Total flavonoids extracted from Penthorum chinense Pursh inhibits oxidative stress and inflammation in liver cancer cells through the JAK2/STAT3 pathway

Introduction

Liver cancer, a malignant tumor (1), plagues global people with increasing incidence and mortality rate (2,3). In addition to strengthening the early cancer screening, liver cancer-related research has increasingly focused on screening chemoprophylaxis drugs (4). Chemopreventive drugs typically require long-term administration and are associated with more significant side effects, thereby posing greater challenges for clinical application (5). Drug-induced toxicity is a major obstacle in the treatment of liver cancer, underscoring the need to develop effective and safe therapeutic alternatives. Traditional Chinese medicine has a long history and is the medical treasure in China, which is widely recognized for minimal side effect and remarkable curative efficacy (6,7). Therefore, discovering potential anti-tumor drugs from natural herbs has become an effective approach to treat liver cancer (8). In recent years, an increasing number of natural products from plants have demonstrated positive effects in the chemoprevention of liver cancer (4,9).

Penthorum chinense Pursh (PCP), the dried aerial part of the plant of the genus Penthorum, has been used in the treatment of jaundice and edema (10). The studies have revealed that PCP has pharmacological effects such as hepatoprotective, antiviral, anti-inflammatory, anti-tumor, and diuretic properties, and can effectively suppress the growth and differentiation of hepatic stellate cells (10). Current research on PCP’s hepatoprotective components mostly focuses on its total extract and total flavonoids (11). Flavonoids are primary bioactive components of PCP, including quercetin, Thonningianins A, luteoloside, and prunin, etc., which may have anti-inflammatory and antioxidant effects. For example, in vitro experiments and animal models have shown that quercetin exhibits a wide range of biological activities, including anti-cancer, anti-inflammatory, anti-viral, and anti-oxidative stress effects (12). Luteoloside has also been revealed to exert anti-inflammatory and antioxidant effects in methylglyoxal-induced human dental pulp cells (13). However, the effects of total flavonoids extracted from PCP (PCPTF) on the liver cancer are still not clear.

In our previous study, we found that the pharmacological effect of PCP on liver cancer may be related to the inhibition of oxidative stress and inflammatory response in vivo. As an important signaling pathway, the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway is widely involved in collective immune regulation, inflammatory response, and apoptosis (14-16). In recent years, an increasing number of studies have emphasized the crucial role of the JAK2/STAT3 signaling cascade in the development of liver cancer. For example, bufothionine induces autophagy in hepatoma-bearing mice by inhibiting the JAK2/STAT3 pathway, thus exerting anti-cancer effects (17). In liver cancer cells, the stimulation of the JAK2/STAT3 signaling axis promotes cell proliferation and inhibits programmed cell death, thereby facilitating the growth of malignant tumors (18). Total flavonoids extracted from PCP (PCPTF) can improve liver fibrosis via activating toll-like receptor 4 (TLR4)/myeloid differentiation primary response protein 88 (MyD88)-mediated inflammatory nuclear factor-κB (NF-κB) pathway and regulating liver metabolism, so as to improve liver function and morphology (19). In addition, quercetin impacts apoptosis through regulating the expression of certain genes and represses the activation of STAT3 in liver cancer cells (20). PCP improves aflatoxin B1-induced spleen immune imbalance in broilers through the JAK2/STAT signaling pathway (21). Also, ubiquitin-specific peptidase 9X-linked (USP9X) promotes the proliferation, invasion, and metastasis of liver cancer cells by regulating the JAK2/STAT3 signaling pathway (22).

Therefore, we speculated that PCPTF may inhibit oxidative stress and inflammatory response via regulating the JAK2/STAT3 pathway, and thus mitigate liver cancer. To test this conjecture, we established a nude mouse model of subcutaneous tumor formation of liver cancer and a model of lipopolysaccharides (LPS)-induced inflammation of liver cancer cells. After PCPTF treatment, we assessed the levels of JAK2/STAT3 pathway proteins, inflammatory factors, and oxidative stress. We hope to clarify the protective mechanism of PCP against hepatocarcinoma. We present this article in accordance with the ARRIVE reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1525/rc).

Methods

Animal ethics

A total of 18 specific pathogen-free (SPF) BALB/c female nude mice (4–6 weeks old, 18 to 22 g) were purchased from Hangzhou Medical College, and fed in the SPF animal laboratory with constant humidity (40–60%) and temperature (26–28 ℃). The experiment procedures were commenced after a 1-week acclimation. All feeding and operation procedures have been approved by the Institutional Animal Care and Use Committee, Zhejiang Baiyue Biotechnology Co., Ltd. (No. ZJBYLA-IACUC-20241129). The animal studies were carried out in accordance with the regulations of the Committee for the Protection and Use of Animals.

Animal modeling and experiments

A protocol was prepared before the study without registration. Human liver cancer cells SMMC-7721 (AW-CELLS-H0339) were procured from Shanghai Anwei Biotechnology Co., Ltd. (Shanghai, China). Cells were identified by short tandem repeat. SMMC-7721 cells with good growth state were washed twice with phosphate-buffered saline (PBS; ST477, Beyotime, Shanghai, China) and digested in a cell incubator (Forma Steri-Cult, Thermo Fisher, Waltham, MA, USA) at 37 ℃ for 2 minutes with 2 mL pancreatic enzyme (C0201, Beyotime). Cells were collected and resuspended with normal saline (ST341, Beyotime). Two hundred µL (2.5×10⁴ cells/µL) cell suspension was inoculated into the subcutaneous skin of the nape of neck of each nude mouse to establish a subcutaneous tumor model of liver cancer (23). One week after the cells were inoculated, when tumors were detected, the nude mice were randomly divided into Model, PCPTF-L (low-dose), and PCPTF-H (high-dose) groups, with six mice in each group. Mice in PCPTF-L and PCPTF-H groups were orally given 100 and 500 mg/kg/day of PCPTF, respectively (19), while those in the model group were given equal volume of normal saline. The PCPTF was purchased from Chenguang Biotechnology Group Co., Ltd. (Baoji, China), with reference to reported extraction methods (19). The drug was administered continuously for 4 weeks, and the change of the tumor volume was recorded once a week. The weight of the mice was measured on the last day of administration, and blood samples from the eyeballs were taken to separate the serum. The levels of inflammatory factors, superoxide dismutase (SOD), malondialdehyde (MDA), and reactive oxygen species (ROS) were detected. Tumor tissue of mice was collected to weigh the tumor. Hematoxylin and eosin (HE) staining was used to detect the pathological characteristics of tumor tissue. The proliferation and apoptosis of tumor cells were detected, and the protein level was detected by western blot. The animals were anesthetized with pentobarbital sodium (STY667, Zzsiji, Zhengzhou, China), and then blood samples were collected from the abdominal aorta. The experimental animals were euthanized by cervical dislocation and dissected to obtain liver tissues.

Cell modeling and experimental grouping

Human hepatocellular carcinoma cells SMMC-7721 were cultured in Dulbecco’s modified Eagle medium (DMEM; PM150312A, Pricella, Wuhan, China) containing 10% fetal bovine serum (FBS; A5256701, Thermo Fisher), and cell passage was performed when the cell adhesion reached more than 80%. The 4^th generation of logarithmic growing SMMC-7721 cells were selected with the density adjusted to 2×10⁵ cells/mL, and inoculated into six-well plates with 2 mL/well. Later, the cells were divided into the control group, LPS (ST1470, Beyotime) group, LPS + PCPTF group, and LPS + PCPTF + coumermycin A1 (CA1; HY-N7452, MCE, Princeton, NJ, USA) group. The control group had no drug treatment, and other groups adopted treatment using LPS (1 mg/L) and DMEM culture medium containing 10% FBS (24). In the PCPTF group, 60 µg/mL PCPTF was added for co-culture. In the LPS + PCPTF + CA1 group, 30-minute treatment with 10 µM CA1 was performed (25). Then, cells were cultured in a 5% CO₂ incubator at 37 ℃. After 24 hours, cells and cell supernatant were collected for testing.

HE staining

Tumor tissue was fixed in 4% paraformaldehyde (P0099, Beyotime) for more than 24 hours. Different concentrations of ethanol (C06915101, Nanjing Reagent, Nanjing, China) and xylene (C04305302, Nanjing Reagent) were used for dehydration. Paraffin embedding was performed after dehydration. The tissues were cut into 5 µm thick slices and stained separately with HE reagent (C0105S, Beyotime), and the histopathological changes were observed under a 400× microscope (THUNDER Imager Tissue, Leica, Wetzlar, Germany).

Immunohistochemistry

The tumor tissue was soaked in 4% paraformaldehyde for fixation and embedded with paraffin. The embedded tissue was cut into slices about 5 µm thick, dewaxed with xylene, and dehydrated with gradient ethanol. Then, the tissue was incubated overnight with primary antibody marker of proliferation Ki-67 (Ki67; ab15580, Abcam, Cambridge, UK) at 4 ℃. The next day, culture with goat anti-rabbit immunoglobulin G (IgG) heavy & light (H&L) (ab6702, Abcam) was conducted for 20 minutes. The samples were then stained with 3,3'-diaminobenzidine (DAB; D12384, Sigma-Aldrich, Saint Louis, MO, USA) and re-stained with hematoxylin (C0105S, Beyotime). After dehydration and drying, the sections were fixed with neutral gum (G8590, Solarbio, Beijing, China) and photographed under a 400× microscope.

Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining

Tumor tissue sections were dewaxed, dehydrated with gradient ethanol, and treated with protease K solution (ST532, Beyotime). TUNEL apoptosis detection kit (C1086, Beyotime) was used for TUNEL staining, and cell nuclei were dyed with 4',6-diamidino-2'-phenylindole (DAPI; C1005, Beyotime). Finally, the apoptosis of the cells was observed under a 400× fluorescence microscope (Ni-U, Nikon Corporation, Tokyo, Japan).

Enzyme-linked immunosorbent assay (ELISA)

Tumor necrosis factor-α (TNF-α; BMS607-3TEN, Thermo Fisher), interleukin-1β (IL-1β; BMS6002-2, Thermo Fisher), and interleukin-6 (IL-6; BMS603-2, Thermo Fisher) are inflammatory cytokines that were detected in tissue or cells using ELISA kits. As directed by the kit’s instructions, the concentration of each cytokine was detected using the given formula. To be specific, 100 µL of samples or standard solutions of varying concentrations were dispensed into the corresponding wells, followed by the addition of 100 µL of biotinylated antibody to each well. The plate was sealed with an adhesive film and incubated at room temperature for 1 hour. After incubation, the plate was washed five times with wash buffer. To ensure complete removal of residual liquid, the plate was thoroughly blotted by inverting it onto a stack of absorbent papers. Subsequently, 100 µL of horseradish peroxidase-conjugated Streptavidin was added to every well. The plate was resealed and incubated at room temperature in the dark for 20 minutes. Following a final wash cycle, 100 µL of TMB Substrate Solution was added to each well, and the plate was incubated at room temperature in the dark for 30 minutes to allow for color development. Finally, the reaction was ceased by the terminating solution. Absorbance was measured at 450 nm using a microplate reader (Varioskan LUX, Thermo Fisher).

Detection of SOD, MDA, and ROS levels

Serum samples and cell samples were collected. According to instructions of SOD kit (BC0175, Solarbio), MDA kit (S0131S, Beyotime), and oxiSelect in vitro ROS/reactive nitrogen species (RNS) assay kit (STA-347, Cell Biolabs, San Diego, CA, USA), the SOD, MDA, and ROS levels were measured. The detection of SOD was conducted in serum directly and in the cells after treatment with the extraction solution. The MDA level in serum and in cells after lysis with cell lysate (P0013, Beyotime) was measured. The ROS/RNS level in cell and serum samples was determined using fluorescent probe DCFH-DiOxyQ. The absorbance of SOD (560 nm), MDA (530 nm), and ROS/RNS (530 nm) was detected by a microplate reader.

Western blot

Following the extraction of total proteins from tissue or cells, radioimmunoprecipitation assay buffer (RI-PA) lysate (R0010, Solarbio) was added, and the protein concentration was detected using the bicinchoninic acid (BCA) (BCA1, Sigma-Aldrich) assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (P0012A, Beyotime) was used to separate the proteins. After wet transfer of the protein to a polyvinylidene fluoride (PVDF) membrane (YA1700, Solarbio), the membrane was sealed with 5% skim milk at room temperature for 1.5 hours and cultivated (overnight, 4 ℃) with primary antibodies β-actin (#4967, 45 kDa, 1:1,000, CST, Danvers, MA, USA), phospho- (p-)JAK2 (#4406, 125 kDa, 1:1,000, CST), JAK2 (#3230, 125 kDa, 1:1,000, CST), p-STAT3 (#52075, 86 kDa, 1:1,000, CST), STAT3 (#12640, CST, 79 kDa, 1:1,000, CST), cyclin D1 (#55506, 36 kDa, CST), vascular endothelial growth factor A (VEGFA; ab46154, 44 kDa, Abcam), Bcl-2 (#3498, 26 kDa, CST), and Bax (ab32503, 21 kDa, Abcam). Then, incubation with the corresponding secondary antibody (ab6721, 1:2,000, Abcam) was conducted for 2 hours. The gel imaging apparatus was used to visualize the bands, and ImageJ software was used to evaluate the gray values.

Colony formation experiment

To gather cell precipitate, the cells at the logarithmic growth phase were digested and centrifuged. After precipitation and counting, the cells were diluted to 1×10³ cells/mL. Cells at a gradient density of 200 cells/well were seeded in six-well plates and cultivated in incubators. The following day, pharmacological treatment was carried out in accordance with the experimental group following cell adhesion. After 2–3 weeks of incubation, the reaction was terminated upon observation of visible clones in the petri dish. The culture media were removed, and the cells were carefully washed with PBS two or three times, fixed with 1 mL of methanol (C06901102, Nanjing Reagent) for 15 minutes, and stained with 1 mL of Giemsa dyeing solution (C0133, Beyotime) for 10–30 minutes. The dyeing solution was removed with running water. In the end, clones were imaged using a camera (Nikon D90; Nikon Corporation) and counted.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 8.0. Measurement data were expressed as mean ± standard deviation. Two-way analysis of variance (ANOVA) or one-way ANOVA was applied for comparison among multiple groups, and followed by Dunnett’s post-hoc test or Tukey’s post-hoc test. P<0.05 was considered to be statistically significant.

Results

Effect of PCPTF on subcutaneous tumor formation of liver cancer in mice

In order to investigate the effect of PCPTF on liver cancer, a subcutaneous tumor formation model of liver cancer was established, followed by treatment with different doses of PCPTF. It was observed that high doses of PCPTF significantly increased the weight of mice compared to the model mice (Figure 1A, P<0.001). We observed a progressive increase in tumor volume over time after cancer cell transplantation, and both high and low doses of PCPTF could reduce the growth rate of the tumor (Figure 1B, P<0.001). Also, PCPTF treatment also decreased tumor weight (Figure 1C, P<0.001). By observing the pathological changes of tumor tissue, we found that mice in the model group had obvious lesions, closely arranged tumor cells, enlarged nuclei, and deep staining (Figure 1D). After PCPTF treatment, the pathological changes were improved visibly, with loose tumor cell arrangement and nuclear contraction or rupture, and high-dose PCPTF exerted more pronounced effects (Figure 1D). We also examined the proliferation and apoptosis of tumor cells. The results showed that PCPTF significantly inhibited proliferation and promoted apoptosis of cancer cells (Figure 1E-1H, P<0.05).

Figure 1 PCPTF improved the function and pathological changes of liver cancer mice. (A) Measurement of body weight of mice with liver cancer. (B) Tumor volume change. (C) Measurement of tumor weight. (D) HE staining was used to detect the pathological characteristics of tumor tissue in mice (400×). Scale bar =50 µm. (E,F) Tumor cell proliferation was detected by immunohistochemistry (400×). Scale bar =50 µm. (G,H) TUNEL was applied to detect apoptosis of tumor cells (400×). Scale bar =50 µm. Each group had six mice. *, P<0.05; **, P<0.01; ***, P<0.001 vs. model. H, high-dose; HE, hematoxylin and eosin; L, low-dose; PCPTF, total flavonoids extracted from Penthorum chinense Pursh; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

The relevant indices in the serum of mice were also detected. We determined that both high and low doses of PCPTF apparently down-regulated serum levels of IL-1β and TNF-α inflammatory factors (Figure 2A,2B, P<0.001), while only high doses of PCPTF down-regulated IL-6 (Figure 2C, P<0.05). In the serum of model mice, SOD levels were increased after PCPTF treatment, while MDA and ROS/RNS levels were decreased (Figure 2D-2F, P<0.05). Protein levels of p-JAK2, JAK2, p-STAT3, and STAT3 in tumor tissues were detected by western blot, and the data unveiled that PCPTF treatment evidently diminished the ratios of p-JAK2/JAK2 and p-STAT3/STAT3 (Figure 2G-2I, P<0.05). In summary, PCPTF inhibited oxidative stress and inflammation in mice with liver cancer through the JAK2/STAT3 pathway.

Figure 2 PCPTF regulated inflammatory factors, oxidative stress, and protein levels in mice. (A-C) Serum levels of inflammatory factors IL-1β, TNF-α, and IL-6 were detected by ELISA. (D-F) The contents of SOD, MDA, and ROS in serum of mice were detected. (G-I) The expressions of JAK2, STAT3, p-JAK2, and p-STAT3 proteins in tumor tissues were determined by western blot. β-actin was the internal parameter. Each group had six mice. *, P<0.05; **, P<0.01; ***, P<0.001 vs. model. ELISA, enzyme-linked immunosorbent assay; H, high-dose; IL-1β, interleukin-1β; IL-6, interleukin-6; JAK2, Janus kinase 2; L, low-dose; MDA, malondialdehyde; p-, phospho-; RNS, reactive nitrogen species; ROS, reactive oxygen species; SOD, superoxide dismutase; STAT3, signal transducer and activator of transcription 3; TNF-α, tumor necrosis factor-α.

Effects of PCPTF on oxidative stress and inflammation in LPS-stimulated human hepatocellular carcinoma cells via the JAK2/STAT3 pathway

To verify the mechanism of PCPTF improving liver cancer, we also conducted cell experiments. We established cell models by exposing human hepatocellular carcinoma cells to LPS, and then treated model cells with PCPTF and JAK2 agonist CA1. We found that LPS stimulation activated the JAK2/STAT3 pathway and increased ratios of p-JAK2/JAK2 and p-STAT3/STAT3. In LPS-treated cells, PCPTF inhibited the JAK2/STAT3 pathway and down-regulated the ratios of p-JAK2/JAK2 and p-STAT3/STAT3, but CA1 reversed the effect of PCPTF (Figure 3A-3C, P<0.05). Cell proliferation experiment results showed that PCPTF repressed the colony formation of model cells, which was offset by CA1 (Figure 4A,4B, P<0.05).

Figure 3 Western blot assay. (A-C) The expressions of p-JAK2, JAK2, p-STAT3, and STAT3 proteins in hepatocellular carcinoma cells were measured by western blot. β-actin was the internal parameter. The experiment was repeated three times. ^, P<0.05; ^^, P<0.01 vs. control. ^###, P<0.001 vs. LPS. ⁺⁺, P<0.01 vs. LPS + PCPTF. CA1, coumermycin A1; JAK2, Janus kinase 2; LPS, lipopolysaccharides; p-, phospho-; PCPTF, total flavonoids extracted from Penthorum chinense Pursh; STAT3, signal transducer and activator of transcription 3.

Figure 4 Effect of PCPTF on human hepatocellular carcinoma cells stimulated by LPS. (A,B) Colony formation was performed to examine the proliferation of liver cancer cells (stained with Giemsa dyeing solution). (C-E) The contents of inflammatory factors IL-1β, TNF-α, and IL-6 were detected by ELISA. (F-H) The contents of SOD, MDA, and ROS were measured. All experiments were repeated three times. ^^, P<0.01; ^^^, P<0.001 vs. control. ^##, P<0.01; ^###, P<0.001 vs. LPS. ⁺, P<0.05; ⁺⁺, P<0.01; ⁺⁺⁺, P<0.001 vs. LPS + PCPTF. CA1, coumermycin A1; ELISA, enzyme-linked immunosorbent assay; IL-1β, interleukin-1β; IL-6, interleukin-6; LPS, lipopolysaccharides; MDA, malondialdehyde; PCPTF, total flavonoids extracted from Penthorum chinense Pursh; RNS, reactive nitrogen species; ROS, reactive oxygen species; SOD, superoxide dismutase; TNF-α, tumor necrosis factor-α.

Levels of oxidative stress and inflammatory factors in model cells were measured. PCPTF treatment downregulated IL-1β, TNF-α, and IL-6 in LPS-induced model cells, while CAI reversed the regulatory effects of PCPTF on these inflammatory factors (Figure 4C-4E, P<0.001). The detection of oxidative stress levels showed that PCPTF diminished MDA and ROS/RNS levels and elevated SOD levels in model cells (Figure 4F-4H, P<0.01). However, CA1 stimulation attenuated the role of PCPTF in oxidative stress levels in model cells (Figure 4F-4H, P<0.01). Our cell experiments demonstrated that PCPPTF mediated oxidative stress and inflammation levels in LPS-treated cells through the JAK2/STAT3 pathway.

Effects of PCPTF on the downstream genes related to the JAK2/STAT3 pathway and apoptosis protein in LPS-stimulated human hepatocellular carcinoma cells

In addition, we detected the effect of PCPTF on the expression of cyclin D1, VEGFA, Bcl-2, and Bax. The expression of cyclin D1 and VEGFA was increased in LPS-induced model cells (Figure 5A-5C, P<0.01), while PCPTF treatment decreased the expression of cyclin D1, VEGFA, and Bcl-2, but increased the expression of Bax in LPS-induced model cells (Figure 5A-5E, P<0.001). In addition, CA1 stimulation attenuated the role of PCPTF on these protein expressions in model cells (Figure 5A-5E, P<0.001). These results indicated that PCPTF promoted apoptosis in LPS-treated cells through the JAK2/STAT3 pathway and inhibited the downstream genes related to the JAK2/STAT3 pathway.

Figure 5 Effects of PCPTF on the downstream genes related to the JAK2/STAT3 pathway and apoptosis protein in LPS-stimulated human hepatocellular carcinoma cells. (A-E) The expressions of cyclin D1, VEGFA, Bcl-2, and Bax in hepatocellular carcinoma cells were measured by western blot. β-actin was the internal parameter. The experiment was repeated three times. ^^, P<0.01; ^^^, P<0.001 vs. control. ^###, P<0.001 vs. LPS. ⁺⁺⁺, P<0.001 vs. LPS + PCPTF. CA1, coumermycin A1; JAK2, Janus kinase 2; LPS, lipopolysaccharides; PCPTF, total flavonoids extracted from Penthorum chinense Pursh; STAT3, signal transducer and activator of transcription 3; VEGFA, vascular endothelial growth factor A.

Discussion

Liver cancer, a highly malignant tumor, faces challenges in treatment due to drug resistance, metastasis, and recurrence (26). To develop therapeutic strategies, increasing emphasis has been laid on traditional Chinese medicine. PCP has many pharmacological activities, such as hepatoprotective, antiviral, lipid-lowering, anti-mutagenic, and anti-cancer effects, and has been widely used in the treatment of viral hepatitis, liver fibrosis, and fatty liver (27). In addition, Gansu granules made from PCP have a definite curative effect on acute and chronic liver diseases and various complications (28). Modern pharmacological studies have shown that Gansu granules can block the proliferation and differentiation of hepatic stellate cells, reduce the expression of profibrotic cytokines, regulate intracellular and extracellular signal transduction pathways, suppress the secretion of type 1 collagen, and prevent the formation of extracellular matrix, thereby playing an anti-fibrosis role (28). However, the literature review found that the mechanism of PCPTF in liver cancer has been less discussed. In this study, we demonstrated that PCPTF can inhibit oxidative stress and inflammatory response by regulating the JAK2/STAT3 pathway, thus hindering liver cancer progression.

Activation of the JAK2/STAT3 pathway is a common contributor to liver cancer, and overexpression of STAT3 is often detected in hepatocellular carcinoma (29). STAT3, as a driver, plays a key role in the occurrence, progression, metastasis, and immunosuppression of hepatocellular carcinoma, and is associated with poor prognosis (30,31). Phosphorylated JAK2 can activate the dimerization of STAT3 into p-STAT3 into the nucleus, and then activate the transcription and expression of corresponding genes, resulting in abnormal proliferation and malignant transformation of tumor cells (32). Activation of the JAK2/STAT3 signaling pathway promotes the development of liver cancer and inhibits cell apoptosis and autophagy, thus promoting the occurrence of breast cancer (33-35). IL-6 can activate JAK2/STAT3 signal transduction and promote metastasis of non-small cell lung cancer (36). Besides, PCP improves aflatoxin B1-induced spleen immune imbalance in broilers through the JAK/STAT signaling pathway (21). Accordingly, we stimulated hepatocellular carcinoma cells with the JAK2 activator CA1 and treated them with PCPTF. The data suggested that PCPTF dampened the proliferation of cancer cells by inhibiting JAK2/STAT3 phosphorylation.

In addition, cytokines such as IL-1β, TNF-α, and IL-6 are mainly produced in lymphocytes and mononuclear macrophages, and participate in pathological changes such as inflammatory lesions (37,38). The development and progression of liver cancer is also accompanied by a series of inflammatory reactions. Dysregulation of the IL-6-mediated JAK/STAT3 signaling pathway is closely related to the development of diverse human solid tumors (39). It has been reported that prostate apoptosis response protein-4 promotes the malignant behavior of hepatocellular carcinoma cells through the IL-6/STAT3 signaling pathway (40). In this study, we observed down-regulation of IL-1β, TNF-α, and IL-6 following PCPTF treatment, implying that the intervention may impact upstream genes, potentially at the level of cytokine receptors (e.g., IL-6R) or their associated negative regulators (e.g., SOCS proteins), to initially suppress JAK2 activation. Moreover, our results demonstrated that PCPTF significantly downregulated the expression of key downstream effectors of the JAK2/STAT3 pathway, including cyclin D1 and VEGFA, in LPS-stimulated hepatocellular carcinoma cells. Cyclin D1, as a key regulator of the cell cycle process, and its downregulation further confirms that PCPTF can inhibit the proliferation cycle of liver cancer cells by suppressing the JAK2/STAT3 signaling pathway. At the same time, the downregulation of VEGFA indicates that PCPTF may also inhibit tumor angiogenesis through this pathway, thereby limiting the tumor’s nutrient supply and metastatic potential. These findings collectively demonstrate that PCPTF not only inhibits the release of inflammatory factors but also suppresses the progression of liver cancer through the regulation of genes related to proliferation and angiogenesis in multiple dimensions.

SOD is the main enzymatic defense system against oxygen-free radicals in the body. If SOD activity is insufficient and cannot play the antioxidant role in time, cells succumb to oxygen-free radicals, causing cell damage (41). The increase of oxygen-free radicals in cells can trigger peroxidation with polyvalent unsaturated fatty acids in the phospholipids on the plasma membrane, thus producing peroxides that further lyse into cytotoxic MDA (42). MDA is a metabolic product of lipid peroxidation reaction with unsaturated fatty acids in biofilm, and changes in its content indirectly reflect the content of oxygen-free radicals in tissues and the degree of cell damage (42). High levels of ROS induce programmed cell death through an oxidative damage response (43,44). In this study, we found that PCPTF promoted SOD and decreased MDA and ROS levels. Furthermore, this study revealed that PCPTF treatment significantly reduced the expression of Bcl-2, while increasing the level of Bax, indicating that it can promote apoptosis of liver cancer cells. These findings suggest that PCPTF may exert its effect by inhibiting the transcriptional activity of STAT3, thereby reducing its regulation of downstream anti-apoptotic genes (such as Bcl-2), and thereby enhancing cell apoptosis.

Beyond a linear pathway, the broader signaling network must be considered. The JAK2/STAT3 pathway is known to engage in extensive cross-talk with other oncogenic pathways pivotal in liver cancer, such as PI3K/AKT, MAPK, and NF-κB (45,46). The therapeutic efficacy of PCPTF may stem from its coordinated modulation of this interconnected signaling network. Thus, future work should focus on delineating the precise upstream triggers, the key downstream effector genes, and the critical cross-talk mechanisms to fully elucidate the polypharmacological action of PCPTF against liver cancer.

While our findings demonstrate the anti-tumor efficacy of PCPTF in preclinical models, several limitations must be acknowledged to fully assess its clinical translational value. Key translational parameters, including the optimal therapeutic dosage, the most efficacious administration route (such as oral bioavailability), and the potential toxic side effects of PCPTF, remain undefined. The dose and intraperitoneal injection used here are appropriate for our initial mechanistic investigation, but may not be ideal for clinical translation. Furthermore, the lack of clinical data means the direct relevance to human liver cancer treatment remains to be established. Therefore, future studies are imperative to systematically investigate the pharmacokinetics, dose-response relationships, chronic toxicity, and safety profile of PCPTF. Subsequent research should also validate these findings in more clinically relevant models and, ultimately, in human trials.

Conclusions

In summary, our study explores the mechanism by which PCPTF inhibits the occurrence of liver cancer, and demonstrates in vivo and in vitro that PCPTF may suppress oxidative stress and inflammatory response of liver cancer by regulating the JAK2/STAT3 pathway. These preclinical findings not only elucidate a novel molecular mechanism but also underscore the potential of PCPTF as a promising therapeutic candidate for liver cancer. Given the central role of JAK2/STAT3 in tumorigenesis and therapy resistance, our work provides a strong rationale for further investigation of PCPTF as a potential targeted therapy. Future research will be essential to validate these effects in clinical settings, optimize its pharmacological profile, and ultimately translate these findings into a novel treatment option for patients battling against liver cancer.

Acknowledgments

We thank Zhejiang Baiyue Biotechnology Co., Ltd. for providing the animal experimental platform.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1525/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1525/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1525/prf

Funding: This work was supported by the Zhejiang Province Traditional Chinese Medicine Science and Technology Project (No. 2023ZL344) and the Hangzhou Medical Health Technology Project (No. B20241730).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1525/coif). The authors have no conflicts of interest to declare.

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. Animal experiments were performed under a project license (No. ZJBYLA-IACUC-20241129) granted by the Institutional Animal Care and Use Committee, Zhejiang Baiyue Biotechnology Co., Ltd., in compliance with the regulations of the Committee for the Protection and Use of Animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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