Hedyotis diffusa Willd suppresses hepatocellular carcinoma tumor-stromal interactions by inactivating cancer-associated hepatic stellate cells

Abstract

Background

Hedyotis diffusa Willd (HDW) has the effects of clearing the heat, removing the toxins, restoring the circulation, eliminating the stasis and promoting the process of diuresis. Variety formulations of HDW various formulas involving HDW show remarkable clinical effects and have become second-lines anticancer drugs in China. However, its underlying mechanism against hepatocellular carcinoma (HCC) remains unclear. The objective of this study is to investigate the functional effect of HDW in the treatment of HCC, and to clarify the mechanism of HDW in suppression of tumor-stromal interaction.

Methods

Therapeutic effects of HDW on HCC cells and cancer-associated hepatic stellate cells (HSCs) were investigated in vitro and in vivo. Potential targets of HDW were predicted and signaling pathway analyses were conducted using network pharmacology. The HDW-target gene set (HDW-TGS) was systematically screened to investigate the mechanisms by which HDW influences tumor-stromal interactions and to identify target genes with significant clinical value.

Results

HDW induced HCC cells apoptosis via reactive oxygen species (ROS) induction. HDW-TGS significantly correlated to prognosis in The Cancer Genome Atlas (TCGA) HCC samples. HDW suppressed activated-HSC migration and invasion by regulating expression of fibrosis markers α-SMA and p-ERK1/2. In an HCC cell and HSC splenic co-transplanted xenograft mouse model, HDW suppressed liver tumor formation by downregulating fibrosis, indicating that HDW inhibited tumor-stromal interactions in vivo. Finally, we revealed a potential downstream target of HDW in HCC. We proposed that HDW might interrupt tumor-stromal interaction via ADH4.

Conclusions

This study provides a new perspective for the treatment of HCC with HDW, demonstrating the feasibility of ADH4 as an HCC treatment target.

Highlight box.

Key findings

• Hedyotis diffusa Willd (HDW) interrupts tumor-stromal interaction and fibrosis of hepatocellular carcinoma (HCC) in vitro and in vivo through suppressing activated-hepatic stellate cells (HSCs) migration and invasion by regulating the expression of fibrosis markers α-SMA and p-ERK1/2. As a potential therapeutic target of HDW, ADH4 may be involved in extracellular matrix remodeling and fibrosis of HCC.

What is known and what is new?

• The anti-tumor effect of HDW has been demonstrated, and HDW has been shown to enhance the efficacy and reduce the side effects of sorafenib and 5-fluorouracil in HCC treatment.

• This study is the first to investigate the effect and mechanism of HDW in cancer-associated HSCs, and screen therapeutic targets of HDW which are involved in HCC tumor-stromal interaction.

What is the implication, and what should change now?

• Through our research, the functional role of HDW in tumor-stromal interaction of HCC has been identified. The combination therapeutic strategy of HDW and classic chemotherapy drugs for HCC has great research value and is highly anticipated.

Introduction

Hepatocellular carcinoma (HCC) accounts approximately 90% of all cases of primary liver cancer and is the second leading cause of cancer-related death worldwide (1). In China, HCC exhibits the highest incidence rate among all cancers, with a median survival of 23 months and 5-year overall survival rate of 12% (2). Most HCC patients are diagnosed at an advanced stage (3), rendering curative resection unfeasible. Cancer-associated fibroblasts (CAFs), which originate from activated hepatic stellate cells (HSCs), secrete extracellular matrix (ECM) proteins and growth factors, promoting tumor-stromal interaction and fibrosis of HCC (4). As a consequence, the efficacy of the current chemotherapy drugs is unsatisfactory, and the overall high recurrence rate has not improved (3). Therefore, it is urgent to develop new therapeutic approaches to optimize HCC clinical therapy.

The multitarget and coordinated intervention effects of Chinese herbal medicine (CHM) have made it a promising option for treating HCC (5). Extensive molecular and biological studies have demonstrated the potential of CHM-based compounds as anti-HCC treatments (6). CHM reduces the proliferation of HCC cells both in vivo and in vitro, triggers cell death, modulates immunity, curbs metastasis, diminishes inflammatory responses, and enhances antiviral activity (7). Recent retrospective cohort studies have indicated that using combined CHM therapy can prolong the survival time of patients with unresectable HCC following transcatheter arterial chemoembolization and radiofrequency ablation (8,9). CHM prescriptions including Hedyotis diffusa Willd (HDW) have been reported to play an important role in lowering serum α-fetoprotein and increasing serum albumin levels in the clinical treatment of HCC patients. Moreover, HCC patients treated with CHM had significantly higher overall survival rates and liver-specific survival rates, with an approximately 3-month increase in median survival time and a reduced risk of all-cause mortality (10).

According to the Chinese Pharmacopoeia, HDW is a medicinal herb used in Chinese medicine for its properties of heat clearance, toxin removal, circulation restoration, stasis elimination, and diuresis promotion. In China, HDW has been commonly used as an adjunct treatment for liver cancer for decades (11). Research has demonstrated the anti-tumor effect of HDW in several cancer types including colorectal cancer, cervical cancer, glioblastoma, lung cancer, breast cancer and leukemia (12-17). Various formulations of HDW have been developed, including injections and granules. HDW injections have been introduced as new second-line anticancer medications in China, with impressive clinical outcomes. Studies have shown that HDW exhibits anticancer properties by inducing apoptosis and boosting immune functions (18-20). HDW was reported to induce apoptosis of HCC cells by regulating AKT/mTOR and JNK/Nur77 signaling pathways (21,22). Additionally, HDW enhanced the efficacy and reduced the side effects of sorafenib and 5-fluorouracil in HCC treatment (23,24). Further exploration of the components and molecular mechanisms of HDW against HCC may help improve clinical treatment.

In the present study, we investigated the functional effect of HDW in tumor-stromal interaction of HCC. We demonstrated that HDW induced HCC cell apoptosis and cellular reactive oxygen species (ROS) induction. Furthermore, HDW effectively inhibited liver tumor formation in a xenograft mouse model. We established an HDW-target gene set (HDW-TGS) to explore genetic alterations of HDW treatment using network pharmacology and bioinformatics analyses. We identified evidence for an involvement of HDW in interrupting tumor-stromal interaction and fibrosis of HCC in vitro and in vivo. We further identified the potential therapeutic target ADH4, one of the genes in HDW-TGS, which may be involved in extracellular matrix remodeling and fibrosis of HCC. Our results indicated that HDW has the potential as a new therapeutic agent for HCC. We present this article in accordance with the ARRIVE reporting checklist (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-753/rc).

Methods

Cell lines, cell culture, and treatment

The HCC cell lines used in this study included LX-2, Hep3B, Huh7, MHCC97H, and HepG2 (Chinese Academy of Medical Sciences, Beijing, China) and they were described before (25). Cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin at 37 ℃ with 5% CO₂. Cells (2×10⁵ cells/well in 6-well culture plates) were treated with HDW (sterilized aqueous solution extracted from HDW, national medicine permission number: Z34020595, Anhui, China) for 48 h, and RNA was extracted for subsequent analyses. For collection of conditioned medium, HCC cells were washed twice with PBS and incubated with fresh medium for 48 h. The medium was collected, filtered with a 0.22-μm syringe filter, and centrifuged (UFC9030, Amicon Ultra-15, Merck). The concentrated medium was collected and mixed with fresh medium containing 10% FBS at a ratio of 50 µL: 1 mL.

Immunohistochemistry

Immunohistochemical staining of tissue sections was performed as described in previous studies (25,26). Sections were incubated with anti-Ki67 (A20018, ABclonal, Wuhan, China) and anti-α-SMA (19245S, Cell Signaling Technology, Danvers, USA) with an SP-POD Kit (SP0021, Solarbio, Beijing, China). Image acquisition was performed using a Leica microscope (DMi8, Wetzlar, Germany).

ROS detection analysis

ROS production was measured using dichlorofluorescin diacetate (DCFH-DA, S0033, Beyotime, Shanghai, China) in accordance with manufacturer’s instruction. Cells were washed with PBS and treated with 10 µM DCFH-DA at 37 ℃ for 0.5 h. The cells were washed twice with PBS and analyzed at emission 525 nm and excitation 488 nm wavelengths using the Multiskan GO (ThermoFisher Scientific, Waltham, USA).

Apoptosis detection

HCC cells were seeded in 24-well plates and treated with control or HDW-containing medium for 48 h at 37 ℃ with 5% CO₂. Cells were washed with PBS and stained with Annexin V-FITC and propidium iodide (PI) using the Annexin V-FITC Apoptosis Detection Kit (C1062S, Beyotime) following the manufacturer’s instructions. Fluorescent images were acquired using a Leica microscope (DMi8).

Migration and invasion assays

Migration and invasion of HCC cells were assessed using uncoated or Matrigel-coated transwell chambers (8 µm pore size) as previously described (25,27). For co-culture assays, LX-2 cells were seeded and cultured in the lower chambers of the transwell chambers; control HCC cells or 5% HDW pre-treated HCC cells (5×10⁴) were seeded and cultured in the upper chamber. After 24 or 48 h, the migrated or invaded cells were fixed with 70% ethanol and stained with hematoxylin and eosin. The cell number was counted in five random fields (100× magnification).

Cell viability assay

Cells (2×10³ cells/well) were seeded in 96-well plates. At 24 h after seeding, medium was replaced with 100 µL of control or 5% HDW medium. After 48 h of incubation, cell viability was examined using the CCK-8 kit (C0038, Beyotime).

In vivo experiments

BALB/c athymic female nude mice (4 weeks old) were obtained from GemPharmatech (Nanjing, China). Mice were raised in a specific pathogen-free animal room (SPF) of Shenzhen Peking University, Hong Kong University of Science and Technology Medical Center. Splenic co-transplanted xenograft mouse model was performed as described previously (25). After 7 days of acclimation, mice were splenic co-transplanted with 5×10⁵ Huh7 cells and 5×10⁵ LX-2 cells and randomized into PBS or HDW treatment group (n=5 per group). At 7 days after transplantation, mice received intraperitoneal vehicle or 10% HDW PBS twice a week for 3 weeks. The mice were sacrificed under anesthetized conditions, and livers were collected for subsequent analyses.

Ethical statement

The animal study protocol was approved by the Animal Welfare and of Peking University Shenzhen Hospital (Shenzhen, China, No. 2023-140, June 21, 2023), in compliance with the animal use regulations of the U.S. National Institutes of Health (NIH) for the care and use of animals.

Bioinformatics

The HDW-target genes were acquired from the BATMAN^1.0 database (28). The online bioinformatics analysis tool is specifically designed for studying the molecular mechanisms of Traditional Chinese Medicine and is based on Traditional Chinese Medicine ingredient target prediction. DAVID (29) was used for Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and Gene Ontology (GO) enrichment analysis. The protein interaction information was obtained using the STRING database (30). UALCAN (31), GEPIA2 (32), GSCA: Gene Set Cancer Analysis (33) and SRplot (34) were used for gene expression, survival, and bioinformatics analyses.

Molecular docking

To further investigate the interaction between compounds and targets, molecular docking was performed. Crystal structures of the core targets were downloaded from the PDB database (https://www.pdbus.org/) (35), while small molecule ligand structures were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/). CB-Dock2 was used to process the structures and conduct molecular docking simulations (36). The highest Vina score conformation was extracted, analyzed, and visualized.

Statistical analysis

Data are shown as mean ± standard error of mean (SEM). The log-rank test and Kaplan-Meier analyses were used for survival analyses. P<0.05 indicated statistical significance. GraphPad Prism and R language were used for statistical analysis.

Results

HDW induced apoptosis and ROS production in HCC cells

To investigate the effect of HDW on HCC cells, we performed cell viability assays in Hep3B, Huh7, MHCC97H and HepG2 cells treated with HDW. The results showed that HDW at concentrations ranging from 5% to 20% inhibited cell viability (Figure 1A). From these results, we selected 10% for use in further experiments, which induced cell viability repression at a low dose. We found that HDW induced cell apoptosis compared with controls (Figure 1B). We also observed significantly increased intracellular ROS induction in cells after HDW treatment compared with the controls (Figure 1C).

Figure 1.

HDW induced apoptosis and ROS production in HCC cells. (A) HDW at the indicated doses inhibited the cell viabilities of HCC cells. (B) HDW triggered apoptosis of HCC cells. Green: Annexin V-FITC; red: propidium iodide. Scale bar =100 µm. (C) HDW increased intracellular ROS induction in HCC cells. *, P<0.05; **, P<0.01; ***, P<0.001. HCC, hepatocellular carcinoma; HDW, Hedyotis diffusa Willd; LIHC, liver hepatocellular carcinoma; NC, negative control; ROS, reactive oxygen species.

Screening the specific targets of HDW in HCC

To investigate the potential targets of HDW against HCC, we searched the BATMAN^1.0 database for HDW to identify active compounds of HDW and screen the correlated target genes. The analysis of Enriched Online Mendelian Inheritance in Man (OMIM) diseases showed that the target genes of HDW had a significant association with HCC, providing potential evidence for HDW’s effects in HCC treatment (Table 1). The target prediction analysis identified 4, 51, 117, and 46 potential targets for the active ingredient oleanolic acid-28-O-beta-D-glucopyranoside, oleanolic acid, stigmasterol, and ursolic acid, respectively. After combining duplicates, the total number of predicted targets for HDW was 168 (Figure 2A and Table S1). Using GEPIA2, 2207 differential genes related to The Cancer Genome Atlas (TCGA) HCC tumors were searched. A Venn diagram of HDW targets and HCC differential genes was drawn, and 24 intersecting genes were identified (Figure 2B). The 24 intersecting genes were analyzed using GO enrichment analysis and KEGG enrichment analysis. The results revealed 31 biological process (BP) terms, 10 cell component (CC) terms, and 19 molecular function (MF) terms (Figure S1A-S1C). Additionally, KEGG enrichment analysis revealed that the 24 intersecting genes were correlated to ROS (Figure S1D), which is consistent with HDW treatment of HCC cells. We generated a PPI network map for Homo sapiens using the STRING database and identified protein interaction relationships (Figure S1E).

Table 1. Enriched OMIM diseases of HDW-targeted genes are related to HCC.

OMIM ID	Disease name	Adjusted P value	Targets*
114500	Colorectal cancer	4.18e−06	11
211980	Lung cancer alveolar cell carcinoma, included	4.26e−07	10
114480	Breast cancer	2.07e−03	8
114550	Hepatocellular carcinoma	1.17e−003	29
167000	Ovarian cancer, epithelial, included	2.4e−002	27
259500	Osteogenic	4.70e−006	25
137215	Gastric cancer, hereditary diffuse	2.20e−002	19

*, adjusted P value smaller than 0.05. HCC, hepatocellular carcinoma; HDW, Hedyotis diffusa Willd; OMIM, Enriched Online Mendelian Inheritance in Man.

Figure 2.

Screening the specific targets of HDW in HCC. (A) Schematic overview of active compounds and related target genes of HDW. (B) Screening HDW-TGS by intersecting HDW-target genes and HCC differential genes. (C) Kaplan-Meier survival analysis of overall survival and progression-free survival of HCC patients in accordance with HDW-TGS by TCGA GSVA score. High GSVA scores of HDW-TGS were associated with shorter survival times (log-rank P=0.005 and <0.001). (D) Cancer-related pathway analysis revealed HDW-TGS significantly correlated to hormone ER (P=0.57), RASMAPK (P=0.52), and RTK (P=0.49) pathways. *, P≤0.05; ^#, FDR ≤0.05. DEGs, differentially expressed genes; FDR, false discovery rate; GSCA, Gene Set Cancer Analysis; GSVA, Gene Set Variation Analysis; HCC, hepatocellular carcinoma; HDW, Hedyotis diffusa Willd; HDW-TGS, HDW-target gene set; OS, overall survival; PFS, progression-free survival; TCGA, The Cancer Genome Atlas.

To further explore the potential mechanisms, the 24 genes were used as a gene set (HDW-TGS) for Gene Set Cancer Analysis (Table S2 and available online: https://cdn.amegroups.cn/static/public/jgo-2025-753-1.xlsx). The HDW-TGS significantly correlated to progression-free survival and overall survival of HCC patients in TCGA (Figure 2C). Gene Set Variation Analysis (GSVA) pathway analysis revealed a high correlation between HDW-TGS and signaling pathways including hormone ER (P=0.57), RASMAPK (P=0.52), and RTK (P=0.49) (Figure 2D and Table S3).

HDW inhibited HSC activation and HCC-HSC interaction

Activated HSCs are the major origin of CAFs within the TME (4). We next examined the effects of HDW in on activated HSC cells. Compared with control cells, LX-2 cells activated by TGF-β1 recombinant protein (10 ng/mL) or cancer cell conditioned medium showed significantly increased sensitivity to HDW treatment (Figure 3A). In transwell assays, 5% HDW did not affect LX-2 migration or invasion capacity. In co-culture transwell assays, we found the co-culture of MHCC97H or Huh7 cells increased the migration and invasion capacities of LX-2 cells, and 5% HDW inhibited these effects (Figure 3B). Next, we investigated markers for HSC activation after HDW treatment. We found that p-ERK1/2 and α-SMA were significantly downregulated in the presence of MHCC97H or Huh7 cell conditioned medium (Figure 3C), suggesting the effect of HDW in suppressing HSC activation.

Figure 3.

HDW suppressed HCC-HSC interaction by inactivating HSC. (A) TGFβ1-activated and cancer supernatant-activated LX-2 cells were more sensitive to the treatment of HDW compared with LX-2 cells. *, P<0.05; **, P<0.01; ***, P<0.001. (B) Migration and invasion assays of LX-2 cells were performed for 24 and 48 h with cancer cell supernatant and/or HDW. Graphs show the numbers of cells calculated from five fields. Scale bars =100 µm. *, P<0.05; ***, P<0.001. (C) The protein levels of p-ERK1/2 and α-SMA in LX-2 cells following the indicated treatment. HCC, hepatocellular carcinoma; HDW, Hedyotis diffusa Willd; HSC, hepatic stellate cell; NC, negative control.

HDW suppressed HCC progression in vivo

To investigate the effect of HDW against tumor-stromal interaction of HCC in vivo, we performed a splenic co-transplanted xenograft mouse model (25) by implanting Huh7 cells and LX-2 cells of nude mice. After one week, mice received intraperitoneal injections of PBS or HDW twice weekly for three weeks (Figure 4A). At the end of drug administration, the livers were collected (Figure 4B). Treatment of HDW remarkably decreased tumor nodules in livers (Figure 4C). Immunohistochemistry of serial sections revealed a downregulation of Ki67 and α-SMA expressions in the HDW-treated group compared with a control group (Figure 4D). These findings indicated that HDW suppressed HCC fibrosis and tumor formation in vivo.

Figure 4.

HDW suppressed HCC tumor formation in vivo. (A) Scheme of xenograft experiments. Nude mice were co-transplanted with Huh7 cells and LX-2 cells into spleens and randomized into two groups (n=5/group). At 7 days after implantation, mice were intraperitoneally administrated twice a week with vehicle or HDW (20% in PBS) for 3 weeks; dosing occurred from day 14 to day 35. At day 36, the mice were sacrificed and their livers were harvested. (B) Gross pathology showed that HDW effectively suppressed tumor formation (arrowheads: tumor lesions). (C) The maximum size of lesions of liver tumors was decreased in the HDW treatment group. *, P<0.05. (D) H&E staining and immunohistochemical staining showed histopathology characteristics of lesions and significant reductions in Ki67 and α-SMA expression. Quantification of protein expression from five fields. Scale bars =100 µm. **, P<0.01; ***, P<0.01. HCC, hepatocellular carcinoma; HDW, Hedyotis diffusa Willd; HE, hematoxylin and eosin; NC, negative control.

Identification of ADH4 potential clinical value in HCC

We further characterized the gene targets in HDW-TGS as potential therapeutic targets for HCC. Among the 24 genes in HDW-TGS, eight genes were independently correlated to overall survival in TCGA HCC samples, including ADH1B, ADH4, ATP1A1, APOE, CYP2E1, ESR1, GJA5 and IGF1 genes (Figure S2A). ROC curves showed that the expression level of all eight genes accurately discriminated between HCC tumor and normal tissues (all with AUC >0.7): ADH1B (AUC =0.848), ADH4 (AUC =0.895), ATP1A1 (AUC =0.708), APOE (AUC =0.743), CYP2E1 (AUC =0.773), ESR1 (AUC =0.916), GJA5 (AUC =0.876), and IGF1 (AUC =0.731) (Figure 5A, Figure S2B,S2C). Among these genes, ADH4 gene expression significantly correlated with tumor grade (P=0.003), cancer stage (P=0.002), overall survival (P=0.005), progression-free interval (P=0.03) and disease-specific survival (P<0.001) of TCGA HCC samples, suggesting its correlation with HCC progression (Figures 5B and Figure S3). Furthermore, we found that ADH4 was significantly correlated to cancer stage (Stage 1 vs. Stage 4, P=2.98E−06; Stage 2 vs. Stage 4, P=9.23E−04; Stage 3 vs. Stage 4, P=9.94E−03), tumor grade (Grade 1 vs. Grade 2, P=5.60E−06; Grade 1 vs. Grade 3, P=2.07E−04; Grade 2 vs. Grade 3, P=2.50E−03), and TP53 mutation (normal vs. TP53 mutant, P=1.62E−12; normal vs. TP53 non-mutant, P=1.62E−04; TP53 mutant vs. TP53 non-mutant, P=1.88E−03) of HCC samples (Figure 5C-5E). Taken together, the results suggested the clinical value of ADH4 as a marker in HCC.

Figure 5.

Identification of the potential clinical value of ADH4 in HCC. (A) AUC analysis of HDW-TGS between normal and pancreatic tumor tissues. (B) Relative expression level of the eight genes in HDW-TGS in stage I, stage II, and stage III–IV HCC. (C-E) Correlation of ADH4 and cancer stage, tumor grade, and TP53 mutation status in normal and HCC tumor tissues (TCGA and GTEx databases). AUC, area under the curve; FPR, false positive rate; GTEx, Genotype-Tissue Expression; HCC, hepatocellular carcinoma; HDW, Hedyotis diffusa Willd; HDW-TGS, HDW-target gene set; TCGA, The Cancer Genome Atlas; TPR, true positive rate.

Expression of ADH4 associated to ECM-related pathways in HCC

Using the Human Protein Atlas database, we detected varying expressions of ADH4 in normal liver and HCC tissue samples, including negative, weak, and medium expression (Figure 6A,6B). The expression of ADH4 is notably specific to the liver (Figure S4A). ADH4 expression exhibits heterogeneity in HCC tissues. While the expression of ADH4 is significantly higher in liver cancer tissue than in normal liver tissue, we also found overexpression of ADH4 in some normal liver tissue samples (Figure S4B). To investigate the functional role of ADH4 in HCC cells, we performed Gene Set Enrichment Analyses (GSEA). The results indicated that the expression of ADH4 is associated with the enrichment of CAF and ECM-related signaling pathways (Figure 6C). HDW inhibited ADH4 mRNA expression in Huh7 cells (Figure 6D). We performed downregulation of ADH4 in Huh7 cells using short interfering RNA (siRNA) (Figure 6E). High-throughput transcriptome sequencing (RNA-seq) was used to examine gene expression pattern in cells treated with HDW and downregulated for ADH4 expression. We found that either downregulation of ADH4 or treatment of HDW induced alteration of ECM-related signaling pathways, such as “extracellular matrix structural constituent”, “collagen trimer and collagen containing”, and “extracellular matrix” (Figure 6F,6G).

Figure 6.

Expression of ADH4 correlated to extracellular matrix pathways in HCC. (A,B) Expression of ADH4 in normal liver and HCC tissues. Scale bar =100 µm. Original images, staining method and antibody are available at: https://www.proteinatlas.org/ENSG00000198099-ADH4. (C) GSEA enrichment analysis of ADH4-correlated genes. (D) HDW inhibited ADH4 mRNA expression in Huh7 cells. (E) siRNA-mediated downregulation of ADH4 in Huh7 cells. (F,G) Downregulation of ADH4 or HDW treatment induced alterations of ECM-related signaling pathways including extracellular matrix structural constituent (NES =−1.694, P=0.02, FDR =1.019), collagen trimer (NES =−1.780, P=0.03, FDR =0.030) and collagen-containing extracellular matrix (NES =−1.436, P=0.14, FDR =0.141). ECM, extracellular matrix; FDR, false discovery rate; GSEA, Gene Set Enrichment Analyses; HCC, hepatocellular carcinoma; HDW, Hedyotis diffusa Willd; NES, normalized enrichment score.

Molecular docking of HDW-compounds to ADH4

The binding of ADH4 in HDW-compounds stigmasterol, oleanolic acid, ursolic acid and oleanolic acid-28-O-beta-D-glucopyranoside were examined through molecular docking (35,36). All binding energies were below -7 kcal/moL, indicating stigmasterol, oleanolic acid, ursolic acid and oleanolic acid-28-O-beta-D-glucopyranoside all had strong binding with ADH4 (Figure 7 and Table S4).

Figure 7.

Molecular docking. Model diagram between ADH4 protein and compounds of HDW. HDW, Hedyotis diffusa Willd.

Discussion

In this study, we comprehensively examined the mechanism of HDW in the treatment of HCC in vitro and in vivo. We established the HDW-TGS and uncovered evidence for HDW’s potential mechanism in tumor-stromal interaction of HCC. Among genes in the HDW-TGS, we identified ADH4 with potential high clinical value and proved its involvement in HCC tumor-stromal interactions. In recent years, ADH4 has been incorporated into numerous liver cancer prediction models (37,38). Our RNA sequencing data indicated that the inhibition of ADH4 significantly downregulated ECM-related signaling pathways in HCC cells. These findings align with previous research demonstrating that ADH4 is implicated in liver fibrosis and the regulation of retinoic acid during HCC progression (39). Furthermore, studies have reported a substantial infiltration of B cells in HCC patients exhibiting low ADH4 expression, suggesting its role in modulating distinct immune microenvironments (40). These insights offer a novel perspective on the role of ADH4 within the TME of HCC. Nonetheless, further investigation is warranted to elucidate the functional mechanisms of ADH4 in HDW-induced ROS, ERK signaling, and HSC activation.

HCC primarily arises in the liver with fibrosis, which plays a key role in the tumor occurrence, tumor development, and the TME (41). As the main element of HCC-related fibroblasts, activated HSCs produce ECM proteins and are vital in the development of liver fibrosis (42). In the HCC TME, stimulation from HCC cells promotes HSC changes from a quiescent state to activated CAFs. CAFs are considered to originate from activated HSCs. As the primary cell type in the production and remodeling of ECM, activated CAFs secrete cytokines and chemokines and produce ECM proteins to promote the progression of the HCC TME (43). Our previous study indicated that interrupting the activation of HSCs to suppress tumor-stromal interactions may be an effective therapeutic strategy for HCC (25). Unlike previous studies that focused on HDW’s anti-tumor effects on HCC cells, the present study focused on the effect of HDW on HSCs in tumor-stromal interactions. We demonstrated that HDW significantly inhibited activated HSC proliferation, migration, and invasion. HDW-TGS significantly correlated to prognosis in TCGA HCC samples. Recent research suggested that activated HSCs contribute to HCC resistance against chemotherapy agents, including 5-fluorouracil, regorafenib, cisplatin, and sorafenib (44,45). In other studies, HDW was reported to enhance the effectiveness of sorafenib or minimize the side effects in cancer patients receiving chemotherapy (23,24). Given the above findings, the combination therapeutic effect of HDW and classic chemotherapy drugs for HCC has great research value and is highly anticipated.

The integration of modern scientific methodologies with CHM has opened new frontiers in research, offering transformative potential for both theory and practice. Emerging approaches such as multi-omics technologies (genomics, proteomics, metabolomics) enable systematic analysis of the holistic mechanisms of CHM, bridging the gap between empirical knowledge and evidence-based science (46,47). Network pharmacology further deciphers the “multi-component, multi-target” synergy of herbal formulas, aligning with CHM’s systemic view of health (48,49). Recent studies have unveiled the therapeutic potential of plant-derived extracellular nanovesicles—a groundbreaking frontier in herbal medicine research. These naturally occurring, bilayer nanoparticles (30–500 nm) from medicinal herbs carry bioactive lipids, proteins, and nucleic acids, mirroring mammalian exosomes in intercellular communication (50,51). In summary, the research in the field of CHM is gradually becoming more comprehensive and advanced. However, the effector components in these nanovesicles remain to be discovered and the molecular mechanisms remain largely unknown. Further in-depth studies are required to understand the regulatory mechanisms of HDW and its active ingredients in HCC progression and therapy.

Conclusions

Our findings elucidated the functional effects and underlying mechanisms of HDW in the treatment of HCC, specifically highlighting its role in inhibiting tumor-stromal interactions. Furthermore, our study demonstrated the potential of ADH4 as a therapeutic biomarker.

Supplementary

The article’s supplementary files as

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The animal study protocol was approved by the Animal Welfare and of Peking University Shenzhen Hospital (Shenzhen, China, No. 2023-140, June 21, 2023), in compliance with the animal use regulations of the U.S. National Institutes of Health (NIH) for the care and use of animals.

Data Sharing Statement

Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-753/dss

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DOI: 10.21037/jgo-2025-753