Abstract Objective To explore the molecular mechanism of Aidi injection in the treatment of prostate cancer (PCa). Materials and methods CCK-8 and colony formation assays were used to detect the effects of Aidi on PC3 and DU145 cells; effects on the cell cycle and apoptosis of DU145 cells were detected by flow cytometry; effects on migration and invasion of PC3 and DU145 cells were detected by wound healing and transwell assay, respectively. The main active components of Aidi, their corresponding targets, and PCa associated pathways were predicted and analyzed by network pharmacology. Then predicted key targets and related signaling pathways were further verified by western blotting. The potential active components of Aidi were predicted by molecular docking technology. Results Aidi significantly inhibited the proliferation, colony formation, migration, and invasion of PC3 and DU145 cells; Aidi induced apoptosis and cell cycle G2/M phase arrest of DU145 cells. Network pharmacology analysis yielded 36 potential core targets of Aidi against PCa, and the top 10 signaling pathways including MAPK, PI3K-Akt, and HIF-1α and so on were enriched. Western blotting confirmed that Aidi upregulated the expression levels of p-JNK, p-p38, p-ERK, and ERK in DU145 cells. Molecular docking study showed that kaempferol, (Z)-1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one, 7-O-methylisomucronulatol, calycosin, and N-salicylidene-salicylamine can be well binding with JNK and p38. Conclusion Aidi could inhibit PCa cell proliferation and metastasis through induction of apoptosis and cell cycle arrest, which may be related to activating JNK and p38 signaling pathway. Keywords: Prostate cancer, Aidi injection, Network pharmacology, JNK and p38 signaling pathway 1. Introduction Prostate cancer (PCa) is a malignant tumor that occurs in prostatic epithelial tissues and is one of the main malignant tumors in elderly men worldwide. There were about 1,276,000 new cases of PCa worldwide in 2018, and more than 359,000 patients died of PCa [[41]1]. The incidence of PCa in China is less than the global average, but with the aging of the population and changes in diet and lifestyle, it has shown a significant upward trend in recent years, and the outlook is not optimistic [[42]2,[43]3]. The main treatment strategies for PCa are radiotherapy, chemotherapy, androgen deprivation therapy, endocrine therapy, targeted therapy, and immunotherapy [[44]4]. Given the life expectancy of patients and the development of the disease, most patients can only be treated with maximum androgen blockade. In the early stages of androgen deprivation, symptoms can be relieved to some extent; however, after a period of remission (lasting an average of 22.5 months), almost all PCa will eventually develop into castration resistant PCa (CRPC) [[45]5,[46]6]. At present, the main treatment methods for CRPC include enzalutamide, abiraterone, carbataxel, and docetaxel, as well as anti-bone-metastasis therapy and immunotherapy [[47]7]. However, these drugs can only control the tumor for a limited period of time, and the treatment is often accompanied by side effects. With the continuous development of traditional Chinese medicine (TCM), combinations of TCM and Western medicine have shown advantages in the treatment of cancer, and the importance and status of TCM in the treatment of cancer are gradually increasing. A systematic review of the relevant literature [[48]8] showed that TCM could be used as one of the main treatment methods for CRPC and could improve the quality of life of patients, delay further development of the disease, improve the curative effect, and prolong survival times. Aidi injection, a Chinese patent formulation, is made by purification of effective components of Mylabris phalerata Pallas (Mylabris), Panax ginseng C.A.Mey. (Ginseng Radix et Rhizoma), Eleutherococcus senticosus (Rupr.&Maxim.) Maxim (Acanthopanacis Senticosi Radix Et Rhizoma Seu Caulis) and Astragalus mongholicus Bunge (Astragali Radix). Aidi has been used clinically for the treatment of a variety of tumors, including lung cancer, liver cancer, colorectal cancer, gastric cancer, and ovarian cancer [[49]9]. Although Aidi has been widely used and studied in the clinical treatment of tumors, there has not been any experimental study of its anti-PCa effects at the cellular or animal levels, and there has been a lack of investigation of its molecular mechanism, which currently limits the clinical applications of Aidi in PCa. As a system biology-based technology, network pharmacology provides an effective method to evaluate the multi-pharmacological effects of TCM at the molecular level [[50]10]. Therefore, in this study, the activity and underlying molecular mechanism of Aidi against PCa were investigated by integrating network pharmacology prediction and molecular biology experimental validations. Molecular docking was employed to validate the binding mode between the active components and their potential targets. These results may provide new insights into the mechanism of action of Aidi in the treatment of PCa. 2. Materials and methods 2.1. Drugs and reagents 2.1.1. Experimental cell lines and drugs The human PCa cell lines PC3 and DU145 were provided by the Key Laboratory of Longevity and Age-related Diseases of Chinese Ministry of Education and the Translational Medicine Research Center of Guangxi Medical University. Benign prostatic hyperplasia cells BPH-1 were obtained from Wuhan Sevier Biotechnology Co., Ltd. Aidi injection (batch no. 20190419, and 20230406) was purchased from Guizhou Yibai Pharmaceutical Co., Ltd. Paclitaxel (PTX) injection(Cat No.E161002), were purchased from Jiangsu Aosaikang Pharmaceutical Co., Ltd.(Jiangsu, China). 2.1.2. Main experimental reagents and consumables Roswell Park Memorial Institute (RPMI)-1640 medium (Cat No. 350-006-CL), penicillin/streptomycin (Cat No. 325-041-EL), were purchased from Beijing Vicente Biotechnology Co., Ltd. (Beijing, China). Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, USA). Annexin V-FITC/PI Apoptosis Detection Kit (Cat No. 550825) and 7-AAD staining solution (Cat No. 340242) were purchased from BD Biosciences (San Jose, CA, USA). Cell Counting Kit-8 (Cat No. C0040) was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). The primary antibodies against the p44/42 MAPK (extracellular-signal-regulated protein kinase [ERK]1/2, Cat. 4695T), p-ERK (Cat. 4370T), C-Jun N-terminal kinase (JNK, Cat. 9252S), p-JNK (Cat. 9255S), p38 (Cat. 9212S), p-p38 (Cat. 4511), β-actin (Cat. 8457), and GAPDH (Cat. 2118L) were purchased from Cell Signaling Technology (Danvers, USA). PD98059(Cat No.E161002), were purchased from Shanghai Yuanye Biotechnology Co., Ltd.(Shanghai, China). 2.2. Experimental method 2.2.1. Study on the chemical composition of Aidi injection 2.2.1.1. Sample solution Take an unopened Aidi, open the bottle and pass it through 0.22 μm the test solution is obtained by filtering through a microporous filter. 2.2.1.2. Test conditions for ultra high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometer (UPLC-Q-TOF/MS) * (1) Liquid phase conditions The chromatographic analysis of the test solution was carried out in the ultra-high performance liquid chromatography analysis system (waters), using the ACQUITY UPLC HSS T3 C18 column (100 mm) as the chromatographic column × 2.1 mm, 1.8 μm i.d., column temperature: 30 °C, mobile phase consisting of aqueous phase (A) containing 0.1 % formic acid and 5 mM ammonium formate, and organic phase of acetonitrile (B), elution conditions: 0–0.3 min: 98 % A; 0.3–7.0 min: 98 %–2 % A; 7.0–9.5 min: 2 %–98 % A; 9.5–10.0 min: 98 % A. * (2) Mass spectrometry conditions The mass spectrometric analysis of the test solution was carried out in the XEVO G2-S QTOF mass spectrometer (Waters Corp, Manchester, United Kingdom) equipped with an electric spray ion source (ESI). The mass spectrometry detection parameters are set to: collision voltage, 3.0 kV; Sample and extraction voltage, 30 and 4.0 V. Desolvent gas rate and temperature, 600 L/h and 350 °C; Source temperature, 100 °C; Scanning time, 0.15 s; Scanning delay, 0.02 s, we used leucine enkephalin as a locking block (m/z 556.2771 in cationic mode and m/z 554.2615 in anionic mode), with a concentration of 0.5 mg/mL and a flow rate of 10 μL/min. Using the MSE collection mode for mass spectrometry data detection, the primary mass spectrometry data collection range is m/z 100–1200, while the secondary mass spectrometry data collection range is m/z 10–1200. 2.2.1.3. Analysis and Identification of Aidi's main chemical components Wash the test solution according to the conditions of ultra-high performance liquid chromatography time-of-flight mass spectrometry to obtain primary and secondary mass spectrometry data. Analyze the chemical composition by comparing the measured mass to charge ratio with the chemical composition mass spectrometry information collected in databases and literature. 2.2.2. Cell proliferation assay The CCK-8 method was used to detect the proliferation of PC3, DU145, and BPH-1 cells treated with different drugs. We adjusted the concentration of PC3, DU145, and BPH-1 cells to 3 × 10^4/mL, cell suspension at 100 μL/well was inoculated into 96-well plates and incubated overnight. Then, cells were treated with different concentrations (Aidi: 0, 25, 50, 75, 100, 150, 200, and 300 μL/mL; PTX: 0, 2.5, 5, 10, 20, 40, 60, 80, 120, and 160 ng/mL) of drugs, and culture was continued for 24 h, 48 h, and 72 h. Besides, Aidi combined with ERK inhibitor PD98059 experiment, PC3 and DU145 cells were pretreated with ERK inhibitor PD98059 (40 μM) for 1 h, and then cotreated with Aidi for 48 h. Then, 10 % CCK-8 medium was added to the culture medium, followed by incubation at 37 °C for 1 h. The optical density (OD) at 450 nm was measured with a microplate reader, and the relative activity of the cells was calculated using the following formula: [MATH: Relativecellactivity(%)=ODvalueofadministrationgroup−ODvalueofblankcontrolgroupODvalueofsolventcontrolgroup−ODvalueofblankcontrolgroup×100% :MATH] GraphPad Prism 8 was used to plot relative activity curves for cells, and SPSS 25 was used to calculate the half-maximal inhibitory concentration (IC[50]) of Aidi against PCa and BPH-1 cells. 2.2.3. Colony formation assay The effect of Aidi on the clonogenic ability of PC3 and DU145 cells was detected by plate clonogenic assay. As previously mentioned [[51]11], PC3 and DU145 cells were inoculated into 6-well plates at a density of 150 cells/well. After 24 h of culture, 2 mL RPMI-1640 complete medium containing different concentrations of Aidi (20 and 40 μL/mL) were added to each well. A negative control group without Aidi was also set up. All groups of cells were cultured in a 37 °C, 5 % CO[2] incubator. The culture was terminated when multiple clonal clumps were observed in the microplates, followed by staining with 0.05 % crystal violet for 30 min. Image J software was used to calculate the number of cell clones, GraphPad Prism 8 was used to draw a statistical chart of the cloning rates, and SPSS 25 was used to analyze the experimental results. 2.2.4. Cell cycle and apoptosis analysis Flow cytometry (FACScan; BD Biosciences) was used to detect the effect of Aidi on DU145 cell cycle and apoptosis. As previously described [[52]12], DU145 cells were inoculated into a 6-cm cell culture dish at a cell concentration of 1.5 × 10^5/mL. After 16 h of culture, 3.5 mL medicated culture medium was added to the cells in the experimental group, and the control group received the same amount of culture medium, for 48 h. Then cell cycle distribution was measured using7-AAD staining, and the proportion of apoptotic cells was determined using an Annexin V-FITC/7-AAD apoptosis detection kit according to the manufacturer's protocol. The raw flow cytometry data were analyzed using Flow Jo software, and the cell proportions in each phase were analyzed with GraphPad Prism 8. 2.2.5. Cell migration assay Cell migration was assessed by wound-healing assay as previously mentioned [[53]13]. Briefly, PC3 and DU145 cells were seeded in 6-well plates according to the cell density of 6 × 10^5 cells/well and 8 × 10^5 cells/well, respectively. The 6-well plates were cultured at 37 °C for 24 h in 5 % CO[2] incubator. Then 20 μg/mL of mitomycin was added to the 6-well plates and incubated at 37 °C for 2 h. Then a vertical wound was created using a sterile 10 μL pipette tip. Cells were treated with serum-free RPMI-1640 medium containing 35 or 70 μL/mL of Aidi for PC3 cells, and 25 μL/mL or 50 μL/mL of Aidi for DU145 cells, and the control group of serum-free medium was set. Cells were photographed using a microscope at three time points (0, 12, and 24 h). The area of migration was measured and analyzed with Image J software. 2.2.6. Cell invasion assay Invasion assay was determined using transwell chambers coated with Matrigel as described previously [[54]14]. Briefly, after 24, 48, and 72 h pre-treatment with different concentrations of Aidi (65 or 130 μL/mL for PC3 cells and 40 or 80 μL/mL for DU145 cells), tumor cells were collected and re-suspended in serum free cell culture media. A total of 8 × 10^4 cells in 100 μL were added in the upper chamber, and 600 μL of medium containing 10 % FBS was added in the lower chamber. The cells were allowed to invade for 24 h, then invaded cells were stained with 0.1 % crystal violet and counted in five random microscopic fields. 2.2.7. Network pharmacology prediction We used TCM Systems Pharmacology (TCMSP) Database ([55]http://old.tcmsp-e.com/tcmsp.php, accessed on February 11, 2020) and TCM Integrated Database (TCMID) ([56]http://119.3.41.228:8000/tcmid/, accessed on February 13, 2020) to collect the active ingredients of Aidi. Target compounds were screened using the criterion of oral bioavailability ≥30 %. Target proteins corresponding to the active ingredients were queried through the TCMSP database. The UniProt database ([57]http://www.unitprot.org/, accessed on February 14, 2020) was used to query the gene name corresponding to the target protein. We then searched the GeneCards database ([58]http://www.genecards.org/, accessed on February 11, 2020) and Online Mendelian Inheritance in Man (OMIM) database ([59]https://www.omim.org/, accessed on February 12, 2020) using the keyword “prostatic cancer” to screen gene targets related to PCa. Furthermore, the overlapped targets both related to the candidate compounds and PCa were kept for network construction and analysis. The protein-protein interaction (PPI) network was constructed by Search Tool for the Retrieval of Interacting Genes/Proteins database (STRING, https://string-db. org/, Version 11.0) with medium confidence 0.400 by default. All the networks were created and visualized via Cytoscape (htttps://cytoscape.org/, Version 3.2.1) a general platform for complex network analysis and visualization [[60]15]. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of intersection genes was carried out. 2.2.8. Western blot analysis Western blot was used to verify the predicted key targets and related signaling pathways. As previously described [[61]16], after treatment with Aidi for 48 h, DU145 cells were collected and the total cell proteins were extracted and quantified by bicinchoninic acid (BCA) method. Protein samples were separated with SDS-PAGE and transferred to polyvinylidene fluoride membranes. Phosphorylated proteins were blocked with 5 % BSA solution, and other proteins were blocked with 5 % skimmed milk for 1 h. Proteins were incubated with primary antibody diluent (dilution ratio 1:1000) for about 12 h, then incubated in secondary antibody diluent (dilution ratio 1:5000) for 1 h, followed by chemiluminescence. Image J software was used to analyze the gray values of the target bands and the internal reference protein. GraphPad Prism 8 was used to plot statistics. The experimental results were statistically analyzed with SPSS 25. 2.2.9. Molecular docking The compound structure (mol2 format) was obtained in the TCMSP database (accessed on April 22, 2021). The protein crystal structure (in pdb format) was extracted from Protein Data Bank (PDB) database ([62]https://www.rcsb.org/, accessed on April 22, 2021). The compound structure and crystal structure were imported into Sybyl X2.0 software for molecular docking. The total score value of Surflex Dock molecular docking module is greater than 6.0 to evaluate the ability of ligands and receptors to bind, screen the possible effective components of Aidi's anti prostate cancer, and visualize the docking mode with Pymol software. 2.3. Statistical method Statistical analysis was performed using SPSS 25. Data were presented as mean ± standard deviation (SD). The two-tailed unpaired Student's t-test was used for comparisons between two groups, the one-way ANOVA was used for comparisons between multiple groups, and the difference was statistically significant at P < 0.05. 3. Results 3.1. UPLC-Q-TOF/MS analysis results 3.1.1. Aidi chemical composition analysis chromatogram After using the UPLC-Q-TOF/MS analyzer to detect Aidi, representative peak intensity chromatograms (BPI) of the collected information in positive and negative ion modes were obtained, as shown in [63]Fig. 1A and B. Fig. 1. [64]Fig. 1 [65]Open in a new tab BPI chromatogram of Aidi injection chemical composition. (A: negative ion mode, B: positive ion mode). 3.1.2. Aidi chemical composition Under positive and negative ion modes, 19 and 23 chemical components of Aidi were identified, including Ginsenoside Rf, Ginsenoside C, Ginsenoside Rh1, Ginsenoside F1, 9,10,13-TriHOME, Pyroglutamic acid, Guanosine, Phenylalanine, Chlorogenic acid, Cryptochlorogenic acid, Astrapterocarpan, etc. 31 compounds were obtained by removing duplicate components (one of which has an unknown compound name). Detailed information on each chemical component is shown in [66]Supplemental Table 1, [67]Supplemental Table 2. 3.2. Aidi inhibited the viability of PCa cells CCK-8 assay was utilized to evaluate the antiproliferative effect of Aidi on PC3 and DU145 cells. As shown in [68]Fig. 2A and B, Aidi injection significantly reduced the cell viability of PC3 and DU145 cells in time- and dose-dependent manners. To further verify the effect of Aidi on non-cancerous cells, the proliferation of prostate hyperplasia cells BPH-1 treated with Aidi was detected by CCK-8 assay. As shown in [69]Fig. 2C and [70]Table 1, the IC[50] values of BPH-1 cells were much greater than those of PC3 and DU145 cells. Besides, The effect of the IC[50] values obtained by PC3 and DU145 cells (Refer to [71]Table 1) on the proliferation of BPH-1 was detected by CCK8 assay. The results showed that the determined IC[50] values of 24 h have a little death effect on non-cancerous cells. Contrary to the above results, the IC[50] values at 48 and 72 h promoted the proliferation in BPH-1 cells, suggesting that it not only inhibited PCa cells but also had a beneficial effect on BPH-1 cells ([72]Supplemental Fig. 1). Fig. 2. [73]Fig. 2 [74]Open in a new tab Aidi inhibited the viability of PCa and BPH-1 cells. (A, B, and C): PC3, DU145 and BPH-1 cells were treated with different concentrations of Aidi (0–300 μL/mL) for 24, 48 and 72 h, and cell viability was detected using the CCK-8 assay. (D and E) PC3 and DU145 cells were treated with different concentrations of PTX (0–160 ng/mL) for 24, 48 and 72 h, and cell viability was detected using the CCK-8 assay. (F) The effects of Aidi on the clonogenic ability of PC3 and DU145 cells were detected by plate cloning assay. (G) The statistical chart of the colony formation rate of PC3 and DU145 cells. **P < 0.01 compared with control group. Table 1. The IC[50] values of Aidi on PCa and BPH-1 cells (μL/mL). Cell lines 24 h 48 h 72 h PC3 251.32 ± 12.47 154.24 ± 3.33 132.71 ± 3.87 DU145 193.65 ± 4.39 135.12 ± 9.8 107.22 ± 4.41 BPH-1 363.95 ± 5.37 231.73 ± 5.96 207.28 ± 5.74 [75]Open in a new tab In addition, the effect of PTX on the proliferation of PC3 and DU145 cells was detected by CCK-8 assay. As shown in [76]Fig. 2D and E, the viability of PC3 and DU145 cells was significantly inhibited by PTX and showed in both dose- and time-dependent manners. After 72 h of PTX treatment, the IC[50] values of PC3 and DU145 cells were 4.38 ± 0.47 ng/mL and 5.97 ± 0.20 ng/mL，respecticely ([77]Table 2). Table 2. The IC[50] values of PTX on PCa cells (ng/mL). Cell lines 24 h 48 h 72 h PC3 292.27 ± 65.61 13.40 ± 1.46 4.38 ± 0.47 DU145 48.95 ± 0.94 35.85 ± 4.43 5.97 ± 0.20 [78]Open in a new tab In order to further evaluate the effect of Aidi on population dependence and proliferation ability in PCa cells, plate cloning experiments were performed. The results are shown in [79]Fig. 2F and G. After treatment with Aidi, the clonogenic ability of PC3 and DU145 cells decreased significantly, and the number and size of clones were less than those of the control group (P < 0.01). The clone formation rates of PC3 and DU145 cells treated with 40 μL/mL of Aidi were 46.66 % and 55.19 %, respectively. 3.3. Aidi induced the apoptosis and cell cycle arrest in DU145 cells To investigate the underlying mechanism by which Aidi inhibited the PCa cell proliferation and cloning formation, we first assessed the effect of Aidi on cell cycle using the flow cytometry and the results were shown in [80]Fig. 3A and B. After treatment with Aidi at a concentration of 40 μL/mL or 80 μL/mL for 48 h, the proportion of DU145 cells in G0/G1 phase decreased and the proportion in G2/M phase increased significantly compared with the control group. These results suggested that Aidi could induce G2/M phase arrest in DU145 cells. Fig. 3. [81]Fig. 3 [82]Open in a new tab Aidi induced the apoptosis and cell cycle arrest in DU145 cells. (A) DU145 cell cycle of flow chart treated by Aidi injection for 48h. (B) The statistical chart of DU145 cell cycle distribution. (C) Flow chart of DU145 cell apoptosis after treatment with Aidi for 48 h. (D) Statistical chart of DU145 cell apoptosis rate. **P < 0.01 compared with control group. Furthermore, flow cytometry was used to verify the influence of Aidi on the apoptosis of DU145 cells, and the results were shown in [83]Fig. 3C and D. After treatment for 48 h with 80 μL/mL of Aidi, the apoptosis rate of DU145 cells was significantly greater than that of the control group, as demonstrated by the fact that the proportion of early apoptotic cells in the lower right quadrant showed a statistically significant increase (P < 0.01). These results suggested that Aidi could induce the apoptosis of DU145 cells. 3.4. Aidi inhibits the migration of PCa cells In order to explore the effects of Aidi on the metastasis of PCa cells, we first observed its effects on the migrative ability of PC3 and DU145 cells by wound healing assay. As shown in [84]Fig. 4, Aidi inhibited the migration of PC3 and DU145 cells to varying degrees: after treatment with Aidi for 12 h and 24 h, the migration rate of PC3 cells was less than that of the control group ([85]Fig. 4A, B, P < 0.01). DU145 cells were treated for 24 h with various concentrations of Aidi. The migration rate of the cells treated with 50 μL/mL of Aidi was significantly less than that of the control group ([86]Fig. 4C, D, P＜0.01). Fig. 4. [87]Fig. 4 [88]Open in a new tab Effects of Aidi injection on migration ability of PCa cells. (A, B) Effects of Aidi on the migration ability of PC3 cells and statistical chart of wound healing. (C, D) Effects of Aidi on the migration ability of DU145 cells and statistical chart of wound healing. **P < 0.01 compared with control group. 3.5. Aidi inhibited the invasion of PCa cells Transwell assay was used to detect the effects of Aidi on the invasive ability of PC3 and DU145 cells. As shown in [89]Fig. 5, Aidi inhibited the invasion of PC3 and DU145 cells to varying degrees: after Aidi treatment for 24 h, 48 h, and 72 h, the invasion rate of PC3 cells was significantly less than that of the control group ([90]Fig. 5A, B, P＜0.01). With the exception of the group treated with Aidi at a low-concentration (40 μL/mL) for 48 h, the invasion rate of DU145 cells in all groups was less than that of the control group, and the difference was statistically significant ([91]Fig. 5C, D, P < 0.01). Fig. 5. [92]Fig. 5 [93]Open in a new tab Effects of Aidi injection on the invasion of PCa cells. (A, B) Effects of Aidi treatment at different concentrations on PC3 cell invasion and statistical chart showing results of transwell assay. (C, D) Effects of Aidi treatment at different concentrations on DU145 cell invasion and statistical chart showing results of transwell assay. **P < 0.01 compared with control group. 3.6. Network pharmacology analysis results 3.6.1. Active ingredients and targets screening To further explore the molecular mechanism underlying the inhibition of proliferation and metastasis by Aidi, we used network pharmacology to predict the possible targets and signaling pathways. A total of 128 ingredients of Aidi were collected according to the ADME threshold of OB ≥ 30 %, which were shown in [94]Supplemental Tables 3 and a herb-ingredient network diagram was shown in [95]Fig. 6A. A total of 430 corresponding targets were predicted to be hit by the 128 ingredients in Aidi, and the detailed information about the targets was shown in [96]Supplemental Table 4. The ingredient-target network was shown in [97]Fig. 6B. Fig. 6. [98]Fig. 6 [99]Open in a new tab Network pharmacology prediction results. A: Herb-ingredient network diagram (diamond nodes indicate herbs in Aidi: red, cantharidin; purple, ginseng; green, Astragalus; yellow, Acanthopanax senticosus) and blue circles indicate the corresponding ingredients. B: Ingredient-target network diagram (purple nodes represent components and blue node represents the corresponding targets). C: Target network diagram for PCa (the yellow node represents PCa and blue nodes represent targets in PCa). D: Venn diagram of target genes and PCa-related genes. E: The network diagram of protein-protein interaction within the common target (the color of nodes in the diagram changes from red to yellow according to the degree of freedom, and the middle node is the selected 36 key common targets). F: Signaling pathways identified by KEGG enrichment analysis (Note: The longer the length of the histogram, the more the number of key genes enriched; The redder the histogram, the smaller the adjusted p-value. (For interpretation of the references to color in this figure legend, the