Introduction

Current evidence suggests that bladder cancer was the 10th most prevalent cancer worldwide in 2020 and the most prevalent malignant tumor of the urinary system, responsible for approximately 17,980 cancer-related deaths in the United States [1]. Smoking has been reported to be the most important risk factor for bladder cancer, followed by exposure to petrochemicals. Additionally, the incidence of this disease shows gender-specific differences, with men having approximately four times higher rates than women [2]. Non-muscle-invasive bladder cancer (NMIBC), which remains confined to the epithelium and lamina propria, accounts for over 70% of newly diagnosed cases, while muscle-invasive bladder cancer (MIBC) constitutes 25% of cases [3]. Patients with early-stage NMIBC commonly undergo transurethral bladder resection, and in some cases, intravesical chemotherapy is recommended. However, two-thirds of patients experience relapse within 5 years of treatment, with this proportion rising to 90% after 15 years [4]. Radical cystectomy is invariably required for MIBC patients due to muscle infiltration of the tumor. Despite the availability of multiple therapies such as chemotherapy, radiotherapy, immunotherapy, and targeted therapy for bladder cancer treatment, approximately 50% of MIBC patients experience metastasis within 2 years [5]. Therefore, further research into potential molecular mechanisms and the design of new strategies to improve the survival of bladder cancer patients is crucial.

In Asia, traditional Chinese medicine (TCM) has been widely accepted as a complementary and alternative form of cancer treatment [6]. It is one of the most popular adjuvant therapies for cancer patients after radical surgery in East Asia, offering distinct advantages, including being multi-component, multi-target, and having minimal side effects due to its natural composition, garnering increased attention worldwide. Both retrospective and prospective studies have demonstrated the therapeutic effect of TCM on cancer [Western blot analysis

Cells were digested and lysed with ice-cold RIPA lysis buffer (containing 1 mM PMSF). The mixture was then centrifuged at 4 ℃ for 20 min at 12,000 ×g. The supernatant protein was collected, and the concentration was determined by bicinchoninic acid assay (BCA). Loading buffer was added to the sample in a ratio of 1:4, and the mixture was boiled for 10 min. The same amount of protein (20 µg) on each sample was separated by 4–15% sodium dodecyl sulfate–polyacrylamide gradient gel electrophoresis (SDS-PAGE) and then transferred to the polyvinylidene fluoride (PVDF) membrane through the transfer box. The PVDF membrane was immersed in the prepared 5% skimmed milk and shaken for 1 h. After being washed, the membrane was placed in a box with the appropriate primary antibody at 4 ℃ overnight. After reacting with the secondary antibody for 1 h, the band was exposed and photographed after drop** the develo** solution.

Statistical analysis

All results were expressed as mean ± standard deviation (SD). Fiji (ImageJ) software was used for image analysis. Data were analyzed using GraphPad Prism 9.0.0, and the significance of the difference was analyzed by one-way analysis of variance analysis. Statistical significance was defined as P < 0.05.

Results

The TCM-compound-target network

45 active compounds in ** with bladder cancer were identified, indicating that this formulation exerts a pharmacological effect on bladder cancer through multiple targets. Quercetin, kumatakenin, polyporusterone G, BDMC, and mandenol were identified as the top five important active compounds. Quercetin is a type of flavonoid with multiple biological activities widely distributed in the plant kingdom. It has been shown to induce apoptosis of bladder cancer cells through the AMPK pathway [29] and yield a sensitizing effect for bladder cancer radiotherapy [30]. Kumatakenin is a natural product first documented in clove, which could induce apoptosis of ovarian cancer cells and inhibit the expression of M2 markers to regulate the tumor microenvironment [31]. Polyporusterone G, a compound belonging to the family of compounds found in Polyporus umbellatus, has been recognized for its cytotoxic properties on leukemia cell proliferation [32]. Besides, Polyporus umbellatus has been shown to promote apoptosis by downregulating AKT in breast cancer [33]. BDMC, a lipid-soluble polyphenolic curcuminoid, yields a range of biological activities, including cytotoxicity in various human cancer cell types, and is more stable than curcumin [34, 35]. Mandenol has been identified as a long-chain fatty acid ethyl ester that acts as a plant metabolite and an anti-inflammatory agent to combat cancer cells [36]. In short, **aozheng decoction is a multi-component prescription with multi-target efficacy, and the relationship between these agents and bladder cancer warrants further investigation.

Five hub target proteins and their related pathways involved in bladder cancer were screened from the compound-target-pathway network analysis. Among these hub targets, ESR1 emerged as the most significant one. Several studies have validated the essential role of ESR1 in bladder cancer risk stratification [37, 38], especially at the tumor grade level [39], though this cancer is not typically regarded as hormone-related. However, upon conducting molecular docking experiments, the affinity of ESR1 was found to be less than ideal, leading us to discontinue further validation for this target. Similarly, the progesterone receptor (PGR), another hormone-related target, was not considered for further validation due to the minimal expression of this protein in bladder cancer [39, 40].

Consistent with the KEGG pathway analysis results, our hub targets showed significant involvement of MAPK signaling-related proteins, particularly MAPK1 and RAF1. The MAPK signaling pathway is a three-tiered signaling cascade composed of RAF, MEK, and ERK, which are serine/threonine-specific kinases that regulate critical cellular processes, such as cell proliferation, differentiation, and migration [41,42,43]. Based on the pharmacological effects observed when blocking RAF1, it is suggested that quercetin and BDMC, derived from **aozheng decoction, may play a crucial role in inhibiting RAF1/MAPK-dependent malignant biological behavior in bladder cancer cells. Both quercetin and BDMC displayed cytotoxic effects in all three bladder cancer cell lines, as demonstrated by the CCK8 assay. Furthermore, BDMC induced cell apoptosis in a dose-dependent manner and exhibited inhibitory effects on colony formation and cell invasion. Another significant target of interest is CDK2, a member of the serine/threonine protein kinases family involved in cell cycle regulation, crucial for driving cell progression through the S- and M-phases of the cell cycle [44]. Interestingly, numerous studies have reported that phytotherapy exerts anti-cancer effects via CDK2-mediated pathways. For example, genistein, an isoflavone derived from soy, was found to promote apoptosis induction in T24 cells, associated with G2/M phase cell cycle arrest and CDK2 inhibition through regulation of the ROS-dependent PI3K/Akt signaling pathway [45]. Similarly, licochalcone A demonstrated a similar mechanism of ROS-mediated cell cycle arrest and apoptosis, involving CDK2 blockade [46]. In the present study, during virtual docking analysis of **aozheng decoction compounds, polyporusterone G exhibited the highest binding affinity to CDK2. However, due to the limited accessibility of polyporusterone G, we could not validate the cell cycle arrest effect of this rare compound derived from **aozheng decoction.

GSK3β, another type of serine/threonine protein kinase, was first described as a component of glycogen synthase regulation via its phosphorylation. As a core downstream component of PI3K/Akt [47], GSK3β mediates many biological processes in tumor cells and promotes the development and metastasis of many types of tumors through EMT [48, 49]. One study has demonstrated that GSK3β is involved in the β-catenin/Snail1 pathway to promote the EMT process in bladder cancer [50]. Moreover, targeting GSK3β with 9-ING-41 (a small molecule inhibitor of GSK3β) has been observed to have multiple anti-bladder cancer effects, including cell cycle arrest, autophagy, and apoptosis in bladder cancer cells. Combined with gemcitabine or cisplatin, 9-ING-41 enhanced the growth inhibitory effects [51]. Our study found that kumatakenin and BDMC have huge potential as GSK3β inhibitors, exhibiting effective inhibition of tumor cell growth. Furthermore, these compounds demonstrated the ability to suppress cell migration and invasion. Western blot analysis revealed that kumatakenin and BDMC dose-dependently inhibited the phosphorylation of GSK3β and β-catenin. The impairment of cell invasion ability was mechanistically elucidated by the downregulation of MMP9, MMP2, and vimentin upon treatment with kumatakenin or BDMC through the EMT process. Flow cytometry and Western blot analysis revealed pro-apoptotic effects of GSK3β inhibition, accompanied by downregulation of the Bcl-2/BAX ratio, indicating the induction of apoptosis.

Conclusion

By employing network pharmacology and conducting in vitro experiments, we have gained preliminary insights into the mechanism through which **aozheng decoction exerts its effects against bladder cancer (Fig. 12). Moreover, this approach allowed us to identify the primary active ingredients and their respective targets, establishing a scientific foundation for further research endeavors.