Based on Network Pharmacology-Quercetin, a Component of Fuzheng Yiliu Decoction Suppressed Prostate Cancer by Regulating PI3K/AKT Pathway

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Introduction
Prostate cancer (PCa) was the second most common cancer in the world's male population and one of the five leading causes of cancer-related death [1]. Through epidemiological examination and clinical research, it was found that the incidence of PCa is still on the rise [2]. Androgen blocking therapy, surgical resection, radiotherapy, and chemotherapy were the main treatments for PCa. However, there were few symptoms in the early clinical stage of PCa. More than 50~80% of patients were often in the advanced stage when visiting a doctor; thus, losing the opportunity of radical surgery [3,4]. Meanwhile, many PCa cases also risk overdiagnosis and overtreatment [5]. This greatly affected the patient's quality of life and brought heavy family burdens [6,7]. In addition, patients might have serious complications, iatrogenic injuries, drug toxicity, and drug resistance [8,9]. Therefore, it was necessary to develop new treatment methods.
Traditional Chinese medicine was widely used to treat various cancers, including PCa [10][11][12]. More evidence showed that the Chinese herbal compound could stimulate therapeutic activities by adjusting multiple targets [13][14][15]. At the same time, traditional Chinese medicine compounds could augment the sensitivity of cancer patients to chemotherapy drugs and reduce the side effects of conventional chemotherapy [16,17]. In addition, the cost of traditional Chinese medicine was relatively low, which could reduce medical expenses. Fuzheng Yiliu decoction (FZYLD) was one of the standard Chinese medicine compounds: Bayu Tian, Hedyotis diffusa, Atractylodes, Poria, Licorice, Cork, raw jaundice, American ginseng, fairy spleen, Zhimu, Quanqi, Raw land, and Tortoiseshell [18]. Previous studies proved that FZYLD could enhance patients' immunity and improve liver function and fibrosis [19]. Meanwhile, FZYLD could be used for cancer treatment, which could enhance the immune function of patients, reduce the recurrence rate, and improve the cumulative survival rate [20]. Our previous studies also showed that FZYLD combined with docetaxel had an antitumor effect on the castrated drug-resistant PCa mice model [18]. However, the specific mechanism of FZYLD on PCa was still unclear.
Because of its multicomponent and multitarget characteristics, it was hard for traditional research methods to systematically clarify its overall action characteristics. The rise of network pharmacology with the advancement of bioinformatics has provided a new direction for studying conventional Chinese medicine compounds [21][22][23]. This was also an effective method to check FZYLD, which could systematically and comprehensively reveal its bioactive components of FZYLD and its relationship with potential targets. In this study, the pharmacological target prediction database was utilized to examine FZYLD, and the protein-protein interaction network (PPI) of FZYLD's major target in the treatment of PCa was built to investigate its main biological activities and signal pathways. Finally, the effects of quercetin, one of the main components of FZYLD, on the proliferation, migration, apoptosis, and PI3K/AKT signaling pathway of PCa were verified in vivo and in vitro.

Materials and Methods
2.1. Query on the Target Information of FZYLD. The chemical constituents of Bayu Tian, Hedyotis diffusa, Atractylodes, Poria, Licorice, Cork, raw jaundice, American ginseng, fairy spleen, Zhimu, and Quanqi were found by TCMSP database. Raw land and Tortoiseshell composition were from Herb (24) and Batman databases. Compounds were screened if their oral bioavailability (OB) was less than 30% and their drug-likeness (DL) was less than 0.18. Although the DL of taurine and trimethylamine of Quanqi did not meet the screening requirements, they were included because of their high OB. The TCMSP database predicted the target points of the included compound components, and all targets were corrected by the uniprot database to remove nonhuman targets.

Disease Target Searching.
With "prostate cancer" as the keyword, the human genes were searched in GeneCards database [24], NCBI gene database, and OMIM database [25]. After searching, 3165 related genes were found in the GeneCards database, 106 in the NCBI database, and 492 targets in the OMIM database. 3509 PCa-related genes were obtained after combining and eliminating the genes in these three datasets. The common targets of drug diseases were input into the String database [26] to construct a proteinprotein interaction (PPI) network, and the biological species was confined as "Homo sapiens" with a reliability greater than 0.9. The PPI network diagram was drawn by Cytoscape 3.8.0 software [27].
2.3. Topological Analysis. The PPI network was imported into Cytoscape 3.8.0 software. NetworkAnalyzer was used to carry out topology analysis. Target proteins with scores higher than average were selected as key targets through degree sorting.
2.4. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Enrichment. The String database was used to GO enrichment analysis (including biological process (BP), molecular function (MF), and cell component (CC)) of the common targets of drug diseases. Next, the enrichment analysis of the KEGG pathway was carried out to explore biological pathways covering related proteins. Screen items with corrected P value less than 0.05. We used the R 4.0.3 software; installed and referred to clusterProfiler, enrichplot, and ggplot2 package; and drew a histogram.

Liquid Chromatography-Mass Spectrometry (LC-MS).
According to previous studies, we used LC-MS to detect the chemical composition of FZYLD [28]. Simply put, we mixed 1 mL methanol : acetonitrile : water (2 : 2 : 1) with 100 mg FZYLD. After the mixture was precipitated for 10 minutes, it was ultrasonicated at 4°C. After standing at -20°C for 1 hour, the mix was centrifuged to get the supernatant. Drain with a freeze concentrator, and then redissolve with 200 μL of acetonitrile : water (1 : 1). The reconstituted samples were analyzed using an LC-MS instrument (LC-MS-MS-8050, Shimazu, Tokyo, Japan), using a Waters HSS T3 column (100 × 2:1 mm, 1.7 μm). Mobile phases A and B were acetonitrile and 0.1% HCOOH-H2O, respectively. The injection volume was 3 μL. MS-DIAL 4.10 software was used to analyze the data [29].
2.8. Transwell Experiment. PC-3 cells were digested and collected with trypsin (AWC0232, Abiowell). After suspending the cells in a serum-free medium of 2 × 10 6 cells/mL, 100 μL cells were introduced to each well in the upper layer of the chamber. 500 μL of F12K medium was placed in the lower layer of the chamber. The cells were placed in an incubator at 37°C for 48 h. Then, we removed the upper chamber, put it in a new hole, washed it with PBS 3 times, and wiped the upper chamber cells with a cotton ball. The cells were dealt with 4% paraformaldehyde and removed from the film. 0.1% crystal violet (G1062, Solarbio) was dyed for 5 minutes and washed with water five times. The membrane was placed on a glass slide, and the cells on the upper outdoor surface were observed under an inverted microscope (DSZ2000X, Beijing Zhongxiang Hengye Instrument) and photographed. The chamber was soaked in 500 μL 10%    The membranes were incubated with SuperECL Plus (AWB0005, Abiowell) and then imaged and measured by a gel imaging system (Chemiscope 6100, Clinx, China).   6 Andrologia docetaxel group was given an intraperitoneal injection of 0.2 mL of docetaxel (0.25 mg/mL) once every 7 days for 21 days. After the treatment, the mice were killed, and the tumors were removed and weighed. The tumor volume was recorded every three days. Tumor volume was calculated using the following formula: volume ðmm 3 Þ = width 2 × length/2. All animal experiments conform to the standard operating procedures approved by the Animal Ethics Committee.
2.13. Statistical Analyses. GraphPad Prism 8 software (GraphPad Software, Inc., USA) was applied to statistical analyses. Each experiment was performed at least 3 times. Statistical analysis of more than two groups and between two groups was performed using ANOVA and Student's t -test, respectively. Data are presented as means ± standard deviations. P value < 0.05 was considered significant.

Network Pharmacological Analysis of FZYLD.
To study the effect of FZYLD on PCa, we analyzed the common targets of FZYLD and PCa through network pharmacology.
Venn diagram showed that FZYLD and PCa had 198 potential targets in common (Figure 1(a)). PPI network was constructed for common targets of drug diseases. The network diagram has 198 nodes and 875 edges (Figure 1(b)). Topology analysis of the PPI network was carried out. Target proteins with scores higher than the average were selected as key targets through degree ranking, and 69 key targets were screened out in total. Figure 1(c) shows the top 20 targets, among which the top five are MAPK1, AKT1, MAPK3, STAT3, and JUN (Figure 1(c)). To better comprehend the complex interaction among components, diseases, and corresponding targets, we built an FZYLD-PCa-target network diagram (Figure 1(d)). Topological analysis was carried out on the network diagram of FZYLD-PCa-target, and degree values of active components were ranked. The higher the degree of active compounds, the greater their importance in FZYLD. Active compounds with a degree value greater than or equal to 25 were listed in

GO and KEGG Pathway Enrichment
Analysis. The common targets of drugs and diseases were enriched by BP, and CC and MF of GO were screened. A total of 2584 BP, 180 MF, and 73 CC were related. Among them, the BP of FZYLD in treating PCa included response to drug, response to steroid hormone, response to lipopolysaccharide, and cellular response to chemical stress. These targets were related to the membrane (raft, microdomain, and region), caveola, plasma membrane raft, vesicle lumen, protein kinase complex, Cyclin-dependent protein kinase enzyme complex, serine/triad protein kinase complex, organelle outer membrane, etc. The MF of these targets was closely related to nuclear receptor activity, ligand-activated, and transcription factor activity. The top 10 enrichment results of BP, CC, and MF were listed separately (Figure 2(a)). The KEGG pathway enriched the common targets of drugs and diseases, the items with corrected P value less than 0.05 were screened, and 176 signal pathways were enriched. The top 20 enrichment results showed the histogram (Figure 2(b)), including endocrine resistance, IL-17 signaling pathway, bladder cancer, Th17 cell differentiation, and PI3K/AKT signaling pathway.  (Figure 3). The node and the edge of the node represented the relationship among active components, target proteins, and pathways that interact with each other.

Quercetin
Affected the Proliferation of PC-3 Cells. By LC-MS detection, we observed that FZYLD was composed of many chemical components (Supplementary figure 1), including active compounds such as quercetin, luteolin, kaempferol, isorhamnetin, naringenin, and beta-sitosterol, which were predicted through network pharmacology (Supplementary Table 1). Next, we tested the influence of quercetin on PC-3 cells. CCK-8 results showed that the cell viability declined continuously with increased quercetin concentration (Figure 4(a)). At 72 hours, the cell viability difference between the quercetin-treated and the control groups was most pronounced, and the quercetin 150 μmol/ L group had the lowest cell viability. Therefore, we chose quercetin to treat PC-3 cells for 72 h for subsequent experiments. Transwell analysis showed that quercetin inhibited the migration of PC-3 cells in a concentrationdependent manner (Figure 4(b)). The inhibition of migration was most pronounced when cells were treated with 150 μmol/L quercetin, in agreement with the CCK-8 results. TUNEL staining showed that the TUNEL-positive rate of PC-3 cells increased with quercetin concentration (Figure 4(c)). Flow cytometry results showed a significant increase in the apoptosis rate of 150 μmol/L quercetintreated PC-3 cells compared to the control group  10 Andrologia (Figure 4(d)). Besides, we also detected apoptosis-related factor levels (BAX and Bcl-2) and tumor metastasis suppressor gene (nm23-H1). The results showed that quercetin reduced the expression of Bcl-2 and promoted the expression of BAX and nm23-H1 (Figure 4(d)). Next, the core targets such as MAPK1, AKT1, STAT3, and JUN were verified by Western blot. The results revealed that quercetin treatment reduced phosphorylated protein levels of MAPK1, AKT1, STAT3, and JUN but not total protein levels in PCa cells. These results demonstrated that quercetin treatment inhibited the vitality and migration of PCa cells and promoted apoptosis.  3.6. The Active Ingredient Quercetin Was Used to Treat PCa through PI3K/AKT Pathway. The network pharmacological analysis found that the PI3K/AKT pathway in the KEGG pathway had the largest number of targets. We speculate that it might be an important target signaling pathway. Therefore, we tested the influence of quercetin on the PI3K/AKT pathway. The results showed that p-PI3K and p-AKT expressions in PC-3 cells were reduced under quercetin treatment, but did not affect PI3K and AKT expressions in PC-3 cells (Figure 6(a)). To further investigate whether the PI3K/Akt pathway was involved in the effect of quercetin on PCa cells, we treated PC-3 cells with quercetin and PI3K/Akt signaling activator IGF-1. The levels of p-PI3K and p-AKT were significantly increased after IGF-1 treatment relative to the quercetin (150 μmol/L) group. Surprisingly, IGF-1 treatment reversed the increase in apoptosis and the decrease in cell proliferation and migration of PCa cells caused by quercetin (Figures 6(b)-6(d)). In addition, IGF-1 treatment inhibited the decrease in Bcl-2 and increase in BAX and nm23-H1 of PCa cells caused by quercetin. From the above, we speculate that quercetin may promote apoptosis and inhibit the proliferation and migration of PCa cells through the PI3K/AKT pathway.

Discussion
In this study, network pharmacology was used to identify the potential targets, biological function, and signal pathways of FZYLD in the treatment of PCa. In addition, in vitro experiments verified that quercetin, one of the active compounds in FZYLD, could inhibit the proliferation and migration of PC-3 cells, inhibit PI3K/AKT pathway, and promote cell apoptosis. The inhibitory effect of quercetin on PCa was further verified by in vivo experiments. As a new method of Chinese herbal prescriptions research, network pharmacology helped identify the targets, active components, and pharmacological mechanisms in traditional Chinese medicine [33,34]. A total of 185 active compounds and 198 targets of PCa-related compounds in FZYLD were identified by network pharmacology. This result suggested that FZYLD exerts its pharmacological effects on PCa through multiple targets. Quercetin, luteolin, kaempferol, anhydroicaritin, and 7-O-methylisomucronulatol were the top five active compounds. Studies showed that quercetin could inhibit the progression of many cancers, including PCa [35][36][37][38]. It was reported that luteolin inhibited the occurrence and development of PCa by inducing apoptosis of prostate adenocarcinoma (TRAP) transgenic rats [39]. As for kaempferol, it promoted cell apoptosis through an androgen-dependent pathway and inhibited angiogenesis simulation and invasion of PCa [40]. Anhydroicaritin has been reported to have toxic effects on hepatocellular carcinoma cells [41]. With regard to 13 Andrologia 7-O-methylisomucronulatol, it had also been suggested in the previous network pharmacology research that it has the potential effect of resisting PCa [22]. On the whole, we speculated that FZYLD was a multicomponent formula with multitarget therapeutic effect. The relationship between these active compounds and PCa should be further studied.
MAPK1, AKT1, MAPK3, STAT3, and JUN were identified as the top five targets related to PCa and FZYLD. Previous studies have reported that downregulation of MAPK1 might significantly inhibit the growth of PCa cells [42]. Similarly, inhibition of the MAPK/ERK signaling pathway could also inhibit the epithelial-mesenchymal transition of PCa [43]. Combination therapy of quercetin and Midkine could reduce the survival of PCa stem cells by inhibiting AKT phosphorylation and decreasing MAPK protein expression [44]. Kaempferol exerted its anticancer effect through STAT3 pathways [45]. An experimental study confirmed that synergistic inhibition of STAT3 activation by KLF5 and HDAC1 could reduce the invasion and metastasis of PCa [46]. Based on the above evidence, we could speculate that FZYLD might act on the target proteins through its active compounds; thus, having a therapeutic effect on PCa.
GO enrichment analysis found that FZYLD was related to major biological processes (e.g., response to steroid hormone, response to metal ion, response to nutrient levels, response to oxidative stress, response to ketone, and response to reactive oxygen species). Steroid hormone was central in maintaining and developing PCa [47]. Quercetin could inhibit the development of PCa induced by testosterone and carcinogens [48]. The treatment of PCa with zinc ion led to increased metallothionein expression, which was significantly related to the resistance of PCa to cisplatin chemotherapy and radiotherapy [49]. Because of gene changes and abnormal growth, the oxidative stress of reactive oxygen species in cancer cells was higher than that in nonmalignant cells [50]. PCa radiation therapy causes cancer cell damage by generating free radicals and oxidative stress [51]. Vitamin C combined with quercetin had a chemopreventive effect on PCa cells through Nrf 2-dependent oxidative stress [52]. Therefore, we speculated that the impact of FZYLD on PCa might be related to the above biological process.
Previous studies have shown that IL-17 promoted PCa through MMP7-induced epithelial-mesenchymal transformation [53]. In addition, inhibiting the expression of AKT and NF-κB related signal proteins and the secretion of tumor necrosis factors could inhibit PCa cells' survival, migration, and invasion [54]. KEGG analyzed the therapeutic effects of FZYLD on PCa through regulating pathways, including the IL-17 signaling pathway, TNF signaling pathway, and PI3K/AKT signaling pathway. Therefore, we conjectured that FZYLD had a therapeutic effect on PCa through the abovementioned active compounds, target proteins, and signal pathways.
The experimental results of this study confirmed that quercetin, one of the active compounds of the FZYLD, inhibited the proliferation and migration of prostate cells and promoted apoptosis. This is consistent with previous research results [44]. At the same time, experiments in vivo also proved the same result. These results showed that quer-cetin had a certain anti-PCa effect. Next, we detected PI3K/ AKT pathway and found that quercetin inhibited the phosphorylation of PI3K and AKT. PI3K/AKT pathway was one of the most deeply studied signaling pathways in tumorigenesis, which might be involved in PCa's occurrence, maintenance, and metastasis [55]. Garcinia acid (GA) inhibited TNF-α-induced invasion of PC-3 cells by inactivating PI3K/AKT signaling pathway [56]. Previous studies showed quercetin reversed docetaxel resistance in PCa through the androgen receptor and PI3K/AKT signaling pathway [57]. These results prompted that quercetin, the active ingredient of FZYLD, might inhibit PCa progression by inhibiting the activation of the PI3K/AKT pathway.
In a word, our research showed that FZYLD contained many effective active compounds, which might directly target the signal pathways related to PCa. Furthermore, quercetin, one of the active compounds in FZYLD, might inhibit the growth and metastasis of PCa by inhibiting the activation of the PI3K/AKT pathway. This research studied the potential pharmacological mechanism of FZYLD on PCa, which could provide a theoretical basis for screening promising cancer candidate drugs.

Data Availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethical Approval
This study was approved by the Ethics Committee of Guangzhou University of Chinese Medicine (IRB number: 20210224025).