Prostate Formula 1 Activates Inflammatory Pyroptosis via the TXNIP/NLRP3 Pathway and Exerts Anti-Prostate Cancer Effects
https://doi-001.org/1025/17637090692672
Xiaochang Fan1 Qingqi Zeng1*
1 Nanjing University of Chinese Medicine, Nanjing, China, 210023.
* Corresponding author
Abstract: To elucidate the molecular mechanism underlying the anti-prostate cancer effects of Prostate Formula 1, this study adopted an integrated research strategy combining network pharmacology, metabolomics, in vivo and in vitro assays, and mechanistic rescue assays. Network pharmacology screened out 27 core active ingredients of Prostate Formula 1 prescription. Metabolomics verified that 12 ingredients such as kaempferol and benzyl benzoate showed high response in Chinese herbal medicine samples, and molecular docking showed that the binding energy of these ingredients with TXNIP and NLRP3 targets was ≤-7.6kcal/mol. In vitro and in vivo experiments confirmed that Prostate Formula 1 can dose-dependently inhibit DU145 and PC-3 cell tumor activity and reduce the transplanted volume in nude mice, at the same time, the expression of TXNIP, NLRP3, GSDMD, c-caspase-1 protein and the concentration of IL-1β and IL-18 were significantly up-regulated, and the mechanistic rescue experiment showed that after TXNIP silencing, the drug-induced NLRP3 pathway activation and anti-prostate cancer effect were significantly blocked. To sum up, Prostate Formula 1 can activate NLRP3 inflammatory focal death pathway through the combination of core active ingredients and up-regulation of TXNIP, and then play a role in anti-prostate cancer, providing experimental basis for its clinical application.
Keywords: Prostate Formula 1 Prescription; Prostate cancer; TXNIP/NLRP3 pathway; Inflammatory pyroptosis; Network pharmacology
Introduction
Prostate cancer (Prostate, PCa) is one of the most common malignant tumors of the male reproductive system, with more than 1.4 million new cases and about 370,000 deaths [1] worldwide every year. In China, with the aging of the population and the westernization of lifestyle, the incidence of prostate cancer is increasing year by year, and about 30% of the patients are in the advanced stage when they are diagnosed, missing the opportunity of radical surgery. [2]. At present, endocrine therapy and chemotherapy are the main treatments for advanced prostate cancer, but drug resistance and recurrence are prone to occur, and the 5-year survival rate is less than 50%. [3]. Therefore, the search for safe and effective new therapeutic drugs and targets has become an urgent need in the field of prostate cancer research.
Chinese herbal medicine has the unique advantages of “multi-component, multi-target and multi-pathway” in the treatment of malignant tumors. It plays a role by regulating tumor cell proliferation, apoptosis, invasion and tumor microenvironment. [4]. Prostate Formula 1 prescription is an empirical prescription based on the theory of “damp-heat betting, blood stasis and toxin binding” in Chinese herbal medicine. It is composed of many kinds of Chinese herbal medicines such as Bixie, dodder, winter melon seed, Lindera, Acorus tatarinowii, etc. It can improve patients’ pain, dysuria and other symptoms and prolong their progression-free survival time [5] when clinically used for adjuvant treatment of prostate cancer. However, the specific molecular mechanism of its anti-prostate cancer is not yet clear, which limits the clinical promotion and dosage form optimization.
Inflammatory pyroptosis (Pyroptosis) is the 1 proinflammatory programmed cell death mode discovered in recent years. It is triggered by the activation of inflammasome (Inflammasome) and is characterized by cell membrane perforation, release of cell contents and secretion of inflammatory factors such as IL-1β and IL-18. It can not only directly remove tumor cells, but also activate anti-tumor immune response. [6]. NLRP3 inflammasome is one of the most widely studied inflammasome. Its activation needs to be triggered by “two signals”: the first signal activates NF-κB pathway and up-regulates the expression of NLRP3 and IL-1β precursors; The second signal induces NLRP3 oligomerization through potassium ion outflow and active oxygen accumulation, and then recruits ASC and pro-caspase-1 to form inflammasome complex, which activates caspase-1 and finally cleaves GSDMD and IL-1β precursors, causes inflammatory pyroptosis. TXNIP (thioredoxin interacting protein) is a key regulator of NLRP3 inflammasome, which can enhance the activation efficiency of inflammasome by directly binding to NLRP3 and promoting its oligomerization.
Network pharmacology is a new research method based on the theory of systems biology. It can rapidly screen the core active ingredients and key action pathways of Chinese herbal medicine compound by constructing a “component-target-pathway” network, providing a direction for mechanism verification [7]. In this study, the active ingredients and potential targets of Prostate Formula 1 prescription were analyzed by network pharmacology, and the key pathways related to prostate cancer and inflammatory pyroptosis were screened out. Subsequently, its anti-prostate cancer effect and its regulatory effect on TXNIP/NLRP3 pathway were verified by in vitro cell experiments and in vivo nude mouse transplantation tumor experiments. Finally, the necessity of this pathway in the anti-prostate cancer of Prostate Formula 1 was confirmed by TXNIP interference experiment, the purpose of this study is to clarify the molecular mechanism of Prostate Formula 1 activating inflammatory pyroptosis through TXNIP/NLRP3 pathway against prostate cancer, and to provide experimental basis for its clinical application.
1 Materials and methods
1.1 experimental materials
1.1.1 Drugs and reagents
Prostate Formula 1 (based on the Chinese herbal medicine theory of “damp-heat accumulation, blood stasis, and toxin retention, composed of Dioscorea Dioscorea, Cuscuta chinensis, Lindera acornis, Weilingxian, Verbena, winter melon seeds, patrinia, coix seed, hirudo (all according to the weight ratio of raw drugs));DMEM medium, fetal bovine serum (FBS, Gibco, USA);CCK-8 kit (Japan Dojindo Company; annexin V-FITC/PI Apoptosis Detection Kit (BD Company, USA);EDU Cell Proliferation Detection Kit (China Biyuntian Biotechnology Co., Ltd.);Transwell Chamber (Corning Company, USA); Rabbit anti-human TXNIP, NLRP3, GSDMD, c-caspase-1 monoclonal antibodies (CST Company, USA); Rabbit anti-human KI67 monoclonal antibody (Abcam Company, UK);HRP-labeled goat anti-rabbit 2 antibody (Proteintech, China);IL-1β, IL-18 ELISA kit (R & D Systems, USA);TXNIP-siRNA and negative control (NC-siRNA, China Gemma Gene Co., Ltd.);Lipofectamine RNAiMAX transfection reagent (Invitrogen, USA);BCA protein quantitative kit, ECL chemiluminescence kit (Thermo Fisher Scientific, China);4% paraformaldehyde, hematoxylin-eosin (HE) staining solution, immunohistochemical kit (Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China).
1.1.2 Cells and animals
Human prostate cancer cell lines DU145 and PC-3 (Shanghai Cell Bank, Chinese Academy of Sciences); 20 SPF Swiss nude nude mice, male, 4-6 weeks old, weighing 18-22g, [provided by the Animal Center of Ocean University], animal license number: SCXK2024-0001.
1.1.3 Instruments and equipment
CO₂ incubator (Thermo Fisher Scientific, USA); Inverted microscope (Japan Olympus Company); Microplate reader (Bio-Tek Company, USA); Flow cytometer (BD FACSCanto II, USA);Transwell culture plate (Corning Company, USA); Protein electrophoresis instrument, film transfer instrument (Bio-Rad Company, USA); Chemiluminescence imaging system (ProteinSimple Company, USA); Paraffin microtome, drying machine, exhibition machine (Leica Company, Germany); pathological section scanner (3DHISTECH, China).
Analysis of 1.2 Network Pharmacology
Enter 10 herbs of Prostate Formula 1 into TCMSP and HERB databases, and screen potential active ingredients with DL≥ 0.18 and OB≥ 30%. After obtaining SMILES structure by PubChem, it is re-screened with Lipinski’s five principles, and then the core active ingredients are obtained through SwissADME platform (GIabsorption = High, ≥ 3 principles meet). The core components are introduced into the SwissTargetPrediction (species: human) to obtain potential targets, and the active ingredient target library is established by Uniprot the name of transgene and de-duplication.
Search “Prostatecancer/PCa” in the GeneCards and OMIM databases to obtain relevant targets, and merge to rebuild the disease target library. Use Venny2.1 to obtain the intersection of active ingredients and disease targets (potential anti-prostate cancer targets); Introduce STRING (species: human, confidence level ≥ 0.9) to build a PPI network, visualize it by Cytoscape3.10.3, use “NetworkAnalyzer” to calculate the value, and screen Top5 core targets. The intersection target was introduced into DAVID (species: human), GO(BP/CC/MF) and KEGG were enriched (P<0.05), and the micro-information platform was mapped, focusing on NOD-like receptor signaling pathway. Screen NOD-like receptor signaling pathways and corresponding components and targets, build a network with Cytoscape3.10.3, and analyze the core regulatory components with degree values.
1.3 in vitro cell assay
DU145 and PC-3 cells were cultured in DMEM containing 10% FBS and double antibody, passaged at 37 ℃ and 5% CO₂ environment, and the cells were taken for logarithmic phase experiment. Cells were inoculated into 96-well plates, and 0-400 μg/mL prostate No. 1 (5 wells) was added. After 48h of culture, CCK-8 was added. The survival rate was calculated by measuring 490nmOD value. IC50 was obtained by fitting the curve. IC50/4, IC50/2 and IC50 were selected as low/medium/high doses.
The cells were inoculated with 6 well plates, and the apoptosis rate was measured by AnnexinV-FITC/PI staining (n = 3) after 48h of drug intervention. 96 well plate inoculation of cells, drug intervention 48h plus EDU incubation, fixed permeability after Apollo, Hoechst staining, fluorescence microscopy counting the positive rate of EDU (n = 3). The 6-well plate cells were scratched after full layer, and the serum-free medium was added. The width was measured at 0/24/48h to calculate the mobility (n = 3).
The upper chamber was Matrigel, the drug cell suspension was added, and the lower chamber was added with FBS medium. After 24h of culture, the invasive cells were fixed and stained, and the invasive cells were counted (n = 3). Drug intervention 48h to take the supernatant, 450nmOD value, calculate IL-1 beta, IL-18 concentration (n = 3). Drug intervention 48h to extract total protein, BCA quantification after electrophoresis transfer membrane, incubation of TXNIP/NLRP3/GSDMD/c-caspase-1/β-actin 1 resistance, 2 resistance, ECL development, ImageJ quantification (n = 3).
1.4 in vivo xenograft tumor experiment in nude mice
DU145 cells were subcutaneously inoculated into nude mice and divided into 4 groups (n = 5): model group and low/medium/high dose group (100/200/400 mg/kg). The body weight and tumor volume were measured by intragastric administration for 2 weeks. Blood and tumor tissue were taken after administration (fixed/frozen).
Paraffin sections of tumor tissue, hematoxylin-eosin staining after dewaxing and hydration, microscopic pathology, calculated the proportion of necrotic area. After the sections were repaired, the TXNIP/NLRP3/KI67 1 anti, 2 anti, DAB color, and the positive cell rate was calculated. Tumor homogenate supernatant, ELISA measured IL-1 beta/IL-18,WB measured TXNIP/NLRP3/GSDMD/c-caspase-1(n = 3).
1.5 statistical analysis
All experimental data were analyzed by SPSS 26.0 software and GraphPad Prism 9.0 software, and the measurement data were expressed as “mean standard deviation (x s). Multi-group comparison using single-factor variance analysis (One-way ANOVA), two-two comparison using LSD-t test; P<0.05 indicates that the difference is statistically significant.
2 Results
Network Pharmacology Analysis Results of 2.1 Prostate Formula 1 Prescription
2.1.1 Compound core active ingredient screening
In order to clarify the material basis for the anti-prostate cancer effect of Prostate Formula 1, the core active ingredients were screened through multiple databases and multiple criteria. First, in the TCMSP (Chinese herbal medicine system pharmacology database) and HERB (herb group identification database), enter “Dodder, winter melon seed, Lindera, Acorus calamus, Patrinia, Clematis, leech, Coix seed, verbena” 10 ingredients, with drug-like (DL)≥ 0.18, oral bioavailability (OB)≥ 30% as the preliminary screening criteria, and initially obtain 128 potential active ingredients; then the initial screening ingredients were imported into the PubChem database to obtain the corresponding SMILES chemical structure, and the corresponding SMILES chemical structure was re-screened according to the Lipinski’s five principles of class drugs, and 65 ingredients that did not conform to the properties of class drugs were eliminated. Finally, through further verification by the SwissADME platform, 27 core active ingredients with high gastrointestinal absorption (GIabsorption) and at least 3 items of the principles of Lipinski, Ghose, Veber, Egan and Muegge5 were screened, these include quercetin, kaempferol, coix, benzyl benzoate and other key ingredients with clear anti-inflammatory and anti-tumor reports.
2.1.2 Screening and intersection analysis of active ingredient targets and disease targets
Based on the 27 core active ingredients obtained by screening, the potential targets of each ingredient are predicted by SwissTargetPrediction the database (species is set as the Homosapiens), and a total of 724 targets are obtained. These targets are introduced into the Uniprot database for gene name standardization, repeated targets and false positive targets are removed, and finally a target library of active ingredients of Prostate Formula 1 containing 342 genes is constructed.
In order to lock the key targets related to prostate cancer, in the GeneCards database and OMIM database, “Prostatecancer” and “PCa” were used as keywords to search for prostate cancer disease-related targets, and a total of 1839 disease targets were obtained. Excel was used to combine the targets of the two databases and remove duplicate values to construct a prostate cancer disease target library containing 1562 genes.
Through the Venny2.1 online tool, the active ingredient target library and the disease target library were mapped and analyzed, and 238 intersection targets were finally obtained (Figure 2-1). These intersection targets are potential key targets related to the occurrence and development of prostate cancer, providing the core direction for subsequent mechanism research.
Figure 2-1 Venny diagram of the intersection of active ingredient targets of Prostate Formula 1 and prostate cancer disease targets
2.1.3PPI network construction and core target screening
In order to analyze the interaction relationship between 238 intersection targets, it is imported into STRING database (set species as Homosapiens, confidence ≥ 0.900), and a protein-protein interaction (PPI) network is constructed. The network includes 238 nodes (representing target proteins) and 889 edges (representing protein interactions). The network structure is complete after hiding isolated nodes, it can clearly reflect the synergistic relationship between targets (Figure 2-2).
Fig. 2-2 PPI network diagram of potential targets of prostate cancer resistance of Prostate Formula 1
Import PPI network data into Cytoscape3.10.3 software for visual processing, use the “NetworkAnalyzer” plug-in to calculate the degree value of each node (Degree, representing the number of interactions between the target and other targets), sort the degree value from high to low, and filter out the TOP5 core targets, SRC (degree value = 89), AKT1 (degree value = 85), HSP90AA1 (degree value = 82), STAT3 (degree value = 78), PIK3R1 (degree value = 75). Among them, SRC is a member of the tyrosine kinase family, which can regulate tumor cell proliferation and invasion; AKT1 is a key node of the PI3K/AKT pathway, which is closely related to tumor drug resistance, suggesting that these core targets may play a central regulatory role in the anti-prostate cancer process.
2.1.4GO and KEGG enrichment analysis
In order to clarify the association between the biological function and signaling pathway of 238 intersection targets, they were imported into DAVID database for GO gene function enrichment analysis and KEGG pathway enrichment analysis, and the screening conditions were P<0.05.
GO function enrichment analysis obtained 769 biological process (BP) entries, 107 cell component (CC) entries and 234 molecular function (MF) entries. The biological processes are mainly concentrated in “epidermal growth factor receptor signaling pathway regulation”, “apoptosis negative regulation”, “PI3K/AKT signaling pathway positive regulation”, “inflammatory response regulation”, etc., which are closely related to tumor cell growth inhibition and inflammatory microenvironment regulation; the molecular function is mainly based on “protein tyrosine kinase activity”, “ATP binding” and “cytokine receptor binding”, reflecting that the target participates in biological processes by regulating enzyme activity and signal binding (Figure 2-3).
Figure 2-3 Functional enrichment analysis of potential targets of prostate cancer for Prostate Formula 1
A total of 143 significantly enriched pathways were obtained by KEGG pathway enrichment analysis. TOP20 pathways were screened out for visualization according to the order of enrichment multiple from high to low (Figure 2-4). Among them, pathways closely related to inflammatory pyroptosis and tumor development include NOD-like receptor signaling pathway (hsa04621), PI3K-Akt signaling pathway (hsa04151), TNF signaling pathway (hsa04668), etc. NOD-like receptor signaling pathway is the core pathway mediating inflammatory pyroptosis, which can induce tumor cell death by activating NLRP3 inflammasome. Combined with the core direction of “inflammatory pyroptosis against prostate cancer” in this study, it is suggested that NOD-like receptor signaling pathway may be the key pathway for Prostate Formula 1 to intervene in prostate cancer.
Fig. 2-4 TOP20 diagram of enrichment analysis of KEGG pathway for potential targets of prostate cancer by Prostate Formula 1
2.1.5 “Component-Target-Pathway” Network Construction and Analysis
In order to further clarify the association between the core active components and key targets of Prostate Formula 1 prescription and NOD-like receptor signaling pathway, NOD-like receptor signaling pathway and corresponding active components and targets were extracted from KEGG enrichment results, and Cytoscape3.10.3 software was introduced to construct the network diagram of “active components-target-pathway” (Figure 2-5).
Figure 2-5 “Core Active Ingredients-Key Targets-NOD-like Receptor Signaling Pathway” Network Diagram of Prostate Formula 1
The network consists of 27 active component nodes (red), 15 target nodes (blue), 1 pathway node (green), and 43 edges (representing interaction relationships). The node degree value was calculated by the “NetworkAnalyzer” plug-in, and the results showed that among the active ingredients, benzyl benzoate (degree value = 8), kaempferol (degree value = 7), and coix (degree value = 6) had the highest degree value, which could act on multiple targets at the same time; among the targets, NLRP3 (degree value = 9), TXNIP (degree value = 8), and CASP1 (degree value = 7) have the highest degree values, which are the core regulatory targets in the pathway. In combination with existing studies, kaempferol can activate inflammatory response by inhibiting NF-κB pathway, and TXNIP can directly bind NLRP3 to promote inflammasome assembly, suggesting that these core components may activate NOD-like receptor signaling pathway by regulating NLRP3, TXNIP and other targets, and then induce inflammatory pyroptosis of prostate cancer cells and play an anti-prostate cancer role.
Correlation Analysis of Metabonomics Verification and Experimental Results in Vitro and in Vivo of 2.2 Prostate Formula 1 Prescription
2.2.1 Core Results of Metabonomics Test for Prostate Formula 1 Prescription
In order to clarify the material basis for the role of Prostate No. 1, the ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) technology was used for metabolomics testing, and a total of 3 Chinese medicine samples were tested, and the experimental process and parameters strictly followed the specifications (Table 2-1). The samples were extracted with methanol, centrifuged at 13000rpm for 5 minutes, separated by Waters Acquity UPLC BEH C18 column, and detected by Sciex TripleTOF 6600 mass spectrometer in positive and negative ion mode. Finally, 29 main chemical components were identified by MS-DIAL 5.5 software and GNPS database.
Table 2-1 Metabonomics Test Parameters of Prostate Formula 1 Prescription
| Experimental link | Specific parameters |
| Sample processing | 50μL Chinese herbal medicine sample + 200μL LC-MS-grade methanol, centrifuge at 13000rpm for 5min, take supernatant and sample |
| chromatographic system | Shimadzu LC-20 ADXR,Waters Acquity UPLC BEH C18 Column (2.1mm × 100mm,1.7 μm) |
| chromatographic conditions | Injection volume 8 μL, column temperature 40 ℃, flow rate 0.3 mL/min; Mobile phase A:0.1 formic acid water, B:0.1 formic acid methanol |
| gradient elution procedure | 0.1-0.15min:1%-99% B ;15-18min:99% B ;18-18.1min:99%-1% B ;18.1-23min:1% B |
| mass spectrometry system | Sciex TripleTOF 6600 mass spectrometer, ESI ion source |
| Mass Spectrometric Parameters | Positive ion spray voltage 5.5KV, negative ion 4.5KV; Capillary temperature 500 ℃; Scanning range 50-1000 m/z |
| Data analysis | MS-DIAL 5.5 software (peak alignment, retention time correction),GNPS database identification |
Among them, there are 12 core active ingredients coinciding with the screening results of network pharmacology, including benzyl benzoate, kaempferol, 8-isopentenyl kaempferol, anethole, coix, diosgenin, yellow vine alkali and so on (Table 2-2). These ingredients show high response intensity in positive and negative ion mode (Figure 2-6, Figure 2-7), which further verifies the reliability of the screening results of network pharmacology, the key material basis is locked for the follow-up mechanism research.
Table 2-2 Coincidence Table of Metabolomics Identification Core Components and Network Pharmacology of Prostate Formula 1 Prescription
| Serial Number | Core Component Name | Metabolomics Detection Response Strength | Metabolomics Detection Response Strength | Network Pharmacology Match | Associated Target |
| 1 | Benzyl benzoate | 3.8 × 10. | 2.5 × 10 | Yes | GSK3B, EGFR |
| 2 | kaempferol | 4.2 × 10. | 3.1 × 10. | Yes | AKT1, STAT3 |
| 3 | 8-Isopentenyl kaempferol | 2.9 × 10. | 1.8 × 10 ﹤ | Yes | NLRP3, CASP1 |
| 4 | anethole | 2.1 × 10. | 1.5 × 10. | Yes | TNF, IL6 |
| 5 | Coix | 3.5 × 10 | 2.2 × 10. | Yes | MAPK1, PI3KCA |
| 6 | Diosgenin | 2.7 × 10. | 1.9 × 10. | Yes | ABCG2, CYP19A1 |
| 7 | yellow vine alkali | 3.0 × 10. | 2.0 × 10. | Yes | ALOX5, PARP1 |
Figure 2-6 Total Ion Current of Core Components in Prostate Formula 1 Square Ion Mode
Figure 2-7 Ion current diagram of core component extraction in negative ion mode of Prostate Formula 1
2.2.2 Verification of association between core components and NLRP3 signaling pathway
Network pharmacology analysis showed that the core components of Prostate Formula 1 prescription were mainly enriched in NOD-like receptor signaling pathway (hsa04621, NLRP3 signaling pathway), which is the key pathway mediating cell inflammatory pyroptosis. Combined with the results of metabolomics identification, the binding ability of core components with key proteins of NLRP3 signaling pathway (TXNIP, NLRP3, CASP1, GSDMD) was further verified by molecular docking. The results showed that the binding energy of kaempferol with NLRP3 protein was the lowest, and the binding energy of benzyl benzoate with TXNIP protein was -7.6kcal/mol, regulation of NLRP3 inflammatory pyroptosis pathway activation.
2.2.3 In vitro experimental results
CCK-8 assays demonstrated that Prostate Formula 1 significantly inhibited the viability of human prostate cancer cell lines DU145 and PC-3, of which the half inhibitory concentration (ICO) on DU145 cells was 86.4 μg/mL and the ICO on PC-3 cells was 92.7 μg/mL. Follow-up experiments were carried out based on the low dose (ICthe/4), medium dose (ICthe/2) and high dose (ICthe/2). The results showed that the apoptosis rate, proliferation rate, migration rate and invasion ability of DU145 cells in the high dose group were significantly changed compared with the control group, and the inhibitory effect increased with the increase of dose (Table 2-3).
Table 2-3 Effects of Prostate Formula 1 Recipe on apoptosis, proliferation, migration and invasion of DU145 cells (x ± s,n = 3)
| Group | Dose (μg/mL) | Apoptosis rate (%) | Proliferation rate (%) | Mobility (%) | Number of Invasive Cells (units/field) |
| control group | 0 | 5.2±0.8 | 98.6±3.2 | 65.4±4.1 | 128.3±8.5 |
| low dose group | 21.6 | 12.5±1.3** | 72.4±2.8** | 48.2±3.5** | 92.5±6.3** |
| middle dose group | 43.2 | 23.8±2.1** | 51.7±3.0** | 35.6±2.9** | 64.8±5.7** |
| high dose group | 86.4 | 38.6±2.5** | 56.3±2.6** | 31.7±2.4** | 53.7±4.9** |
Note: Compared with the control group, **P<0.01; The proliferation rate is calculated as 100 of the control group, and the migration rate = (initial width of scratch-width after 24h)/initial width of scratch × 100%
ELISA experimental results showed that Prostate Formula 1 prescription dose-dependently increased the concentrations of inflammatory pyroptosis-related cytokines IL-1β and IL-18 in the supernatant of DU145 cells. The concentration of IL-1β in the high dose group reached 286.4pg/mL ± 18.7pg/mL, which was 5.3 times higher than that in the control group (45.2pg/mL ± 4.1pg/mL). The IL-18 concentration reached 215.3pg/mL ± 15.2pg/mL, compared with the control group (38.5pg/mL ± 3.6pg/mL), it increased by 4.6 times (P<0.01). See Table 2-4 for specific data and result trend.
Table 2-4 Effect of Prostate Formula 1 Prescription on the Concentration of IL-1β and IL-18 in the supernatant of DU145 Cells (x ± s,n = 3)
| Group | Dose (μg/mL) | IL-1β concentration (pg/mL) | IL-18 concentration (pg/mL) | Relative times of IL-1β (vs control) | Relative fold of IL-18 (vs control) |
| control group | 0 | 45.2±4.1 | 38.5±3.6 | 1.0 | 1.0 |
| low dose group | 21.6 | 98.5±7.2** | 86.3±6.5** | 2.2 | 2.2 |
| middle dose group | 43.2 | 182.6±12.5** | 145.8±10.3** | 4.0 | 3.8 |
| high dose group | 86.4 | 286.4±18.7** | 215.3±15.2** | 6.3 | 5.6 |
Note: **P<0.01 compared with control group
The results of Western Blot(WB) experiment showed that Prostate Formula 1 prescription dose-dependently increased the relative expression of TXNIP, NLRP3, GSDMD and c-caspase-1 proteins in DU145 cells. The expression of these proteins in the high dose group was 2.8 times, 3.2 times, 2.5 times and 3.0 times that of the control group respectively (P<0.01), which was completely consistent with the prediction mechanism of “core component → NLRP3 pathway → inflammatory focal death, specific protein expression bands and quantitative data are shown in Table 2-5.
Table 2-5 Effect of Prostate Formula 1 Prescription on Relative Expression of TXNIP/NLRP3 Pathway Related Proteins in DU145 Cells (x ± s,n = 3)
| Group | Dose (μg/mL) | TXNIP(vs GAPDH) | NLRP3(vs GAPDH) | GSDMD(vs GAPDH) | c-caspase-1(vs GAPDH) |
| control group | 0 | 1.00±0.08 | 1.00±0.07 | 1.00±0.09 | 1.00±0.06 |
| low dose group | 21.6 | 1.52±0.11** | 1.68±0.12** | 1.45±0.10** | 1.62±0.09** |
| middle dose group | 43.2 | 2.15±0.15** | 2.46±0.16** | 1.98±0.13** | 2.25±0.14** |
| high dose group | 86.4 | 2.80±0.18** | 3.20±0.21** | 2.50±0.17** | 3.00±0.19** |
Note: Compared with the control group, **P<0.01; GAPDH was used as the internal reference protein, and the relative expression level of protein = gray value of target protein/gray value of internal reference proteinFigure 2-1 TXNIP, NLRP3, GSDMD, c-caspase-1 protein map
In summary, in vitro experiments confirmed that Prostate No. 1 can inhibit the viability, proliferation, migration and invasion of prostate cancer cells, and activate the TXNIP/NLRP3 inflammatory pyroptosis pathway (up-regulation of pathway proteins and IL-1β, IL-18 expression) to play an anti-prostate cancer effect, and the effect was dose-dependent.
2.2.4 In vivo experimental results
In order to verify the anti-prostate cancer effect of Prostate Formula 1 prescription in vivo and its regulatory effect on TXNIP/NLRP3 inflammatory focal death pathway, DU145 cell subcutaneous transplanted tumor model in nude mice was established. Control group (normal saline), low dose group (100 mg/kg), medium dose group (200 mg/kg) and high dose group (400 mg/kg) were set up, and the relevant indexes were detected as follows: during the administration of nude mice in each group no death, drug group spirit, eating, hair state and control group no difference, no toxic reaction. The final body weight of the control group was (23.5±1.2)g, and the drug group was (21.9±1.1)-(22.8±1.0)g, with no statistical difference between the groups (P>0.05), suggesting that the drug is safe.
There was no difference in tumor volume between the groups before administration (P>0.05). After 14 days of administration, the tumor volume (1286.5±156.3)mm and weight (1.32±0.15)g in the control group, the volume (428.6±85.4)mm and weight (0.42±0.08)g in the high dose group were 66.7 and 68.2 lower than those in the control group respectively (P<0.01), and the inhibitory effect increased with the increase of dose.
2.2.5 Pathological morphology and pathway regulation
HE staining showed that the tumor necrosis area accounted for (5.8±1.2)% in the control group and (35.6±4.2)% in the high dose group (P<0.01), accompanied by inflammatory cell infiltration. Immunohistochemistry and WB results showed that the positive cell rate and protein expression of TXNIP and NLRP3 in the high dose group were significantly higher than those in the control group (P<0.01), the tumor tissue IL-1β(238.6±15.3)pg/mL and IL-18(202.4±12.8)pg/mL in the high dose group were significantly higher than those in the control group (P<0.01) in a dose-dependent manner (Table 2-6).
In summary, Prostate No. 1 can inhibit the growth of transplanted tumor in nude mice in a dose-dependent manner, and the mechanism is related to the activation of TXNIP/NLRP3 inflammatory focal death pathway and the promotion of IL-1β/IL-18 release, and the safety is good.
Table 2-6 Effect of Prostate Formula 1 Prescription on Body Weight, Volume and Weight of DU145 Transplanted Tumor in Nude Mice (x ± s,n = 5)
| Group | Dose (mg/kg) | Initial weight (g) | Terminal weight (g) | Tumor volume (mm³) | Tumor weight (g) | Tumor Inhibition Rate (%) |
| control group | – | 20.3±1.1 | 23.5±1.2 | 1286.5±156.3 | 1.32±0.15 | – |
| low dose group | 100 | 20.5±0.9 | 22.8±1.0 | 892.4±120.5 * | 0.95±0.11 * | 28.0 |
| middle dose group | 200 | 20.1±1.0 | 22.3±0.9 | 654.2±98.7** | 0.68±0.09** | 48.5 |
| high dose group | 400 | 20.4±0.8 | 21.9±1.1 | 428.6±85.4** | 0.42±0.08** | 68.2 |
Note: Compared with the control group, * P<0.05,**P<0.01; Compared with the low-dose group, P<0.05,P<0.01; Tumor inhibition rate =
control group tumor weight-administration group tumor weight)/control group tumor weight x 100%
Table 2-7 Effects of Prostate Formula 1 Prescription on Necrosis Area, Pathway Indexes and Cytokines of Xenograft Tumor in Nude Mice (x ± s,n = 5)
| Group | Dose (mg/kg) | Percentage of necrotic area (%) | Rate of TXNIP positive cells (%) | Rate of NLRP3 positive cells (%) | Tumor tissue IL-1β(pg/mL) | Tumor tissue IL-18(pg/mL) |
| control group | – | 5.8±1.2 | 12.5±2.3 | 10.8±1.9 | 65.8±7.1 | 58.5±6.3 |
| low dose group | 100 | 12.3±2.1** | 28.6±3.5** | 25.3±3.1** | 102.5±8.6** | 85.6±7.2** |
| middle dose group | 200 | 21.5±3.4** | 45.2±4.1** | 42.8±3.8** | 165.3±10.2** | 138.4±9.5** |
| high dose group | 400 | 35.6±4.2** | 68.9±5.3** | 65.4±4.9** | 238.6±15.3** | 202.4±12.8** |
Note: **P<0.01 compared with control group; P<0.05,P<0.01 compared with low dose group
2.3TXNIP/NLRP3 pathway regulatory mechanistic rescue experiment
Combined with network pharmacology prediction, metabolomics verification and in vivo and in vitro experimental results, it is clear that TXNIP, as the upstream key molecule of NLRP3 inflammatory body activation, may be the core target of Prostate Formula 1 to regulate inflammatory pyrodeath anti-prostate cancer. Referring to the mechanism verification idea of high uric acid inducing cell pyroptosis through TXNIP/NLRP3 pathway, the experimental system of “loss of function-drug intervention-phenotype recovery” was designed. The core objectives include: ① verifying the blocking effect of TXNIP silencing on NLRP3 pathway activation induced by Prostate Formula 1 prescription; ② clarifying the necessity of TXNIP in drug regulation of cell proliferation, apoptosis and inflammatory pyroptosis phenotype; ③ Improve the molecular mechanism chain of “core component → TXNIP → NLRP3 pathway → inflammatory pyroptosis.
2.3.1TXNIP silencing blocks drug-induced NLRP3 pathway activation
The qRT-PCR results showed that compared with si-NC + Drug group, si-TXNIP + Drug group decreased TXNIPmRNA expression by 72.3 (P<0.01), while NLRP3 and CASP1mRNA expression decreased by 58.6 and 61.2 respectively (P<0.01)(Fig. 2-2). WB results showed that the relative expression levels of TXNIP, NLRP3, GSDMD and c-caspase-1 proteins in si-NC + Drug group were 2.8 times, 3.2 times, 2.5 times and 3.0 times of those in control group respectively, while the above protein levels in si-TXNIP + Drug group were significantly decreased compared with those in drug group, with NLRP3 protein decreased to 42.7% of that in drug group (P<0.01)(Figure 2-3), suggesting that TXNIP is a necessary molecule for drug activation of NLRP3 pathway.
Figure 2-2 Expression of mRNA
Fig.2 -3 Protein map of TXNIP, NLRP3, GSDMD and c-caspase-1
2.3.2Regulation of TXNIP silencing reversal drugs on inflammatory pyroptosis phenotype
ELISA test showed that the concentration of IL-1β and IL-18 in the cell supernatant of the drug group reached 286.4pg/mL and 215.3pg/mL respectively, which were significantly higher than that of the control group (P<0.01). However, the concentration of IL-1β and IL-18 in the si-TXNIP + Drug group decreased to 112.5pg/mL and 98.7pg/mL, which were 60.7% and 54.2% lower than that in the drug group (P<0.01), LDH release also decreased from 45.2 to 22.3 in the drug group (P<0.01)(Table 2-3). Immunofluorescence results showed that the co-localization signal of NLRP3 and GSDMD-N fluorescence in the drug group was significantly enhanced, while the co-localization signal was significantly weakened after TXNIP silencing, which confirmed that the absence of TXNIP could block the drug-induced inflammatory pyroptosis.
Table 2-8 Effect of TXNIP Silencing on inflammatory focal death Indexes Induced by Prostate Formula 1 Prescription (x ± s,n = 3)
| Detection index | control group | Drug group | si-NC + Drug Group | si-TXNIP + Drug Group | P-value (si-TXNIP + Drug vs Drug) |
| IL-1β(pg/mL) | 45.2±4.1 | 286.4±18.7** | 278.6±16.3** | 112.5±9.8** | <0.01 |
| IL-18(pg/mL) | 38.5±3.6 | 215.3±15.2** | 208.4±13.7** | 98.7±8.2** | <0.01 |
| LDH release rate (%) | 12.5±1.3 | 45.2±3.8** | 43.6±3.5** | 22.3±2.1** | <0.01 |
Note: **P<0.01 compared with control group
2.3.3 cell proliferation and apoptosis phenotype reverting effect
CCK-8 experiments showed that the cell viability of the drug group was 58.3% lower than that of the control group (P<0.01), while the cell viability of the si-TXNIP + drug group was 42.6% higher than that of the drug group (P<0.01). The results of flow cytometry showed that the apoptosis rate of the drug group was 38.6%±2.5%, and the apoptosis rate of the si-TXNIP + Drug group was reduced to 20.3%±1.8%, which was significantly lower than that of the drug group (P<0.01), suggesting that TXNIP silencing can partially reverse the inhibition of prostate cancer cell proliferation and the induction of apoptosis.
Table 2-9 Effect of silencing TXNIP on the viability and apoptosis of DU145 cells regulated by Prostate Formula 1 (x ± s,n = 3)
| Group | Processing method | Cell viability (% vs control) | Apoptosis rate (%) |
| control group | Untransfected +0 μg/mL drug | 100.0±4.2 | 5.2±0.8 |
| Drug group | Untransfected +86.4 μg/mL drug | 41.7±3.1** | 38.6±2.5** |
| si-NC + Drug Group | Transfection NC-siRNA +86.4 μg/mL drug | 43.2±3.5** | 36.9±2.2** |
| si-TXNIP + Drug Group | Transfection TXNIP-siRNA +86.4 μg/mL drug | 79.5±4.5** | 20.3±1.8** |
Note: **P<0.01 compared with control group
3 Discussions
Through the systematic research paradigm of “network pharmacology-metabolomics-in vivo and in vitro assays-mechanistic rescue”, this study for the first time clarified the core material basis and molecular mechanism of Prostate Formula 1 anti-prostate cancer. Network pharmacology screened 27 core active ingredients. Metabolomics showed that 12 ingredients such as kaempferol and benzyl benzoate showed high response intensity in Chinese herbal medicine samples, and molecular docking showed that these ingredients had strong binding activity (binding energy ≤-7.6kcal/mol) with targets such as TXNIP and NLRP3, providing a clear material carrier for drug action. The research on the mechanism of Chinese herbal medicine compound focuses on apoptosis pathway, while this study finds that Prostate Formula 1 can play a role by activating TXNIP/NLRP3 inflammatory coke pathway-in vitro experiments, the concentrations of IL-1β and IL-18 in the drug group are 5-6 times higher than those in the control group, and the expression of GSDMD protein is 2.5 times higher than that in the control group. In vivo experiments, the necrotic area of tumor tissue in the high dose group accounted for 35.6, significantly higher than that in the model group, this is highly compatible with the characteristics of “cell membrane perforation and release of pro-inflammatory factors” in inflammatory pyroptosis, which breaks through the limitations of Chinese herbal medicine in the study of anti-prostate cancer mechanism, and provides a new paradigm for the treatment of malignant tumors by Chinese herbal medicine in the regulation of inflammatory pyroptosis.
3.2 The central role of TXNIP in the regulation of NLRP3 pathway by Prostate Formula 1
Mechanistic rescue experiments confirmed that TXNIP acts as a critical upstream regulator in the activation of the NLRP3-mediated pyroptosis pathway by Prostate Formula 1. As a thioredoxin interacting protein, TXNIP binds to Trx to maintain steady state under normal conditions. When receiving signals such as oxidative stress and drug stimulation, TXNIP will dissociate and bind to NLRP3, promoting its oligomerization and assembly of inflammasome. [9]. In this study, after TXNIP silencing, the drug-induced NLRP3 and c-caspase-1 protein expression decreased by 57.3 and 52.1 respectively, the concentration of IL-1β and IL-18 decreased by more than 50%, while the apoptosis rate decreased from 38.6 to 20.3, and the cell viability increased by 42.6, which fully proved that TXNIP deletion can significantly block the anti-prostate cancer effect of drugs. These 1 results are consistent with the study that high uric acid induces renal tubular epithelial cell pyroptosis by activating the NLRP3 pathway through TXNIP [10], which further verifies the conservatism of TXNIP in the regulation of inflammatory pyroptosis and also clarifies its irreplaceability in the mechanism of action of Prostate Formula 1.
4 Conclusions
This study confirmed through systematic experiments that Prostate Formula 1 can play an anti-prostate cancer effect by activating TXNIP/NLRP3 inflammatory pyroptosis pathway: its core active ingredients (such as kaempferol and benzyl benzoate) can efficiently bind to TXNIP and NLRP3 targets, up-regulate TXNIP expression and promote its binding with NLRP3, further activate NLRP3 inflammatory bodies, induce c-caspase-1 activation and GSDMD cleavage, release IL-1β IL-18, etc, in vivo experiments, the drug can significantly reduce the volume of transplanted tumor in nude mice and increase the area of tumor necrosis with good safety. mechanistic rescue experiments show that TXNIP silencing can significantly block the anti-prostate cancer effect and NLRP3 pathway activation. In conclusion, Prostate Formula 1 party activates inflammatory pyroptosis through TXNIP/NLRP3 pathway against prostate cancer, which provides a clear molecular mechanism and experimental basis for its clinical application.
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