Hua-Zhuo-Jie-Du formula alleviates colitis-associated colorectal cancer via regulating phosphatidylinositol 3-kinase/protein kinase B signaling pathway

Abstract

BACKGROUND

Colitis-associated colorectal cancer (CAC) is a common type of malignant tumor that occurs in the digestive tract and poses a significant threat to human health. The traditional Chinese medicine preparation Hua-Zhuo-Jie-Du formula (HZJDF) has shown clinical efficacy in preventing CAC; however, the potential therapeutic mechanisms of this formula for treating CAC remain under explored.

AIM

To investigate the therapeutic efficacy and mechanism of HZJDF against CAC through applying an integrated research approach.

METHODS

We first established a CAC mouse model using the azoxymethane/dextran sulfate sodium induction method and then assessed the therapeutic effects of various doses of HZJDF on the CAC mice. Subsequently, we utilized a combination of network pharmacology and transcriptomics analyses to help predict the potential mechanisms by which HZJDF acts against CAC. Finally, we sought to validate these prediction findings through animal and cell experiments.

RESULTS

HZJDF significantly mitigated weight reduction and colon length shortening in a dose-dependent manner, enhanced the mice survival rates, lowered the disease activity index scores, diminished the tumor incidence and burden, and improved the pathological condition of the colon tissues in the CAC mice. The findings from the network pharmacology and transcriptomics analyses suggested that the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway might be the potential mechanism through which HZJDF exerts its protective effects against CAC. In vivo studies showed that HZJDF diminished the quantity of Ki67-positive cells within the colon tissues of the CAC mice and lowered the levels of phosphorylated PI3K and AKT proteins. Meanwhile, in vitro studies indicated that serum containing HZJDF decreased the viability, proliferation, and migration of HT-29 and HCT116 cells in a dose-dependent manner, while also lowering the expression of key proteins linked to the PI3K/AKT signaling pathway.

CONCLUSION

HZJDF effectively alleviated CAC tumorigenesis by suppressing the PI3K/AKT signaling pathway, providing reliable evidence to support the appropriate utilization of HZJDF in CAC treatment.

Key Words: Hua-Zhuo-Jie-Du formula; Colitis-associated colorectal cancer; Network pharmacology; Transcriptomics; Phosphatidylinositol 3-kinase/protein kinase B signaling pathway

Core Tip: Hua-Zhuo-Jie-Du formula (HZJDF) has been clinically proven to be effective at preventing colitis-associated colorectal cancer (CAC), but the mechanism for this requires further investigation. This study utilized ultra-high performance liquid chromatography-high-resolution mass spectrometry analysis, network pharmacology, and transcriptomics in an integrated approach to predict the material basis and potential mechanism of action of HZJDF in treating CAC. Furthermore, the prediction results were verified through animal and cell experiments. The results indicated that HZJDF effectively alleviated the tumorigenesis of CAC via inhibiting the phosphatidylinositol 3-kinase/protein kinase B signaling pathway.

INTRODUCTION

Colorectal cancer (CRC) is the third most common type of cancer worldwide and the second leading cause of cancer-related deaths[1]. Colitis-associated CRC (CAC) is a specific pathological type of CRC and represents a significant health concern due to its high rate of recurrence and mortality[2]. Research has shown that the development of CAC progresses through different stages, namely “inflammation-dysplasia-carcinogenesis”. Consequently, inhibition of the “inflammation-cancer transformation” process has emerged as a major focus and research hotspot for prevention and treatment strategies for CAC[3,4]. In particular, the early clinical prevention of CAC is attracting significant attention as a key goal in CRC treatment. While research indicates that drugs such as 5-aminosalicylic acid and immunomodulators can help manage inflammation to some extent, there is still debate over their therapeutic efficacy against CAC[5]. Consequently, there is still a pressing need for the development of safe and effective medications for both the prevention and management of CAC.

It has been reported that traditional Chinese medicine (TCM) offers distinctive benefits in obstructing the inflammation-cancer transformation process, with increasing evidence indicating that TCM has promising potential for the prevention and treatment of CAC[6]. Considering the diverse clinical manifestations of CAC, including abdominal discomfort, persistent diarrhea, and the presence of blood in the stools of CAC patients, it has been posited in TCM that the primary pathological element contributing to the onset and progression of CAC is the presence of turbidity toxins. Specifically, the turbidity toxin theory, proposed by Professor Li DG, a National Master of TCM, posits that the occurrence of various tumors can be attributed to dysfunction of the visceral systems and the internal generation of turbid toxins and their subsequent accumulation in the large intestine, which can lead to the occurrence and development of CAC. This aligns closely with the pathological condition described as inflammation-cancer transformation. Hua-Zhuo-Jie-Du formula (HZJDF) is a representative TCM prescription that has been developed based on the Turbid Toxin Theory[7]. It was created by Professor Yang Q through the modification and innovation of three classical formulas traditionally used to treat ulcerative colitis (UC) and its associated carcinogenesis: Baitouweng Decoction[8], Shaoyao Decoction[9], and Xianglian Pill[10], integrating insights from years of clinical experience. HZJDF has attracted considerable attention in China for its efficacy in preventing and treating CAC and UC. Prior clinical research has demonstrated that HZJDF can significantly reduce symptoms such as abdominal pain and diarrhea, lessen inflammatory reactions, and aid in the healing of intestinal mucosal damage in UC patients. HZJDF treatment also lowers the likelihood of UC recurrence, thus playing a crucial role in preventing CAC. Moreover, several active compounds within HZJDF have recently been identified, including luteolin[11] and quercetin[12], which have been shown to play crucial roles in the formula exhibiting anti-cancer properties. However, for HZJDF to be used in the clinical management of CAC, further investigation of its underlying molecular mechanisms is required.

Ongoing advancements in bioinformatics and high-throughput methodologies have led to the emergence of a new research approach that integrates network pharmacology with transcriptomics, with the potential to serve as a novel tool for uncovering the comprehensive therapeutic mechanisms of TCM formulations. In this study, the azoxymethane (AOM)/dextran sodium sulfate (DSS) induction method was utilized to create a CAC mouse model to evaluate the effects of HZJDF against CAC. Subsequently, a thorough analysis of the active components in HZJDF was conducted using an ultra-high performance liquid chromatography-high-resolution mass spectrometry (UHPLC-Q-Orbitrap-HRMS) method. Following this, we explored the potential mechanisms by which HZJDF mitigates CAC through the integrated network pharmacology and transcriptomics approach. Lastly, we delved deeper into the anti-CAC effects of HZJDF and the mechanism through which it delivers these effects through both animal and cellular experiments.

MATERIALS AND METHODS

Animals

Seventy-two male C57BL/6 mice, aged six weeks and with weights ranging from 18 g to 22 g, were acquired from Beijing SiPeiFu Biotechnology Co., Ltd., located in Beijing, China. The mice were housed at the specific pathogen free-level Experimental Animal Center of Hebei Provincial Hospital of Traditional Chinese Medicine (Shijiazhuang, Hebei Province, China) under the following environmental conditions: A consistent temperature maintained between 23 °C and 25 °C, a 12-hour light and dark cycle, and continuous access to food and water. The animal study was approved by the Ethics Committee for Animal Experimentation at Hebei Provincial Hospital of Traditional Chinese Medicine (approval No. IACUC-HPHCM-2024016).

Reagents and chemicals

AOM (A5486) was sourced from Sigma-Aldrich (St Louis, MO, United States). Mesalazine (220218) was purchased from Sunflower Pharmaceutical Group Co., Ltd. (Harbin, Heilongjiang Province, China). DSS (D10002) was obtained from Psaitong (Beijing, China). Fetal bovine serum (FBS) (11011-8615) was supplied by EVERY GREEN (Zhejiang Province, China). Trypsin solution (KGM27250) and penicillin/streptomycin (KGY0023) were acquired from KeyGEN Biotech. The bicinchoninic acid (BCA) Protein Assay Kit (P0010), bovine serum albumin (BSA) (ST023), and BeyoECL Plus (P0018S) were obtained from Beyotime Biotechnology. Ki67 staining kits (GB111141), McCoy’s 5A medium (GB540), and β-actin (GB15003) were sourced from Servicebio Biotech (Wuhan, Hubei Province, China). The main antibodies utilized included phosphorylated (p)-phosphatidylinositol 3-kinase (PI3K) (A0153), PI3K (BS3678), protein kinase B (AKT) (A0378), and p-AKT (BS43321), and all were provided by Bioworld (Beijing, China).

Preparation of HZJDF

HZJDF granules were sourced from Sichuan Neo-Green Pharmaceutical Technology Development Co., Ltd. Table 1 provides comprehensive details about the 17 herbal components in HZJDF. Following a preparation process established by our research team in earlier work[7], the HZJDF granules were dissolved in 100 °C pure water, then heated and stirred until all the granules had fully dissolved. Subsequently, the solution was concentrated to 1 g/mL.

Table 1 Composition of Hua-Zhuo-Jie-Du formula.

Chinese name	Latin name	Amount (g)	Place of origin	Lot number
Huang Lian	Coptis chinensis Franch.	0.55	Chongqing, (China)	21100496
Sha Ren	Wurfbainia villosa	0.29	Yunnan Province, (China)	21100297
Bai Jiang Cao	Thlaspi arvense L.	0.75	Hebei Province, (China)	21100396
Qin Pi	Fraxinus chinensis Roxb.	1.09	Sichuan Province, (China)	21100423
Di Yu	Sanguisorba officinalis L.	1.36	Gansu Province, (China)	21100653
Wu Wei Zi	Schisandra chinensis (Turcz.) Baill.	0.6	Jilin Province, (China)	21100823
Feng Wei Cao	Pteris multifida Poir.	1.5	Sichuan Province, (China)	21090074
Yi Yi Ren	Coix lacryma-jobi L.	1.33	Yunnan Province, (China)	21100343
Bai Tou Weng	Pulsatilla chinensis (Bunge) Regel	0.57	Sichuan Province, (China)	21100066
Er Cha	Senegalia catechu	1.2	Yunnan Province, (China)	21100201
Chai Hu	Bupleurum falcatum L.	0.55	Gansu Province, (China)	21100244
Qian Shi	Euryale ferox Salisb.	1	Guangxi Province, (China)	21100533
Dang Gui	Angelica sinensis (Oliv.) Diels	1.6	Gansu Province, (China)	21100398
Bai Zhu	Atractylodes macrocephala Koidz.	1.71	Hebei Province, (China)	21100342
Tu Si Zi	Cuscuta japonica Choisy	0.59	Gansu Province, (China)	21100463
Mu Xiang	Dolomiaea costus (Falc.)	0.86	Yunnan Province, (China)	21100549
Bai Shao	Paeonia lactiflora Pall.	0.95	Anhui Province, (China)	21100361

Establishment of the CAC mouse model

Similar to an earlier published study, a CAC mouse model was constructed using the AOM/DSS induction method[13]. In this method, after adaptive feeding, the mice were given a single intraperitoneal injection of 10 mg/kg of AOM. One week later, the mice received three cycles of DSS treatment, each cycle lasting for three weeks. The dosing schedule for each cycle was as follows: In the first week, the mice drank a 2% DSS solution, and then for the following two weeks, they had access to regular drinking water and were allowed to drink freely.

Experimental grouping and drug intervention

After one week of acclimatization, the 72 mice were randomly divided into 6 distinct groups (n = 12/group): Control, model, low-dose HZJDF (HZJDF-L), medium-dose HZJDF (HZJDF-M), high-dose HZJDF (HZJDF-H), and mesalazine groups. Except for the control group, the mice in the other groups were established as CAC mouse models. The mesalazine group received a gavage of 227.5 mg/kg of mesalazine solution, while the HZJDF-L, HZJDF-M, and HZJDF-H groups were administered 1.25 g/kg, 2.5 g/kg, and 5 g/kg of HZJDF via gavage, respectively. Concurrently, the other groups of mice received the same volume of normal saline by gavage. All the treatments commenced on the first day of the model’s establishment. The dosages of HZJDF and mesalazine were calculated based on a clinical equivalent dose formula, with the adult daily dosage of HZJDF being 16.5 g/60 kg and for mesalazine 1.5 g/60 kg. For the mice, the dosage was adjusted to 9.1 times the adult clinical dosage[14]. Consequently, the HZJDF-M group was set at 2.5 g/kg/day, while the HZJDF-H and HZJDF-L groups were established at double and half the dosage of the HZJDF-M group, respectively.

Beginning with the administration of AOM, weekly observations were made regarding the survival rates, body weights, and occurrences of diarrhea and bloody stools in each mouse. Additionally, the disease activity index (DAI) score was utilized to assess colonic damage across the different groups, with the specific calculation methods utilized detailed in Supplementary Table 1. Once the drug treatment phase had concluded, all the mice were anesthetized via an intraperitoneal injection of sodium pentobarbital, and their colon length was then measured. The colon tissues were then washed with chilled phosphate-buffered saline (PBS) and sliced along the colon length using sterile scissors. The total count of tumors along with the tumor burden (total highest diameters of the colorectal tumors) were evaluated and recorded. Following this, the colon tissues were divided into three equal parts. The segment from the distal colon was utilized for further experimental analysis, while the remaining portions were preserved at -80 °C in a freezer.

Histopathological examination

After the colorectal tissues were fixed with paraformaldehyde, 5 μm thick paraffin sections were prepared. These were treated successively with xylene and different concentrations of ethanol. Finally, they were stained with hematoxylin-eosin (HE) and sealed with neutral gum, and then observed under a microscope. In line with previously published literature[15], the pathological scores derived from the HE staining were assessed, with the scoring criteria detailed in Supplementary Table 2.

Immunohistochemical staining

The paraffin sections were treated by dewaxing and hydration, and the antigen was prepared using citrate buffer. Also, 3% hydrogen peroxide solution was used to inhibit the activity of endogenous peroxidase, and then 5% BSA was used as a block for 1 hour. The sections were incubated with Ki-67 primary antibody (1:1000, GB111141, Servibio Biotech) at 4 °C overnight. After this incubation, a secondary antibody was added for 1 hour, followed by 3,3’-diaminobenzidine staining. After hematoxylin staining, the sections were observed under an optical microscope.

Western blot

In line with the method outlined in our earlier study[16], the total proteins of the colorectal tissues and cancer cells were extracted using radio immunoprecipitation assay lysis buffer supplemented with 1% phenylmethylsulfonyl fluoride. The concentration of proteins in each sample was assessed using the BCA protein assay kit. The proteins from each sample were diluted to ensure they matched in volume and concentration using distilled water. The proteins were then subjected to electrophoresis and transferred onto a polyvinylidene fluoride membrane. Following this, we performed a 5% BSA blocking step, along with incubation with the antibodies, and treatment with chemiluminescent reagents. Finally, the ChemiDoc MP system was used for imaging the blots.

Transcriptomic analysis

Acquisition and analysis of the transcriptomic data were performed by Biotree Biotech Co., Ltd. (Shanghai, China). Colorectal tissues were collected from the control, model, and HZJDF-H groups. Total RNA was isolated from each sample utilizing TRIzol, and its purity was assessed using a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, United States). The integrity of the RNA was assessed using an Agilent 5400 Fragment Analyzer system (Agilent Technologies, CA, United States). After confirming the samples met the quality control standards, the transcriptome library was prepared following the NEB Next UltraII RNA Library Prep Kit for Illumina guidelines. Finally, sequencing was conducted on an Illumina Novaseq Xplus PE150 platform.

Raw data underwent filtration with fastp (v0.23.4) to yield clean data, which served as the basis for all the further analyses. The reference genome index was created using HISAT2 (v2.2.1), allowing for alignment of the paired-end clean reads. Following this, StringTie2 (v2.1.4) was employed to reconstruct transcripts and assess the relative gene expression levels in each sample. For differential expression analysis between the two groups, DESeq2 (v1.38.3) software was utilized. Genes were classified as differentially expressed genes (DEGs) based on the criteria: |log₂ fold change| > 1.0 and a P < 0.05. ClusterProfiler (version 4.6.2) was used to conduct Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses on the DEGs. The P value was determined using the hypergeometric distribution method, whereby the significantly enriched terms and pathways for the DEGs were those identified with a P value below 0.05. In addition, gene set enrichment analysis (GSEA) was carried out using the clusterProfiler tool, which does not necessitate a specific threshold for DEGs; instead, it ranks all genes in both sample groups according to their differential expression levels before performing a statistical assessment.

Network pharmacology analysis

In accordance with the methodology described in our earlier study, we analyzed the HZJDF constituents utilizing a UHPLC-Q-Orbitrap-HRMS system. Chromatographic separation was carried out on a Waters ACQUITY UPLC HSS T3 C18 column (2.1 mm × 100 mm, 1.8 μm), using a mobile phase composed of water containing 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B), delivered at a flow rate of 0.2 mL/minute. The mass spectrometry parameters were set as follows: Full scan resolution at 70000, dd-MS2 resolution at 17500, scan range from 100 m/z to 1500 m/z, and spray voltage of 3.2 kV (positive mode) or -3.0 kV (negative mode). Data acquisition and processing were conducted using Compound Discoverer 3.2 software. Compounds were identified by querying the mzVault database, and those with a match score ≥ 80 and ppm < 5 were assigned to the chemical constituents of HZJDF.

In order to improve the extraction of the key active ingredients present in HZJDF, the recognized chemical components were uploaded to the PubChem database to retrieve the SDF files corresponding to their two-dimensional molecular structures. Subsequently, these files were processed through the SwissADME database to obtain detailed pharmacokinetic data for each compound. To pinpoint the primary active substances in HZJDF, we implemented strict absorption, distribution, metabolism, and elimination (ADME) selection criteria: (1) At least 2 of the “drug likeness” rules (Lipinski, Ghose, Veber, Egan, Muegge) must be marked as “yes”; and (2) A “high” classification for “gastrointestinal absorption” in terms of the pharmacokinetics.

The PubChem database was utilized to convert every compound recognized by ADME into its standard SMILES format. Subsequently, these SMILES were submitted to SwissTargetPrediction to identify possible targets. The selection of “homo sapiens” was made with a probability threshold exceeding 0. To compile targets related to CAC, the phrase “colitis-associated colorectal cancer” was used across three different databases: GeneCards, Online Mendelian Inheritance in Man (OMIM), and PharmGkb. These common targets of HZJDF and CAC were then submitted to the STRING database to create a protein-protein interaction (PPI) network. To enhance this network, the cytoNCA plugin was utilized, followed by an evaluation of the topological features of each node by calculating six parameters: “Betweenness centrality”, “closeness centrality”, “degree centrality”, “eigenvector centrality”, “network centrality”, and “local average connectivity”[15]. Finally, the previously identified intersection targets were examined using the clusterProfiler tool within R software for both the GO and KEGG analyses. The top 20 GO enrichments and KEGG pathways, based on their frequency, were visualized and then subjected to further analysis.

Cell culture

Human CRC cell lines HT-29 (catalog number TCH-C213) and HCT116 (catalog number TCH-C185) were obtained from Hysigen Bioscience. The cells were cultured in McCoy’s 5A medium supplemented with 10% FBS and 1% penicillin/streptomycin and stored in a humidified incubator at 37 °C and 50 mL/L carbon dioxide.

Preparation of HZJDF-containing serum

A total of 40 healthy Sprague-Dawley rats, each weighing between 200 g and 250 g, were sourced from Beijing SiPeiFu Biotechnology Co., Ltd. (Beijing, China). This animal study was approved by the Ethics Committee for Animal Research at Hebei Provincial Hospital of Traditional Chinese Medicine (approval No. IACUC-HPHCM-2024058).

Following one week of acclimatization, the rats were divided into two groups: A control group and HZJDF group. The recommended daily therapeutic dosage of HZJDF for the adult rats was 16.5 g per 60 kg of body weight. Based on the body surface area method, the intragastric dose for the rats was calculated as 6.25 times the clinical dose[17]. In the HZJDF group, the rats were given 1.72 g/kg of HZJDF through intragastric administration, while the rats in the control group received the same volume of saline. This treatment was performed daily over a span of seven days. Prior to the final dose, the rats underwent a 12-hour fasting period. After 2 hours post-treatment, the rats were anesthetized via an intraperitoneal injection of pentobarbital sodium. Blood was obtained from the abdominal aorta and allowed to sit at room temperature for 2 hours before being centrifuged. The supernatant obtained was incubated at 56 °C for 30 minutes for inactivation and subsequently sterilized through a 0.22 μm microporous membrane.

Cell viability assay

Cells were collected and their density adjusted to 5 × 10⁴ cells/mL. Next, 100 μL of the cell suspension was added to a 96-well plate. After culturing for 24 hours, different concentrations of serum containing HZJDF (0%, 2.5%, 5%, 10%, or 20%) were added and the cells were incubated for 48 hours. Next, cell counting kit-8 reagent was added with a further incubation at 37 °C for 2 hours. Finally, a microplate reader was used to measure the optical density of the samples at 450 nm.

Cell colony formation evaluation

HT-29 and HCT116 cells were collected and diluted to a cell concentration of 1 × 10⁵ cells/mL. The cell suspension was inoculated into a six-well plate, with 2 mL volume per well. After 24 hours cultivation with serum-free medium, the six-well plate was washed with PBS solution, and then different concentrations of HZJDF (0 mg/mL, 2.4 mg/mL, 4.8 mg/mL, or 9.6 mg/mL) were added and the cells were further incubated for 48 hours. Subsequently, the cell suspensions from each group were collected and the cell concentration was diluted to 1 × 10³ cells/mL using complete medium. The cells were next inoculated into a 6-well plate at a volume of 2 mL per well and cultured in complete medium for 10 days. When obvious clusters of bacteria could be observed under the microscope, the 6-well plate was rinsed with PBS solution. Then, the clones were fixed with methanol and stained with crystal violet.

Wound-healing assay

Initially, two straight reference lines were drawn on the base of each 6-well plate. Subsequently, the cell suspension was added to each well. Once the cells reached approximately 90% confluence, they were carefully scraped off using a 200 μL pipette tip, following the orientation of the marked lines. Varying concentrations of HZJDF-containing serum (0%, 5%, 10%, or 20%) were then introduced. Observations and photographs of the cells in each group were taken at both 0 hour and 48 hours.

Statistical analysis

All the data are presented herein as the mean ± SD. Statistical evaluation was conducted using SPSS software. One-way analysis of variance analysis was employed to evaluate variations between different groups. A P value less than 0.05 indicated that the difference was statistically significant.

RESULTS

HZJDF inhibited the tumorigenesis of CAC

Figure 1A shows the experimental design of this study. Throughout the DSS-treatment phase, there was a significant reduction in the body weight of the CAC mice. However, once they resumed normal water consumption, their weight began to recover gradually. After the experimental period had ended, compared with the control group, the body weights of the rats in the model group were found to be significantly decreased. However, the administration of HZJDF and mesalazine helped mitigate the weight loss observed in the mice (Figure 1B). Next, survival curves for each group during the treatment period were plotted and are shown in Figure 1C. At the end of the study, the survival rate for the model group was recorded as 58%, while the corresponding rates for the HZJDF-L, HZJDF-M, HZJDF-H, and mesalazine groups were 75%, 83%, 83%, and 75%, respectively. Starting from the fourth day post-DSS treatment, the CAC mice displayed various symptoms, such as diarrhea and hematochezia, along with an increase in DAI scores. Compared with the model group, both the HZJDF and mesalazine treatments significantly reduced the DAI scores (Figure 1D).

Figure 1 Hua-Zhuo-Jie-Du formula inhibited the tumorigenesis of colitis-associated colorectal cancer. A: Schematic diagram of the animal experiment operating procedures; B: Percentage change in body weight; C: Rate of survival; D: Disease activity index scores; E: Representative images of the colorectal tissues of the colitis-associated colorectal cancer mice; F: Colon lengths; G: Distribution of tumor sizes; H: Count of the colorectal tumors; I: Representative images for hematoxylin-eosin staining; J: Histological scores. Data are shown as the mean ± SD. ^bP < 0.01 vs control group. ^cP < 0.05 vs model group. ^dP < 0.01 vs model group. Control group (n = 12), model group (n = 7), low-dose Hua-Zhuo-Jie-Du formula group (n = 9), medium-dose Hua-Zhuo-Jie-Du formula group (n = 10), high-dose Hua-Zhuo-Jie-Du formula group (n = 10), and mesalazine group (n = 9). DSS: Dextran sodium sulfate; AOM: Azoxymethane; DAI: Disease activity index; HZJDF: Hua-Zhuo-Jie-Du formula; HZJDF-L: Low-dose Hua-Zhuo-Jie-Du formula; HZJDF-M: Medium-dose Hua-Zhuo-Jie-Du formula; HZJDF-H: High-dose Hua-Zhuo-Jie-Du formula.

Following euthanasia of the animals, the colon lengths and tumor development in each mouse group were measured and evaluated. Figure 1E presents representative images of the colorectal tissues from each group of mice. Compared with the control group, the colon length of the mice in the model group was significantly shortened. The HZJDF and mesalazine treatments were found to be effective in mitigating this reduction (Figure 1F). A decrease in colon length is a common indicator of intestinal injury in CAC mice. Furthermore, compared to the model group, both the number and burden of the colorectal tumors were significantly lower in the groups receiving HZJDF and mesalazine (Figure 1G and H). During the experiment, no significant adverse events were observed in any of the groups of mice.

The results from HE staining showed that the colon tissue structure in the control group mice had a normal appearance, featuring an intact mucosal layer, a well-organized arrangement of epithelial cells and glands, and an absence of inflammatory cell infiltration. In contrast, the colon tissues of the model group mice displayed significant damage, marked by a chaotic arrangement of glands, loss of crypts, decrease in epithelial cell numbers, high-grade intraepithelial neoplasia, and the presence of inflammatory cells. Treatment with HZJDF and mesalazine notably alleviated the intestinal damage induced by AOM/DSS (Figure 1I). Additionally, histological scoring revealed that the model group had a significantly elevated histological score compared to the control group, while the scores for the HZJDF- and mesalazine-treatment groups were considerably lower than those of the model group (Figure 1J). Detailed analysis of different dosages within the HZJDF treatment groups revealed that higher doses of HZJDF typically led to more significant effects. In summary, our findings indicate that HZJDF significantly suppressed tumor development in our CAC mouse model triggered by AOM/DSS.

Use of network pharmacology to predict the mechanism of HZJDF in treating CAC

First, we conducted a thorough qualitative analysis of the elements within HZJDF using an UHPLC-Q-Orbitrap-HRMS system. The total ion chromatograms for the both positive and negative ion modes of HZJDF are illustrated in Figure 2A and B, respectively. In total, 322 distinct components were identified. Detailed information regarding the chemical structures, ion patterns, retention times, and other pertinent characteristics of each component can be found in Supplementary Table 3. Additionally, 120 of these components fulfilled the ADME screening criteria and were recognized as the key active ingredients of HZJDF, including luteolin, quercetin, and kaempferol. Supplementary Table 4 provides extensive details on these 120 active substances.

Figure 2 Network pharmacological analysis of Hua-Zhuo-Jie-Du formula against colitis-associated colorectal cancer. A: Total ion chromatograms (TICs) of Hua-Zhuo-Jie-Du formula (HZJDF) obtained in the positive ion mode; B: TICs obtained in the negative ion mode; C: Venn diagram illustrating therapeutic targets associated with colitis-associated colorectal cancer (CAC); D: Venn diagram showing the overlap between the targets associated with HZJDF’s active ingredients and CAC-related targets; E: Network visualization of HZJDF’s active ingredients and their corresponding CAC targets. Red circles represent HZJDF’s active ingredients, while the green triangles denote the target proteins; F: Topological screening of the protein-protein interaction network; G: Bubble chart depicting the outcomes of the Gene Ontology enrichment analysis; H: Bubble chart illustrating the outcomes of the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis for the targets. OMIM: Online Mendelian Inheritance in Man; HZJDF: Hua-Zhuo-Jie-Du formula; CAG: Colitis-associated colorectal cancer; BC: Betweenness centrality; CC: Closeness centrality; DC: Degree centrality; EC: Eigenvector centrality; LAC: Local average connectivity; MAPK: Mitogen-activated protein kinase; PI3K: Phosphatidylinositol 3-kinase; AKT: Protein kinase B; TNF: Tumor necrosis factor; AGE-RAGE: Advanced glycation end products-receptor for advanced glycation end products; HIF: Hypoxia inducible factor; PD-1: Programmed cell death 1; PD-L1: Programmed cell death ligand 1; EGFR: Epidermal growth factor receptor.

The SwissTargetPrediction database was employed to forecast the targets associated with the 120 key active components of HZJDF. After removing any duplicate entries, a total of 1224 unique targets were identified. The CAC targets were sourced from three distinct databases: 166 from OMIM, 3848 from GeneCards, and 1717 from PharmGkb. By merging these targets and discarding duplicates, 5306 targets related to CAC were compiled (Figure 2C). Additionally, the relevant targets for both HZJDF and CAC were visualized in a Venn diagram using Cytoscape software, revealing 640 common targets (Figure 2D and Supplementary Table 5). A target network associated with the active components of HZJDF-CAC was also created (Figure 2E), indicating that HZJDF exerted anti-CAC effects through various ingredients and targets.

In order to pinpoint the primary targets of HZJDF for CAC treatment, we developed a PPI network based on the 640 overlapping targets identified earlier. This network included 532 nodes and 5120 connections. Following this, a topological analysis was performed on the network. Ultimately, 52 targets emerged as the key targets of HZJDF in combating CAC. The process of selection is illustrated in Figure 2F, and comprehensive details regarding these 52 main targets can be found in Table 2. We next conducted GO and KEGG functional enrichment analyses on these 640 intersection targets. The biological process findings highlighted key areas such as the enhancement of the mitogen-activated protein kinase (MAPK) cascade, reactions to foreign substances, and the stimulation of kinase activity. The cellular component results featured components like membrane rafts, membrane microdomains, and focal adhesions. The molecular function results predominantly included activities related to protein serine kinases, protein tyrosine kinases, and binding to transcription coregulators (Figure 2G). The KEGG analysis uncovered a total of 190 signaling pathways. Among these, the top 30 enriched pathways were presented, with the PI3K/AKT signaling pathway identified as the most significant, indicating its potential role as a key mechanism through which HZJDF may act against CAC (Figure 2H).

Table 2 Fifty-two core targets of Hua-Zhuo-Jie-Du formula for colitis-associated colorectal cancer.

No.	Target	Degree	No.	Target	Degree	No.	Target	Degree
1	SRC	68	19	HSP90AA1	36	37	TNF	26
2	AKT1	64	20	ERBB2	36	38	MAPK14	26
3	TP53	64	21	MAPK8	34	39	CCND1	26
4	ESR1	60	22	EP300	34	40	MAPK12	26
5	STAT3	54	23	HIF1A	34	41	PRKCD	26
6	JUN	48	24	CREBBP	32	42	AR	24
7	EGFR	46	25	FOS	32	43	FYN	24
8	MAPK1	46	26	IGF1R	30	44	IKBKB	24
9	PIK3CA	44	27	GRB2	30	45	PLCG1	24
10	MAPK3	42	28	PTK2	30	46	PTPN6	22
11	PIK3R1	42	29	CDC42	30	47	CHUK	22
12	CTNNB1	40	30	STAT1	30	48	VAV1	22
13	AKT2	38	31	BCL2	28	49	MAP3K7	20
14	RELA	38	32	MAPK9	28	50	IL6	20
15	PTPN11	36	33	MDM2	28	51	IKBKG	20
16	NFKB1	36	34	HSP90AB1	28	52	CASP3	20
17	PIK3CD	36	35	RAF1	28
18	PIK3CB	36	36	HDAC1	26

Use of transcriptomics to elucidate the mechanism through which HZJDF acts against CAC

To further explore how HZJDF functions in treating CAC, we conducted transcriptome sequencing on colon tissues from the mice in the control, model, and HZJDF-H groups. Our analysis revealed 4256 genes with significant differences between the control and model groups (Figure 3A), while 2244 genes were found to differ between the model and HZJDF groups (Figure 3B). Additionally, 1729 genes were common in both comparisons (Figure 3C). Figure 3D illustrates a cluster analysis of the genes that were expressed differently among the control, model, and HZJDF groups.

Figure 3 Transcriptomics analysis of Hua-Zhuo-Jie-Du formula against colitis-associated colorectal cancer. A: Volcano plot of the differentially expressed genes (DEGs) between the control group and model group; B: Volcano plot of the DEGs between the model group and the Hua-Zhuo-Jie-Du formula (HZJDF) group; C: Venn diagram showing the overlapping genes in the control group vs model group and the model group vs HZJDF group; D: Heatmap from cluster analysis of the DEGs among three different groups; E: Bubble plot from Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the genes that were upregulated in the model group compared to the control group; F: Bubble plot from KEGG enrichment analysis of the genes that were downregulated in the HZJDF group compared with the model group; G: Gene set enrichment analysis (GSEA) analysis for comparison of the control group vs model group; H: GSEA analysis for comparison of the model group vs HZJDF group. HZJDF: Hua-Zhuo-Jie-Du formula; MAPK: Mitogen-activated protein kinase; PI3K: Phosphatidylinositol 3-kinase; AKT: Protein kinase B; JAK-STAT: Janus tyrosine kinase/signal transducer and activator of transcription.

Subsequently, we concentrated on the genes that were significantly upregulated in the model group but downregulated in the HZJDF group. Through KEGG enrichment analysis of these genes, we discovered that, compared to the control group, the genes significantly upregulated in the model group exhibited a strong association with the PI3K/AKT signaling pathway (Figure 3E). Furthermore, the genes that were downregulated in the HZJDF group, in comparison to the model group, also demonstrated significant enrichment in the PI3K/AKT signaling pathway (Figure 3F). The GSEA results indicated that when comparing the control and model groups (Figure 3G), the primary genes linked to the PI3K/AKT signaling pathway were largely enriched in the model group [normalized enrichment scores (NES) > 0, P < 0.05]. Similarly, comparing the model and HZJDF groups (Figure 3H), these critical genes were mainly found in the model group (NES < 0, P < 0.05).

The PI3K/AKT signaling pathway emerged as a key discovery in both the network pharmacology and transcriptomic analyses. A variety of research has shown that this pathway is crucial in controlling the growth, survival, and movement of tumor cells. Furthermore, its activation is strongly linked to CAC. Several TCM remedies have shown effectiveness in treating CAC by targeting the PI3K/AKT pathway. As a result, this pathway is considered a central mechanism in CAC treatment with HZJDF and will be selected for additional validation in future studies.

HZJDF prevented the advancement of CAC by inhibiting the PI3K/AKT pathway

A fundamental characteristic of cancer is the unrestrained growth and unchecked advancement of tumor cells. To evaluate cell proliferation in the colon tissues in the present study, Ki67 immunohistochemistry was employed. The results showed a significant rise in Ki-67-positive cells in the model group compared to the control group, while a substantial reduction was observed in the HZJDF and mesalazine groups. Additionally, these effects were more pronounced with the higher doses of HZJDF (Figure 4A and B).

Figure 4 Hua-Zhuo-Jie-Du formula inhibited tumor proliferation and the phosphatidylinositol 3-kinase/protein kinase B pathway in colitis-associated colorectal cancer mice. A: Representative images of Ki67 staining; B: Quantitative analysis of Ki67-positive cell staining; C: Detection of the proteins associated with the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway by Western blot analysis; β-actin served as a loading control; D: Quantitative analysis of the expression levels of phosphorylated-PI3K/PI3K; E: Quantitative analysis of the expression levels of phosphorylated-AKT/AKT. Data are shown as the mean ± SD. ^bP < 0.01 vs control group. ^cP < 0.05 vs model group. ^dP < 0.01 vs model group. HZJDF-L: Low-dose Hua-Zhuo-Jie-Du formula; HZJDF-M: Medium-dose Hua-Zhuo-Jie-Du formula; HZJDF-H: High-dose Hua-Zhuo-Jie-Du formula; PI3K: Phosphatidylinositol 3-kinase; p-PI3K: Phosphorylated-phosphatidylinositol 3-kinase; AKT: Protein kinase B; p-AKT: Phosphorylated-protein kinase B.

Integrating insights from the network pharmacology and transcriptomics analyses, we next conducted Western blot analysis to assess the levels of PI3K/AKT-related proteins in the colon tissues of the various mouse groups. The results revealed there was a significant increase in the phosphorylated forms of PI3K and AKT in the model group compared to the control group. After administering HZJDF, the levels of these proteins showed a reduction that was dependent on the dosage (Figure 4C-E). These results suggest that HZJDF may hinder the progression of CAC by downregulating the PI3K/AKT signaling pathway in vivo.

HZJDF-containing serum reduced the viability, proliferation, and migration of CRC cells by inhibiting the PI3K/AKT pathway

HT-29 and HCT116 cell lines were employed to investigate the effects and mechanisms of HZJDF in treating CAC. We first assessed how different concentrations of serum containing HZJDF (2.5%, 5%, 10%, or 20%) influenced the survival of these two CRC cell lines. As shown in Figure 5A and B, after 48 hours of treatment, HZJDF decreased the viability of HT-29 and HCT116 cells. The ability of cells to form clones serves as an indicator of their proliferation capacity. To evaluate the impact of HZJDF on the proliferation of CRC cells, we treated HT-29 and HCT116 cells with varying concentrations of HZJDF serum (5%, 10%, or 20%) for 48 hours, followed by performing a cell clone formation assay. The results indicated that HZJDF significantly suppressed the proliferation of both cell lines in a dose-dependent fashion (Figure 5C-E). To further explore the effect of HZJDF on cell migration, HT-29 and HCT116 cells were subjected to 5%, 10%, and 20% HZJDF serum for 48 hours, after which a wound-healing assay was conducted. The findings revealed that HZJDF inhibited the migratory capabilities of HT-29 cells in a dose-dependent manner (Figure 5F and G). Lastly, to confirm the molecular mechanisms by which HZJDF acts against CAC, we analyzed the expression of critical proteins in the PI3K/AKT signaling pathway using Western blotting. The results demonstrated that HZJDF significantly reduced the levels of p-PIK/PI3K and p-AKT/AKT in both HT-29 (Figure 5H-J) and HCT116 cells (Figure 5K-M). These results suggest that HZJDF-containing serum may hinder the viability, proliferation, and migration capabilities of CRC cells by targeting the PI3K/AKT signaling pathway.

Figure 5 Hua-Zhuo-Jie-Du formula-containing serum inhibited cell viability, proliferation, migration, and the phosphatidylinositol 3-kinase/protein kinase B signaling pathway in vitro. A: Effects of varying concentrations of Hua-Zhuo-Jie-Du formula (HZJDF)-containing serum on the viability of HT-29 cells; B: Effects of varying concentrations of HZJDF-containing serum on the viability of HCT116 cells; C: Effects of HZJDF-containing serum on the clonogenic ability of HT-29 and HCT116 cells; D: Quantitative assessment of the quantity of HT-29 cell clones generated in each group; E: Quantitative assessment of the quantity of HCT116 cell clones generated in each group; F: Effect of HZJDF-containing serum on the migratory capacity of HT-29 cells; G: Quantitative assessment of the wound-healing area in each group; H: Expressions of proteins associated with the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in HT-29 cells detected by Western blot analysis; I: Quantitative analysis of the expression levels of phosphorylated-PI3K (p-PI3K)/PI3K in HT-29 cells; J: Quantitative analysis of the expression levels of phosphorylated-AKT (p-AKT)/AKT in HT-29 cells; K: Expressions of proteins associated with the PI3K/AKT signaling pathway in HCT116 cells detected by Western blot analysis; L: Quantitative analysis of the expression levels of p-PI3K/PI3K in HCT116 cells; M: Quantitative analysis of the expression levels of p-AKT/AKT in HCT116 cells. β-actin was utilized as a loading control. Data are shown as the mean ± SD. ^aP < 0.05 vs 0% Hua-Zhuo-Jie-Du formula group. ^bP < 0.01 vs 0% Hua-Zhuo-Jie-Du formula group. HZJDF: Hua-Zhuo-Jie-Du formula; PI3K: Phosphatidylinositol 3-kinase; p-PI3K: Phosphorylated-phosphatidylinositol 3-kinase; AKT: Protein kinase B; p-AKT: Phosphorylated-protein kinase B.

DISCUSSION

CAC ranks among the most common gastrointestinal cancers and shows a significant link to inflammatory bowel disease[18]. At present, anti-inflammatory medications, particularly 5-aminosalicylic acid compounds like sulfasalazine and mesalazine, are utilized to reduce inflammation and thereby aid the prevention of CAC. However, it is worth noting that the long-term use of these drugs may induce various adverse effects, including allergic reactions and hepatorenal toxicity, and their preventive efficacy against CAC remains controversial. Consequently, much effort has been directed toward investigating alternative medications that can more safely and efficiently prevent the progression of colitis to cancer, as well as lower the occurrence of CAC[19]. TCM provides benefits in CAC prevention and treatment. These advantages encompass an effective disruption of the transition from colitis to carcinoma, a reduction in the toxicity linked to conventional therapies, and a notable enhancement in life quality[20,21]. Consequently, TCM has emerged as a vital approach in modern cancer prevention and treatment strategies.

In recent years, advances in modern biotechnology have led to many revelations, including the molecular mechanisms by which traditional herbal formulas and monomeric compounds exert therapeutic effects against CAC. For instance, it was revealed that Qi-Qin-Hu-Chang formula induces apoptosis in CRC cells and suppresses their migratory and invasive abilities, mechanisms associated with activation of the c-Jun N-terminal kinase/p38 MAPK signaling pathway[22]. Tong-Xie-Yao-Fang has been shown to attenuate CAC progression by inducing mitophagy in colon cancer cells and reversing epithelial-mesenchymal transition, mediated through regulation of the PINK1/parkin pathway[23]. Furthermore, it was reported that scutellarin ameliorates CAC by inhibiting the Wnt/β-catenin signaling pathway[24]. The continued exploration and mechanistic elucidation of herbal formulas capable of effectively preventing and treating CAC hold significant clinical relevance.

The turbidity toxin theory, introduced by Professor Li DG, a distinguished figure in TCM, represents a novel perspective in TCM. This theory posits that the development of CRC stems from a deficiency in the spleen and stomach, leading to functional disruptions that produce internal pathogenic elements and issues, such as damp-heat, phlegm-turbidity, and blood stasis. These harmful substances accumulate in the large intestine, where their prolonged presence can lead to the formation of turbidity toxins, which are implicated in the onset of cancer. The turbidity toxin theory has been identified as a key mechanism behind the emergence and advancement of CAC treatment, with the principle of “eliminating turbidity and detoxifying” serving as a crucial therapeutic approach. HZJDF, a well-known formula that was developed based on this theory, has been utilized in clinical settings for more than 40 years. Previous studies have demonstrated HZJDF’s effectiveness in managing inflammatory bowel disease and its related cancer risks. However, the essential molecular mechanisms behind HZJDF’s actions are not yet fully understood.

We initiated our study using a CAC mouse model to assess the effectiveness of different dosages of HZJDF in managing CAC. We found that HZJDF significantly reduced weight loss and colon length in the mice, improved survival rates, alleviated diarrhea and bloody stools, lowered the DAI scores, and diminished both the number and burden of colorectal tumors. Additionally, HZJDF helped repair the intestinal damage induced by AOM/DSS. Previous studies have established mesalazine as a standard control treatment[25]. Our investigation demonstrated that mesalazine had a notable therapeutic effect on CAC, consistent with other studies[3]. Furthermore, we found that high-dose HZJDF showed a slight advantage over mesalazine in enhancing the malignant phenotype indicators of CAC. These results underscore the substantial effectiveness of HZJDF in the treatment of CAC.

Analysis using a UHPLC-Q-Orbitrap-HRMS system revealed 322 distinct components in HZJDF. Following the ADME screening process, 120 key active components were identified, which included luteolin, quercetin, and kaempferol. The potential targets of these components were further predicted using the SwissTargetPrediction database. After removing duplicates, 1224 unique targets were identified. To compile CAC-related targets, the GeneCards, OMIM, and PharmGKB databases were consulted, yielding a total of 5306 targets after duplicates were filtered out. The intersection of the two target sets was analyzed, resulting in 640 common targets deemed potential candidates for HZJDF in CAC treatment. Additionally, a network diagram illustrating the relationship between HZJDF’s active components and CAC-related targets was created, which indicated that HZJDF may exert its anti-CAC effects through various components and targets. Also, a PPI network was constructed, and a topological analysis of these targets led to the identification of 52 core targets for HZJDF in CAC therapy. Ultimately, KEGG enrichment analysis emphasized that the PI3K/AKT signaling pathway was the most prominent among the predicted pathways, indicating it plays a crucial role in HZJDF’s therapeutic effects on CAC.

To elucidate the mechanism by which HZJDF functions in the treatment of CAC, we performed transcriptomic analysis of colon tissues obtained from the control, model, and HZJDF-H groups. We discovered 1729 genes that were common between the control and model groups, as well as between the model and HZJDF groups. We conducted KEGG enrichment analysis on the genes that were elevated in the model group relative to the control group and were reduced in the HZJDF group compared to the model group. Notably, the PI3K/AKT signaling pathway emerged as a significant finding in both these KEGG analyses. By integrating the transcriptomic data with the results from the network pharmacology analysis, we identified the PI3K/AKT signaling pathway as a crucial target and key element in the mechanism through which HZJDF inhibits CAC.

The PI3K/AKT pathway is a highly conserved signal transduction cascade that is chiefly mediated by PI3K and AKT. This pathway serves as a master regulator of many critical biological functions, such as cell growth, proliferation, and survival. Under physiological conditions, it acts as a central switch for normal cellular homeostasis. However, its aberrant activation is a common feature across many cancers, where it promotes oncogenic phenotypes including uncontrolled proliferation, resistance to apoptosis, and enhanced metastatic potential. Due to this central role in tumorigenesis, the PI3K/AKT pathway represents a crucial therapeutic target in oncology[26].

The PI3K/AKT signaling pathway has also been recognized as a crucial mechanism in tumors associated with inflammation, with its activation closely linked to CAC. This pathway is viewed as a promising target for CAC therapy, as it influences several vital biological functions, such as tumor cell growth, programmed cell death, and cell movement, by modulating downstream signaling[27]. Recent research has indicated that various TCM formulations can inhibit CAC by targeting the PI3K/AKT pathway. For instance, Shaoyao Decoction, a well-known remedy for UC, was shown to reduce CAC cell proliferation and enhance apoptosis through the suppression of the PI3K/Akt pathway[15]. Likewise, Huangqin Tang[6] and Wumei Wan[28] have demonstrated effectiveness in preventing the initiation and advancement of CAC by inhibiting the PI3K/AKT signaling pathway. Furthermore, we further clarified that luteolin, quercetin, and kaempferol are the main active components of HZJDF. Numerous studies have established that these compounds influence cancer development by modulating the PI3K/AKT pathway. Luteolin (C₁₅H₁₀O₆, molecular weight: 286.25), found in both Feng Wei Cao and Bai Jiang Cao within HZJDF, has been shown to inhibit the growth of various malignancies, including non-small cell lung cancer[29] and melanoma[30], through regulation of the PI3K/AKT pathway. Quercetin (C₁₅H₁₀O₇, molecular weight: 302.25), present in Huang Lian, Bai Jiang Cao, Di Yu, and Feng Wei Cao in HZJDF, can reduce the invasion and migration of advanced metastatic colon cancer cells by inhibiting the PI3K/AKT pathway[31]. Kaempferol (C₁₅H₁₀O₆, molecular weight: 286.24), which is found in Bai Shao, Bai Jiang Cao, Di Yu, Er Cha, Tu Si Zi, and Chai Hu in HZJDF, has been demonstrated to inhibit the growth of the human colon cancer cell lines HCT-8 and HCT-116 in a dose-dependent manner, and induce apoptosis, and its action is linked to the inhibition of the PI3K/AKT signaling pathway[32]. Consequently, the role of the PI3K/AKT signaling pathway will be further investigated in upcoming experiments.

Finally, we performed experiments on animals and cells to validate the results obtained from the network pharmacology and transcriptomic analyses. We discovered that HZJDF diminished the region stained for Ki67-positive cells in a manner that depended on the dosage, while also suppressing the activity of the PI3K/AKT signaling pathway within the colon tissues of the mice with CAC. Further cellular studies demonstrated that serum containing HZJDF could decrease the viability, proliferation, and migration of HT-29 and HCT116 cells, in addition to suppressing the PI3K/AKT signaling pathway. Thus, our research has established that HZJDF significantly mitigates CAC tumor development by targeting the PI3K/AKT signaling pathway.

Our research is pioneering in demonstrating the therapeutic potential of HZJDF for CAC and in shedding light on its underlying mechanisms. Nonetheless, there are several limitations to our study that should be noted. First, it is possible that multiple signaling pathways may influence the impact of HZJDF on CAC; however, our analysis concentrated exclusively on the PI3K/AKT pathway. Future studies should undertake more extensive pharmacological analyses to unravel these complex molecular interactions. Additionally, we did not employ PI3K/AKT pathway inhibitors for functional validation, which could strengthen future trials and enhance the robustness of the findings. Despite these limitations, our work lays important scientific groundwork for clinical justification for the use of HZJDF in CAC prevention and treatment, which could offer a safer and more effective therapeutic option than is currently available.

CONCLUSION

This research thoroughly demonstrated the significant therapeutic benefits of HZJDF in combating CAC and explored its possible mechanisms of action using an integrated approach comprising transcriptomics, network pharmacology, and experimental validation. We found that HZJDF effectively mitigates CAC by targeting the PI3K/AKT signaling pathway. Our findings offer a solid scientific foundation for the potential clinical use of HZJDF in preventing and treating CAC.