Unveiling the therapeutic potential of HZQYF: exploring the inhibitory impact of a clinical herbal formula on gastric cancer through network pharmacology and transcript analysis.

Introduction
Gastric cancer (GC) is a prevalent global health concern, ranking as the fifth most common malignancy and the third leading cause of cancer-related deaths worldwide [1]. Treatment options for this condition encompass surgical interventions, radiotherapy, chemotherapy, and targeted therapy [2–8]. Unfortunately, the clinical treatments involving radiotherapy and chemotherapy often come with significant adverse effects due to the disease's tendency to be diagnosed in advanced stages. Traditional Chinese medicine (TCM), known for its holistic approach and evidence-based therapies, offers promise in improving patient symptoms and mitigating the occurrence of adverse reactions [9–11].
Prescriptions in TCM are typically derived from the clinical practical application of TCM, guided by TCM theory. This involves consolidating individual medication experiences in clinical practice and progressively elucidating the appropriate population, dosage, therapeutic characteristics, and clinical benefits. The culmination of this process is to develop a standardized prescription, which can evolve into a new TCM suitable for a specific group of patients. This patient-centered approach focuses on clinical value, reflecting the distinctive role of TCM. It makes the best of the clinical advantages of TCM and is executed through various methods, such as integrating diseases and evidence, focusing on particular diseases and medications, or evidence-based TCM. These strategies clarify the clinical benefits for patients, thereby ensuring the efficacy of the treatment.
Based on the clinical application of TCM theory, we have identified and formulated a clinical formula called Hezi Qingyou Formula (HZQYF) to treat gastrointestinal disorders. Our research has demonstrated its efficacy in managing gastric discomfort caused by Helicobacter pylori infection [12] and mitigating the side effects induced by chemotherapeutic drugs. Helicobacter pylori-induced gastric cancer is believed to be a result of chronic inflammation that can lead to atrophic gastritis, gastric intestinal metaplasia, dysplasia, and eventually gastric adenocarcinoma [12, 13].
HZQYF is composed of Chebulae Fructus, Ficus hirta Vahl, and Cloves. Chebulae Fructus has been considered to possess antioxidant, hepatoprotective, neuroprotective, gastroprotective, antidiabetic, and anti-inflammatory activities [14]. Additionally, our previous study proved that Chebulae Fructus inhibited Helicobacter pylori-induced inflammatory response [15]. Cloves, also known as Syzygium aromaticum (L.) Merr. & L.M Perry, is traditionally used to support gastrointestinal function, alleviate pain, and treat stomach disorders such as vomiting, flatulence, and nausea [16]. Ficus hirta Vahl is the dried root of Ficus hirta Vahl. Studies have shown that Ficus hirta Vahl has the functions of liver protection, cancer prevention, and enhancement of immunological function [17–19]. Another study proved that it exerts antiproliferative and growth-inhibitory effects on Hela cells [20].
Herein, this study aimed to investigate its anti-gastric cancer effects and explore its possible mechanisms.

Material and Methods

The preparation of HZQYF water extracts
Chebulae Fructus and Ficus hirta Vahl (Root) were obtained from Guangzhou Zhining Pharmaceutical Co., Ltd. Cloves were offered by Guangzhou Junyuan Chenxiangshan Traditional Chinese Medicine Herbal Pieces Co., Ltd. 10 g HZQYF (60% Ficus hirta Vahl, 30% Chebulae Fructus and 10% Cloves) were extracted with tenfold water and boiled three times for 1 h at 90 ℃, then filtered, concentrated, and freeze-dried to powder. HZQYF water extracts were stored at − 20 ℃ and deposited in the International Pharmaceutical Engineering Lab of Shandong Province.

Ultra-high performance liquid chromatography-MS/MS (UHPLC-MS/MS)
As described in our previously published article [22], 1 mg/mL HZQYF water extract was filtered through 0.22 μm membrane and analyzed by UHPLC-MS/MS (Vanquish Q Exactive, Thermo Fisher, USA). The mobile phase was water-acetic acid (100:0.2, solvent A) and methanol (solvent B). The gradient condition was 0 min: 95% A, 5% B; 5 min: 85% A, 15% B; 10 min: 75% A, 25% B; 20 min: 40% A, 60% B; 35-45 min: 10% A, 90% B; 45.1- 60 min: 95% A, 5% B. The flow rate was 0.2 mL/min and the detected UV spectra was 270 nm. The Chromatography Column was ACQUITY UPLC BEH C18, 2.1 * 100 mm, 1.7 μm. Monitoring mode: Full MS ddMS2; Spray voltage: (+) 3500 V (-) 3000 V; Auxiliary gas flow rate: 7 Arb; Auxiliary gas heater temperature: 300 °C; Sheath gas flow rate: 35 Arb; Capillary temperature: 325 °C.

Network Pharmacology and TCGA data analysis
Potential drug targets were screened from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, OB ≥ 30% and DL ≥ 0.18, http://www.tcmspw.com/tcmsp.php), SWISS Target Prediction Database (http://www.uniprot.org/) and Traditional Chinese Medicine Integrated Database (TCMID, http://www.megabionet.org/tcmid). GC RNA-seq data were obtained from The Cancer Genome Atlas-Stomach Adenocarcinoma (TCGA-STAD). Differentially expressed genes (DEGs) of GC were analyzed by the R Foundation with a fold change cut-off value of 1.3 and a P value of less than 0.05. The Venn Diagram was drawn online (https://bioinformatics.psb.ugent.be/webtools/Venn/). The protein–protein association network was analyzed by the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database in 2023 (https://cn.stringdb.org/cgi/input?sessionId=b4apxqd43I6Z&input_page_show_search=on).

GO and KEGG analysis
Enrichment GO term (BP/CC/MF) and KEGG were plotted by https://www.bioinformatics.com.cn (last accessed on 10 Nov 2023), an online platform for data analysis and visualization [21].

Reactome pathway
The Reactome Pathway was analyzed using Analysis Tools (https://reactome.org/ ) [22].

Cell viability
Cell viability was conducted by CCK- 8 assay [23]. GC cells (BGC- 823 cells and SGC- 7901 cells) were cultured in RMPI 1640 medium containing 10% FBS in a 37℃ CO₂ incubator with a humid atmosphere. 6000 cells of BGC- 823 and SGC- 7901 were seeded in 96-well plates overnight and co-incubated with different concentrations of HZQYF (0–800 µg/mL) for 48 h. Then 10% CCK- 8 solutions were added into the medium and incubated for 2 h at 37 ℃. The process details of the drug preparation were as follows: Initially, 10 mg of HZQYF water extract powder was precisely weighed and dissolved in 1 mL of distilled water. Subsequently, the solution was filtered through a 0.22 μm membrane to obtain a filtrate for subsequent use. Finally, the 10 mg/mL stock solution of HZQYF was serially diluted with RMPI 1640 medium containing 10% FBS to reach the desired concentrations of 50, 100, 200, 400, and 800 µg/mL. The control group was treated in a way similar to that of the stock HZQYF solution. Specifically, it was initially treated with an equal volume of distilled water and further diluted with RMPI 1640 medium containing 10% FBS.

Cell colony formation
500 cells were seeded and cultured in a 12-well plate overnight. Different concentrations of HZQYF (0, 50, 100, 200 µg/mL) were treated with the GC cells. The medium was changed every three days and the cells were cultured until the number of clones was visible and then fixed with paraformaldehyde for 15 min, stained with 0.1% crystal violet, and photographed.

Data analysis
Statistical analyses were conducted using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Data are expressed as mean ± standard deviation (SD). Differences between groups were assessed using an unpaired two-tailed Student’s t-test, with significance levels denoted as *P < 0.05 and **P < 0.01. To ensure robustness and reproducibility, all experiments were performed in triplicate.

Results

The main compounds were identified by UHPLC-MS/MS
In order to determine the main components of the drug, we performed a UHPLC-MS/MS analysis [22]. The results demonstrated that the primary compounds were identified and characterized, namely Shikimic acid, Chebulic acid, Quinic Acid, Punicalin, Gallic Acid, Galloyl-glucose, Galloylquinic acid, HHDP-glucose, Digalloyl glucoside, HHDP-hexoside, Vanillic acid glucose, Corilagin, Magnolioside, Brevifolin-carboxylic acid, Chebulanin, Trigalloyl-glucoside, HHDP-galloyl glucose, Galloyl chebuloyl-HHDP Glucose, Tetragalloyl glucose, Chebulinic acid, Quercetin- 3-o-Glucuronide, Ellagic acid, Kaempferol, kaempferol- 3-Oglucuronide, Quercetin- 3-Oglucuronide, 6’’-methylester, 3-Methylquercetin (Isorhamnetin), Psoralen, Angelicin and Bergapten (Fig. 1, S-Table 1).

The potential targets of HZQYF and GC were identified via network pharmacology and transcript data analysis
As shown in Fig. 2 A, 289 drug targets of HZQYF were screened, consisting of targets of Chebulae Fructus, Ficus hirta Vahl, and Cloves, respectively. Subsequently, 260 genes were co-expressed between HZQYF and GC targets (Fig. 2B). The PPI network diagram of these genes is shown in Fig. 2C.

GO and KEGG pathway analysis
We performed pathway prediction analyses to explore the pathways that may be enriched for these drug-disease targets. From the results of GO analysis, we found that the HZQYF anti-GC effect may be achieved through the regulation of steroid metabolic process, postsynaptic membrane potential, GABA-A/GABA receptor complex, chloride channel complex, postsynaptic membrane, steroid activity, extracellular ligand-gated ion channel activity, and transmitter-gated ion channel activity (Fig. 3A). KEGG results indicated that it may control reactive oxygen species, GABAergic synapse, Nitrogen metabolism (Fig. 3B).

Reactome analysis
In order to further explore the relationship between HZQYF and GC, Reactome analysis was conducted. As displayed in Fig. 4, the results of enrichment analysis suggested that the anti-GC effect of HZQYF might be related to the signal transduction, translocation of olfactory receptors, neutrophil degranulation.

The Inhibitory effect of HZQYF on gastric cancer cells
Next, to investigate its active role, we examined the anti-gastric cancer activity of HZQYF. The experimental results demonstrated that HZQYF at concentrations of 50, 100, 200, 400, and 800 µg/mL significantly inhibited the viability of GC cells (Fig. 5A and 5B). Additionally, HZQYF at concentrations of 50, 100, and 200 µg/mL suppressed the clonogenic formation ability of GC cells (Fig. 5C).

Discussion
Network pharmacology and bioinformatics provide high efficiency for studying the core pathways and targets of their actions, as well as laying the theoretical foundation for subsequent precision therapy. The combined use of network pharmacology and bioinformatics offers favorable evidence in our search for the effects and targets of herbal compounds against gastric cancer. Moreover, this approach provides a better understanding of the valuable efficiency of screening based on big data.
Gastric cancer is a highly lethal malignant tumor [24]. The treatment for advanced gastric cancer has evolved significantly, particularly with the advent of immune checkpoint inhibitors combined with chemotherapy, which has shown remarkable survival benefits for patients with positive PD-L1 expression. Combining immune checkpoint inhibitors and chemotherapy can remarkably prolong survival [25–27]. However, the over-activation of the immune system may attack the normal tissues and organs of the body, leading to immune-related adverse reactions, such as cytokine release syndrome, which may compromise patient safety [28]. Furthermore, the efficacy of immune checkpoint inhibitors is predominantly observed in PD-L1-positive patients, while those with PD-L1-negative expression derive limited benefit [29]. Not all advanced gastric cancer patients can benefit from immunotherapy, and it is necessary to accurately screen the appropriate patient population [30]. In addition, the significant toxic side effects and substantial financial burden associated with current chemotherapeutic and immunotherapeutic agents have prompted the exploration of alternative therapeutic strategies. In light of these challenges, there is growing interest in exploring alternative or adjunctive therapies, particularly traditional herbal formulas, which may offer a safer and more cost-effective approach. It has been reported that certain Chinese herbal drugs, like Sishen Jiedu Decoction, Pingxiao Tablet, Fuzheng Guben Granule, and Buyang Huanwu Tang, are capable of enhancing the five-year survival rates of patients afflicted with upper digestive tract cancers [31].
As a clinical formulation, Hezi Qingyou Formula (HZQYF) represents a novel and promising candidate. While standard treatments like chemotherapy and immunotherapy remain cornerstone therapies, HZQYF offers a complementary approach that may address some of their limitations, such as toxicity and high costs. In this study, we present the first experimental evidence demonstrating the anti-gastric cancer activity of HZQYF in vitro, underscoring its potential as a promising therapeutic agent. To further explore its efficacy and elucidate its mechanisms of action, future studies directly comparing HZQYF with chemotherapy or immunotherapy are essential. Such comparative analyses will not only validate its therapeutic potential but also pave the way for its integration into comprehensive gastric cancer treatment regimens, offering a complementary or alternative approach to current therapies.
In this study, we initially identified potential drug targets of HZQYF by analyzing three medicinal herbs—Chebulae Fructus, Ficus hirta Vahl, and Cloves—using network pharmacology. To ensure the relevance of these targets to gastric cancer, we integrated them with differentially expressed genes identified from TCGA-STAD gastric cancer tissue sequencing data. Through this combined analysis, we identified 260 shared target genes that are likely to play critical roles in gastric cancer pathophysiology. These targets were further prioritized based on their involvement in key biological processes and signaling pathways associated with gastric cancer. This approach ensures that the selected targets are not only relevant to the pharmacological action of HZQYF but also directly linked to the molecular mechanisms underlying gastric cancer progression.
Meanwhile, based on the results of big data, it was predicted that the core probable function of HZQYF might be regulating reactive oxygen species (ROS), GABAergic synapse, and nitrogen metabolism. ROS, which includes superoxide anions, hydrogen peroxide, and hydroxyl radicals, are essential for maintaining normal physiological functions when balanced within and outside cells. However, excessive ROS production or impaired antioxidant defense systems can lead to oxidative stress, closely associated with tumor initiation and progression, including gastric cancer [32]. Excessive ROS can cause oxidative damage to DNA, proteins, and lipids, promoting abnormal cell proliferation and carcinogenesis. Additionally, ROS are involved in various signaling pathways related to gastric cancer, such as inflammation, cell apoptosis, and angiogenesis, further promoting gastric cancer development [32, 33]. Additionally, ROS plays a pivotal role in modulating the PI3 K/AKT signaling pathway. By precisely controlling ROS levels, this pathway can be inhibited, leading to the induction of apoptosis and thereby effectively curbing the advancement of cancer [34]. Elevated ROS levels can induce oxidative stress, resulting in DNA damage and genomic instability, further activating PI3 K/AKT signaling to drive tumor growth [35]. Therefore, controlling ROS levels and maintaining the oxidative-antioxidative balance are crucial for the prevention and treatment of gastric cancer.
GABAergic synapse can impact gastric cancer through its potential influence on the neurotransmitter gamma-aminobutyric acid (GABA) and its associated synaptic pathways [36]. GABA, known as an inhibitory neurotransmitter in the central nervous system, has been observed to affect non-neuronal tissues, including the gastrointestinal system. A prior study indicated that GABA and its receptors may influence various cancer-related processes, such as cell proliferation, apoptosis, migration, and invasion [37]. Moreover, the presence of GABA receptors in gastric cancer cells suggests a potential association between GABAergic signaling and the development of gastric cancer [38]. What’s more, GABA receptors could act as a novel therapeutic target in cancer [39, 40]. GABA has been shown to stimulate the proliferation of human gastric cancer cells through both autocrine and paracrine mechanisms mediated by GABAAR. This process subsequently triggers the activation of the MAPK/ERK signaling pathway and upregulates cyclin D1 expression [41]. Therefore, a comprehensive investigation of the GABA signaling pathway is of paramount importance, as HZQYF may serve as a potential therapeutic target that plays a crucial regulatory role in this pathway. Further studies will be conducted to elucidate its molecular mechanisms and explore its therapeutic potential in gastric cancer treatment.
Nitrogen metabolism can impact gastric cancer through various mechanisms [42]. For instance, changes in nitrogen metabolism can influence the synthesis of nucleotides and amino acids, which are crucial for cell growth and proliferation [43]. Additionally, nitrogen metabolism contributes to the production of reactive nitrogen species, which may lead to oxidative stress and DNA damage, potentially promoting cancer development. Moreover, nitrogen-containing compounds like nitrosamines, formed as a result of nitrogen metabolism in the human body, are implicated in the development of gastric cancer. Overall, disruptions in nitrogen metabolism can affect cellular processes and contribute to the molecular pathways involved in the initiation and progression of gastric cancer.
In addition, we predicted that its possible components that exert anti-tumor effects are likely to be chebulic acid, quinic acid, gallic acid, corilagin, tetragalloyl glucose, chebulinic acid, ellagic acid, kaempferol, isorhamnetin, psoralen, angelicin and bergapten. Studies have shown that gallic acid is a potential anticancer agent [44], inducing cell apoptosis [45], inhibiting cell migration and invasion via blocking the AKT/small GTPase signals pathway, and inhibition NF-kappaB activity [46]. Additionally, research has demonstrated that corilagin and tetragalloyl glucose can induce cancer cell apoptosis [47–50], ellagic acid can suppress the migration and invasion of GC cells [51–53], kaempferol can promote autophagy and cell death, and inhibit tumor growth [54–58]. Isorhamnetin has been found to inhibit proliferation and invasion and induce apoptosis [59–62]. Research revealed that psoralen increases chemotherapeutic sensitivity [63, 64] and angelicin inhibits liver cancer growth [65]. Additionally, research has found that bergapten has anti-cancer and anti-bacterial properties, reduces cell viability, induces cell cycle arrest, and exerts potent anticancer and apoptotic effects through autophagy [66–71].
Our study provides a rational theoretical basis for clinical prescription against tumors. Given the current research context and the scope of our study, we mainly resorted to in-silico methods. Network pharmacology, which can analyze complex drug-target-disease interactions at a systems level, helped us map potential pathways and molecular mechanisms. Transcriptomic analysis, meanwhile, revealed gene expression profiles relevant to our research, allowing us to identify key genes and regulatory networks. These in-silico techniques provided valuable clues and emphasized the predictive value of our findings as a basis for future experiments.
However, this article has some shortcomings. First, pathway-based validation should be performed. At present, pathway-based experimental validation is lacking. In the forthcoming work, we have devised a plan to carry out a comprehensive series of pathway-centered experiments. These will include assays for ROS modulation and investigations into the GABAergic synapse.
Additionally, we have only carried out preliminary investigations through cell experiments. To fully validate the therapeutic efficacy of HZQYF against GC and its potential for clinical translation, further in vivo studies are essential. In the subsequent research, we will conduct animal models. Specifically, we plan to select appropriate animal models for gastric cancer, such as the mouse xenograft tumor model. Prior to the experiments, we will accurately convert the clinical dose into the dosing doses for mice or rats based on well-established conversion principles and meticulously design three dose groups: low, medium, and high. This approach will allow us to systematically analyze the impact of different doses of HZQYF on the treatment effect, with particular emphasis on enhancing efficacy and monitoring the potential toxicity. During the experiments, we will conduct them strictly in accordance with the standard operating procedures. By observing the tumor growth curve, evaluating the changes in tumor volume and weight, and detecting relevant biomarkers and other indicators, we will comprehensively explore the inhibitory effect of HZQYF on gastric cancer in the in vivo environment and its underlying mechanisms. Simultaneously, we will also pay close attention to the overall physiological status of the animals, including changes in body weight, diet, and activity. This comprehensive assessment will help us determine the safety and tolerability of HZQYF.
In addition, advanced molecular biology techniques will be employed. Specifically, gene knockout and overexpression models will be utilized to deliberately disrupt specific pathways. By doing so, the consequent phenotypic alterations can be closely monitored and recorded, which will serve as crucial indicators of the functional changes within the biological system. Subsequently, high-throughput screening approaches will be implemented. 16S rRNA sequencing and proteomics analysis, in particular, will be carried out. These techniques will enable us to meticulously and thoroughly characterize the molecular modifications that occur within the pathways. It is not only anticipated to effectively fill the existing gap in pathway-based experimental validation, which has been a limitation in our current study, but also to generate more accurate, reliable, and conclusive outcomes. Consequently, this will significantly enhance and strengthen the scientific foundation. Moreover, it will expand the translational potential of our research, making it more likely to have practical applications and impacts in relevant fields. Meanwhile, the clinical formula is only based on the in-hospital preparation being used, and we will follow up with multi-clinical center experiments to explore the possibility of developing it into a new drug.
For the development of clinical prescriptions into new drugs for research, there is still a lot of work that needs to be done to follow up with further research. The translation of HZQYF into clinical practice presents both promising prospects and numerous considerations. Navigating the complex pharmaceutical regulatory network is an extremely challenging task. The approval process demands comprehensive documentation, ranging from detailed preclinical data to well-structured clinical trial protocols. To meet regulatory standards, strict adherence to good laboratory practice and good clinical practice is essential. For instance, during the submission of a new drug application, exhaustive information regarding HZQYF's chemical composition analysis, entire production process, and pharmacological properties must be provided. This process is time-consuming and resource-intensive and requires the relevant personnel to have a deep understanding of the constantly evolving regulatory landscape.
Transitioning from small-scale laboratory preparations to large-scale industrial production poses a series of difficulties. Ensuring the stability of raw material supply in large-scale production is crucial, as minor alterations can significantly impact the drug's final efficacy and safety. Scaling up production necessitates comprehensive and meticulous optimization of all aspects to maintain consistent product quality. Key factors include the selection of appropriate production equipment, precise control of temperature and humidity throughout production, and proper establishment of post-production storage conditions. If the formulation originated from a small laboratory, challenges such as batch-to-batch reproducibility and cost-effective procurement of high-quality raw materials must be overcome during industrialization.
A thorough investigation of potential side effects is of utmost importance. Cell and animal experiments conducted in the early stage, as well as any available human data, require careful analysis. Understanding the frequency and severity of potential side effects is not only vital for patient safety but also critical for meeting regulatory requirements and enhancing the overall acceptance of HZQYF in the clinical practice environment.
The translation of HZQYF into clinical practice necessitates a rigorous approach to dosage determination and safety evaluation, both of which are critical to ensuring therapeutic efficacy and patient safety. To systematically evaluate its efficacy, animal studies will employ the body-surface area normalization method to calculate species-specific dosages, ensuring translational relevance. Specifically, the drug’s effects will be investigated at 0.5 ×, 1 ×, and 2 × the human equivalent dose, enabling a comprehensive analysis of the dose–response relationship and providing critical insights for optimizing therapeutic dosages in future clinical trials. Safety assessment is equally paramount, and a robust monitoring framework has been designed for clinical implementation. This includes routine biochemical profiling to evaluate hepatic and renal function, as well as vigilant monitoring for potential allergic reactions. Furthermore, the safety protocol aligns with guidelines from authoritative regulatory bodies such as the FDA and EMA, ensuring compliance with international standards. By integrating dose–response characterization with rigorous safety evaluation and regulatory adherence, this approach establishes a scientifically validated and globally compliant pathway for translating HZQYF from preclinical research to clinical application, thereby bridging the gap between traditional medicine and modern therapeutic development.
Although the translation of HZQYF into clinical practice is faced with hardships and challenges, by adopting a multi-pronged comprehensive strategy to address issues in key areas such as regulation, formulation, and safety, the door to successful clinical application can be opened smoothly.

Conclusion
Based on this, we found that the clinical prescription HZQYF has anti-gastric cancer activity in vitro, while the core pathway proteins of its action may regulation of GABAergic synapse and nitrogen metabolism. Additionally, its core active ingredients are mainly chebulic acid, quinic acid, gallic acid, corilagin, tetragalloyl glucose, chebulinic acid, ellagic acid, kaempferol, isorhamnetin, psoralen, angelicin and bergapten. This provides a theoretical basis for the development of clinical formulas into new drugs.

Supplementary Information