, bavachinin, neobavaisoflavone, methoxsalen, psoradin, bakuchiol, and angelicin may be the main active components of PCL that promote bone formation. PPARc and aryl hydrocarbon receptor (AhR) were verified as targets of PCL in MC3T3-E1 cells, and the western blot and immunofluorescence staining results showed that compared to the control, PCL reduced the expression of these targets. Conclusions. .e active components of PCL and the mechanisms by which they promoted osteogenic differentiation were successfully identified using network pharmacology. 1. Introduction molecular mechanism of PCL that promotes bone formation remains unclear. Psoralea corylifolia Linn (PCL) is commonly used in clinical Traditional Chinese medicine (TCM) is characterized by Chinese medicine. Modern pharmacology shows that PCL having multiple targets and multiple effects, and the tra- has effects including promoting cardiac health; vascular ditional model of “one-drug, one-target” seriously restricts dilatation; and antitumor, antibacterial, and antiworm our ability to scientifically explain the mechanisms of TCM properties. Additionally, PCL is used to treat local hair loss, in the treatment of diseases [4, 5]. Network pharmacology inflammation, vitiligo, leprosy, psoriasis, and eczema [1]. was first proposed by Hopkins in 2007, and this method .e main active components of PCL are coumarin, flavo- establishes a network that maps the action of a drug, in- noids, and flax flavonoids [2]. PCL and some of its active cluding the disease and drug targets [6] Network pharma- components have been shown to promote osteogenic dif- cology aims to study the complex relationships among ferentiation during bone metabolism and can be used as an targets, drugs, diseases, and pathways. .is technique is intervention for osteoporosis [3]. However, the specific novel in drug research and has been proven to be effective in 2 Evidence-Based Complementary and Alternative Medicine the identification of new active components of TCM and measure the ALP activity. .en, the absorbance was mea- their mechanisms of action [7]. sured at 405 nm using a microplate reader and normalized to In this study, network pharmacology was used to identify the total protein concentration. the main active ingredients of PCL and the mechanisms by which they promoted osteogenic differentiation. .en, we 2.5. RT-qPCR. MC3T3-E1 cells seeded in 6-well plates were selected aryl hydrocarbon receptor (AhR) and PPARc to cultured with PCL (0, 40, or 80 μg/mL). Total RNA was perform experimental verification of the network. A flow- extracted using TRIzol reagent according to the manufac- chart of this study is depicted in Figure 1. turer’s instructions. Total RNA samples were reverse tran- scribed into first-strand cDNAs using a ReverTra Ace qPCR 2. Materials and Methods RT kit (Toyobo, Osaka, Japan). Quantitative real-time PCR (qRT-PCR) was performed on a LightCycler 480II real- 2.1. Materials. PCL was purchased from Beijing Heyanling time PCR system (Roche, Mannheim, Germany)® using Pharmaceutical Development Co. Ltd. (Beijing, China) and cDNA as the templates. .e condition of amplification had been identified as the Genuine Medicinal Herb by reactions was as follows: 94°C for 3 min (initial de- Professor Lei Yan in Shandong First Medical University and naturation), 40 cycles of 15 s denaturation at 94°C, and Shandong Academy of Medical Sciences. A BCIP/NBT annealing and extension 64°C for 1 min. All reactions were Alkaline Phosphatase Color Development Kit, DAPI carried out in triplicate. mRNA expression level was cal- Staining Solution, Blocking Buffer for Immunol Staining, culated by the 2–ΔΔCq method with normalization to the Antifade Mounting Medium, a bicinchoninic acid (BCA) GAPDH mRNA level [9]. .e PCR primer sequences are protein assay kit, and penicillin-streptomycin were pur- listed in Table 1. chased from Beyotime (Shanghai, China). A ReverTra Ace Qpcr RT Kit and Taq SYBR Green Qpcr Premix were® purchased from TOYOBO (Shanghai,® China). β-Glycer- 2.6. Western Blot Analysis. Osteoblasts seeded in 75°cm2 ophosphate, P-nitrophenyl phosphate, and ascorbic acid-2- culture bottles were lysed with cell lysis buffer for western phosphate were purchased from Sigma-Aldrich (St. Louis, blotting. .e protein concentration was measured by the MO, USA). TRIzol was obtained from Invitrogen (Carlsbad, BCA assay kit. Protein samples (40 μg/lane) were separated CA, USA). .e anti-GAPDH, anti-PPARc, anti-AhR, and by SDS-PAGE and electrophoretically transferred to a CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG polyvinylidene fluoride (PVDF) membranes (0.45 μm). .e (H + L) antibodies were obtained from Proteintech Group PVDF membranes were blocked in 5% skim milk for 1 h at (Wuhan, China). Alizarin red stain was obtained from room temperature and then were incubated at 4°C overnight Cyagen Biosciences (Guangzhou, China). .e primers were with the primary antibodies against PPARc, AhR, and synthesized by BGI (Shenzhen, China). GAPDH. Subsequently, the membranes were washed three times with TBST and incubated with goat anti-rabbit IgG- HRP secondary antibody for 1 h at room temperature fol- 2.2. Cell Culture. .e MC3T3-E1 (subclone 14) osteoblasts lowed by three wishes with TBST. Blots were visualized cell line was purchased from the Cell Bank of the Type using an enhanced chemiluminescent substrate (ECL) kit. Culture Collection of the Chinese Academy of Sciences .e protein bands were quantified using Image J software (Shanghai, China). .e cells were cultured in α-MEM and were normalized to the density of the respective (HyClone, Logan, UT, USA) containing 10% fetal bovine GAPDH band. serum (Gibco, Waltham, MA, USA), 100 units/mL penicillin ° and 100 μg/mL streptomycin in 5% CO2 at 37 C. Osteogenic induction medium was α-MEM supplemented with ascorbic 2.7. Immunochemical Staining. Osteoblasts seeded in 48-well acid-2-phosphate (50 μg/mL) and β-glycerophosphate plates were fixed with 4% paraformaldehyde at room tem- (10 mM). perature for 15 min followed by three washes with PBS. Cells were permeabilized with 0.3% Triton X-100 for 30 min and blocked with the blocking buffer (Beyotime, Shanghai, China) 2.3. Alizarin Red and Alkaline Phosphatase (ALP) Staining. for 1 h at room temperature. Samples were incubated over- Alizarin red and ALP staining were performed as previously night at 4°C with primary antibody against AhR (1 :100, described [8]. Osteoblasts cultured in 24-well plates were Proteintech). .e next day, the samples were rewarmed at washed with PBS twice and fixed with 4% paraformaldehyde 37°C for 1 h and incubated with an Alexa-Fluor 488-conju- for 30 min at room temperature. Alizarin red strains were gated secondary antibody (1 : 500, Proteintech) for 50 min at prepared with 0.5% Alizarin red S solution (w/v, pH4.2), and 37°C and then stained with DAPI (Beyotime, Shanghai, ALP stains were performed with a BCIP/NBT alkaline China). After three washes, the images were obtained by a phosphatase color development kit. Images of stained laser scanning confocal microscope (Olympus, Tokyo, Japan). samples were captured with a digital camera. 2.8. Preparation of the PCL Water Decoction. .e PCL water 2.4. ALP Activity Measurement. .e BCA protein assay kit decoction was prepared as follows: 50 g of

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