Yiqi Wenyang Jiedu prescription promotes disulfidptosis of gastric cancer by reversing senescent cancer-associated fibroblasts

Abstract

BACKGROUND

Disulfidptosis is a specific form of cell death that occurs in cells with high SLC7A11 expression when glucose supply is insufficient. HAase, a senescence-associated phenotype secreted by senescent cancer-associated fibroblasts (senCAFs), can upregulate GLUT1 in tumor cells, thereby preventing disulfidptosis. Yiqi Wenyang Jiedu prescription (YWJP) is a Chinese herbal formula, and previous studies have shown that it can improve the clinical efficacy in the treatment of gastric cancer.

AIM

To investigate whether YWJP promotes gastric cancer cell disulfidptosis via HAase/GLUT1 pathway by reversing cancer-associated fibroblasts (CAFs) senescence and reducing HAase.

METHODS

CAFs were established using transforming growth factor-β1, and senCAFs were induced by H₂O₂. Senescence proportion was detected via β-galactosidase staining. ELISA and quantitative PCR measured interleukin-6 (IL-6), interleukin-8 (IL-8), hyaluronic acid, HAase, their mRNAs, NADP⁺/NADPH ratio, and disulfide bond levels. Western blot detected GLUT1 and disulfidptosis-related proteins. Propidium iodide (PI) staining quantified dead cells.

RESULTS

Based on previous research methods, we successfully established the CAFs and senCAFs models. The results showed that YWJP effectively reduced the proportion of senCAFs and simultaneously decreased the secretion of IL-6, IL-8, and HAase. Under HAase culture conditions, YWJP significantly downregulated the expression of GLUT1 and disulfidptosis-related proteins (CYFIP1, Rac1, ARP2, and ARP3), increased the disulfide bond level and the proportion of PI-positive cells in MKN45 cells, and thereby promoted disulfidptosis in MKN45 cells.

CONCLUSION

YWJP promotes disulfidptosis in MKN45 cells by reversing the proportion of senCAFs and reducing HAase secretion.

Key Words: Gastric cancer; Cancer-associated fibroblasts; Yiqi Wenyang Jiedu prescription; Disulfidptosis; Traditional Chinese medicine

Core Tip: This study demonstrates that Yiqi Wenyang Jiedu prescription (YWJP) promotes disulfidptosis in gastric cancer MKN45 cells. The mechanism involves YWJP reversing the senescence of cancer-associated fibroblasts (established by transforming growth factor-β1/H₂O₂), thereby reducing the secretion of HAase. The decrease in HAase leads to the downregulation of GLUT1 and key disulfidptosis-related proteins (CYFIP1, Rac1, ARP2, ARP3). This process increases intracellular disulfide bond levels and cell death, confirming that YWJP induces disulfidptosis via the HAase/GLUT1 pathway.

INTRODUCTION

Gastric cancer ranks as the fifth most common malignant tumor worldwide and the third leading cause of cancer-related deaths[1]. Studies have shown that over 60% of gastric cancer patients experience recurrence and metastasis within two years after surgery[2]. Metastasis is the primary cause of death in patients with advanced gastric cancer, the five-year survival rate for patients with advanced gastric cancer who have developed distant metastases is only 10%[3].

Chemotherapy remains the cornerstone of gastric cancer treatment[4]. Platinum-containing dual-drug regimens are currently the first-line chemotherapy options. However, research has indicated that chemotherapy can induce senescence in the tumor microenvironment[5]. Cancer-associated fibroblasts (CAFs) are one of the major cell types in the tumor microenvironment, chemotherapy can also trigger senescence in CAFs. Senescent CAFs (senCAFs) secrete senescence-associated secretory phenotypes (SASP), which remodel the tumor microenvironment[6]. Currently known SASPs include interleukin-6 (IL-6), interleukin-8 (IL-8), transforming growth factor-β (TGF-β)[7], etc. Nevertheless, in our previous study, by establishing an in vitro senCAFs model, we found that senCAFs not only highly express SASPs such as IL-6 and IL-8 but also strongly express HAase (Supplementary material). HAase is a key component of SASP involved in matrix remodeling, and its main function is to degrade hyaluronic acid (HA) in the extracellular matrix[8]. A study demonstrated that HAase can decompose HA, induce the enrichment of GLUT1 on the cytoplasmic membrane of cancer cells, increase glucose uptake, upregulate glycolysis, and thereby accelerate cancer cell migration[9]. Therefore, the secretion of HAase by senescent CAFs, which promotes the expression of GLUT1 and thus upregulates glycolysis in gastric cancer cells, may be a crucial factor contributing to the high recurrence and metastasis rates of gastric cancer.

Disulfidptosis is a novel form of cell death, which mainly occurs in cancer cells with high SLC7A11 expression (e.g., gastric cancer, liver cancer). Under glucose starvation, the supply of NADPH is insufficient to support the process of reducing cystine to cysteine in cells, leading to disulfide stress, subsequent formation of abnormal disulfide bonds, and ultimately disulfidptosis in tumor cells[10-12]. GLUT1 is a key protein that regulates glucose homeostasis between the intracellular and extracellular environments. High expression of GLUT1 enables cancer cells to evade disulfidptosis; therefore, GLUT1 inhibitors are considered effective agents to promote disulfidptosis in tumor cells, and the development of GLUT1-targeted anti-tumor drugs represents a feasible direction for future research[13].

Yiqi Wenyang Jiedu Prescription (YWJP) is a Chinese herbal formula for gastric cancer treatment that has obtained a Chinese National Invention Patent. Previous explorations have confirmed its favorable clinical efficacy, and it has now entered the phase of double-blind, randomized controlled clinical trials[14,15]. Through drug mass spectrometry analysis and network pharmacology analysis, it was found that YWJP exerts regulatory effects on senescence-related targets (e.g., cell cycle) and matrix-related targets (Supplementary material). Pharmacological studies have shown that the active components of YWJP, such as quercetin, flavonoids, resveratrol, and curcumin, can reverse cellular senescence while inhibiting HAase activity and GLUT1 expression[16,17]. Therefore, the anti-gastric cancer effect of YWJP may be associated with the upregulation of disulfidptosis, which is consistent with the mechanism underlying disulfidptosis.

Based on the above, we hypothesize that YWJP can promote disulfidptosis in gastric cancer cells through the HAase/GLUT1 pathway by interfering with the senescence of CAFs and inhibiting the secretion of HAase.

MATERIALS AND METHODS

Cell culture

Human embryonic lung cells (MRC-5 cells, Cat. No. CL-0161) and human gastric cancer cells (MKN45 cells, Cat. No. CL-0292) were purchased from Procell (Wuhan, China). These cells were cultured in DMEM supplemented with 100 mL/L fetal bovine serum (FBS) and maintained in an incubator with 50 mL/L CO₂ at 37 °C under saturated humidity.

CAFs and senCAFs were established in our laboratory; Control group (MRC-5): Cells were cultured in DMEM with 100 mL/L FBS for 48 hours; CAFs group: Based on the control group, cells were treated with 10 ng/mL (TGF-β1, Pepro Tech, NJ, United States) in DMEM containing 100 mL/L FBS for 24 hours; senCAFs group: The induction process was initiated similarly to CAFs. After culturing with 10 ng/mL TGF-β1 in DMEM with 100 mL/L FBS for 24 hours, the original medium was discarded. Cells were washed twice with 1 × phosphate-buffered saline (PBS), then cultured in DMEM with 100 mL/L FBS supplemented with 60 μM H₂O₂ for 24 hours to obtain senescent CAFs (Supplementary material).

Collection of SASP

The culture medium of CAFs and senescent CAFs was discarded. After washing the cells twice with PBS, DMEM basal medium was added for 24 hours of culture. The medium from both groups was collected and filtered to obtain SASP.

Preparation of drug-containing serum for YWJP

Sprague-Dawley rats were acclimated for 3 days under specific pathogen-free conditions: Room temperature 20-26 °C, relative humidity 40%-70%, and a 12 hours light/12 hours dark cycle, with free access to food and water. On the 4^th day, rats were weighed and randomly divided into two groups based on body weight; Blank serum (BS) group: Rats were intragastrically administered 2 mL of pure water twice daily for 3 consecutive days; YWJP-containing serum group: Rats were intragastrically administered 2 mL of YWJP twice daily for 3 consecutive days.

Dose calculation

The clinical adult dose of YWJP is 108 g/d. The body weight conversion coefficient between rats and a 60 kg adult is 6.3. For a rat with an average body weight of 200 g, the equivalent dose was calculated as follows: 108 g/60 kg × 0.2 kg × 6.3 × 2 = 4.536 g/rat/d. After 6 administrations, rats were anesthetized by intraperitoneal injection of pentobarbital (40 mg/kg), and blood was collected from the abdominal aorta. Blood samples were allowed to stand at room temperature for 3 hours, then centrifuged at 3000 g for 15 minutes to extract the upper serum layer. The serum was filtered, inactivated in a water bath at 56 °C for 30 minutes, aliquoted, and stored at -80 °C.

Immunofluorescence staining

Cells for immunofluorescence staining were fixed with 40 mL/L tissue/cell fixative (Servicebio, Wuhan, China) at room temperature for 15 min, then permeabilized with 5 mL/L Triton X-100. After blocking in PBS containing 150 mL/L goat serum (Zsbio, Beijing, China) for 1 hour, primary antibodies against fibroblast activation protein (FAP) and α-smooth muscle actin (α-SMA) were diluted to 1:100 with blocking solution, and cells were incubated overnight at 4 °C. Following a series of washes, cells were exposed to fluorescently labeled secondary antibodies (1:500) and incubated for 1 hour in the dark, then washed again. Cells were mounted with DAPI-containing mounting medium and examined under a Zeiss Instruments confocal microscope.

Senescence assay

Senescent CAFs were fixed with 40 mL/L paraformaldehyde at room temperature for 5 min and washed 3 times with PBS to remove the fixative. A Cellular Senescence β-Galactosidase Staining Kit (Beyotime, Shanghai, China) was used to prepare the staining solution according to the manufacturer’s instructions. 1 mL of staining solution was added to each well of a 6-well plate, and cells were incubated at 37 °C in the dark for 24 hours. After washing twice with PBS to remove residual dye, the proportion of senescent cells was observed under an optical microscope with 10 × to 20 × objectives.

ELISA

Cell culture medium from each group was centrifuged at 1000 g at 4 °C for 20 minutes to remove impurities and cell debris, and the supernatant was collected. The following kits were used: Human IL-6 ELISA kit, Human IL-8 ELISA kit, Human HAase ELISA kit, and NADP⁺/NADPH assay kit (WST-8 method) (Elabscience, Wuhan, China); Human HA ELISA Kit (Boyan Biology, Nanjing, China); protein disulfide bond content assay kit (Servicebio, Wuhan, China). Levels of IL-6, IL-8, HA, HAase, and NADP⁺/NADPH in SASP, as well as protein disulfide bond content in the supernatant of each group, were measured according to the manufacturers’ instructions.

Wound healing assay

Human gastric cancer cells (MKN45 cell line) were seeded into culture plates and cultured until reaching 90% confluence. Linear wounds were created using a sterile pipette tip. Cells were then washed to remove debris and cultured in SASP. Changes in wound width were observed under an inverted optical microscope, and images were recorded at 0 hour, 24 hours, and 48 hours after wound creation.

Colony formation assay

MKN45 cells were adjusted to a concentration of 100 cells/mL, and 2 mL of the cell suspension was added to each well of a 6-well plate. Plates were incubated in a 37 °C incubator with 50 mL/L CO₂. When visible cell colonies formed, the medium was discarded. After washing, cells were fixed with 40 mL/L paraformaldehyde at room temperature for 15 minutes, then stained with 1 mL/L crystal violet. Colonies were photographed under an inverted microscope, and colony area or number was analyzed using software such as Image J.

Western blot analysis

Total protein was extracted from cells using RIPA lysis buffer containing protease and phosphatase inhibitors. Protein concentration was determined using a BCA Protein Quantification Kit (Applygen, Beijing, China). Equal amounts of protein were separated by Tris-Glycine electrophoresis with 200 mL/L methanol, then transferred to a PVDF membrane (Immobilon). The membrane was blocked with protein-free rapid blocking solution at room temperature for 1 hour, then incubated with primary antibodies overnight at 4 °C. After washing, the membrane was incubated with HRP-conjugated goat anti-rabbit secondary antibody at room temperature for 1 hour. Detection was performed using an enhanced chemiluminescence kit, and protein bands were visualized with a molecular imager. The gray values of target proteins and internal reference proteins were quantified using software such as ImageJ to calculate relative expression levels.

Quantitative PCR

Total RNA was extracted using TRIzol reagent (Qiagen, Hilden, Germany). RNA was reverse-transcribed into cDNA using a reverse transcription kit (Qiagen), and cDNA was amplified using SYBR Green PCR Master Mix (Thermo Fisher Scientific, MA, United States). PCR amplification was performed on a Step One Plus Real-Time PCR System (Applied Biosystems, United States). Gene expression levels were normalized using GAPDH as the internal reference gene.

Statistical analysis

Experimental data were analyzed using SPSS 27.0. All data are presented as mean ± SD. Independent samples t-test was used for statistical analysis of data from two groups. One-way analysis of variance (ANOVA) and multiple comparison tests were used for comparisons among multiple groups. Bar graphs were generated using GraphPad Prism 8.0.2. The P < 0.05 was considered statistically significant.

RESULTS

Establishment of the CAFs model

MRC-5 cells grew in an adherent manner and exhibited a spindle-shaped, fibroblast-like morphology. The CAFs model was established by culturing MRC-5 cells in complete medium containing 10 ng/mL TGF-β1 for 24 hours. Results showed that the growth rate of CAFs was faster than that of MRC-5 cells; however, no significant morphological difference was observed between the two groups (Figure 1A). Immunofluorescence analysis revealed that the expression levels of FAP and α-SMA in CAFs were significantly higher than those in MRC-5 cells. This indicates that TGF-β1 induction can cause MRC-5 cells to express CAFs-specific markers, which is consistent with the characteristics of CAFs (Figure 1B and C).

Figure 1 Culture of MRC-5 cells and establishment of the cancer-associated fibroblasts model. A: Morphological characteristics of MRC-5 cells and cancer-associated fibroblasts (CAFs); B: Immunofluorescence detection of fibroblast activation protein in MRC-5 cells and CAFs; C: Immunofluorescence detection of α-smooth muscle actin in MRC-5 cells and CAFs. ^bP < 0.01. Error bars represent the standard error of the data. CAF: Cancer-associated fibroblasts; FAP: Fibroblast activation protein; α-SMA: α-smooth muscle actin.

Effect of YWJP on the intervention of senCAFs

CAFs were treated with 60 μM H₂O₂ to establish a chemotherapy-induced senCAFs model. β-galactosidase staining showed that compared with the CAFs group, more cells in the CAFs group treated with H₂O₂ for 24 hours entered a senescent state, with approximately 60% of cells becoming senescent (cells stained blue-green in the figure are senescent cells), which is consistent with the characteristics of senescent CAFs (Figure 2A).

Figure 2 Establishment of the senescent cancer-associated fibroblasts model and intervention effect of Yiqi Wenyang Jiedu prescription. A: Proportion of β-galactosidase staining-positive cells in the cancer-associated fibroblasts (CAFs) model and senescent CAFs (senCAFs) model under the same scale bar; B: Reversal effect on senCAFs by Yiqi Wenyang Jiedu prescription (YWJP)-containing serum at different concentrations vs blank serum (BS); C: Secretion levels of interleukin-6 (IL-6), interleukin-8 (IL-8), hyaluronic acid (HA), and HAase in senCAFs treated with YWJP at different concentrations vs BS, detected by ELISA; D: MRNA expression levels of IL-6, IL-8, HA, and HAase in senCAFs treated with YWJP at different concentrations vs BS, detected by quantitative PCR. ^aP < 0.05, ^bP < 0.01, NS: Not significant. Error bars represent the standard error of the data. CAF: Cancer-associated fibroblasts; YWJP: Yiqi Wenyang Jiedu prescription; BS: Blank serum; senCAFs: Senescent cancer-associated fibroblasts; IL-6: Interleukin-6; IL-8: Interleukin-8; HA: Hyaluronic acid.

β-galactosidase staining revealed that YWJP-containing serum at 4 concentration gradients (50 mL/L, 100 mL/L, 150 mL/L, and 200 mL/L) all reduced the senescence ratio of CAFs to varying degrees compared with BS of the same proportion. Among them, the 150 mL/L YWJP-containing serum group and 200 mL/L YWJP-containing serum group had the lowest proportion of senescent cells, and there was no statistically significant difference in the proportion of senescent cells between the two groups (Figure 2B). Therefore, 150 mL/L YWJP-containing serum was used in subsequent experiments.

ELISA was used to detect the levels of senescence-associated phenotypes IL-6 and IL-8 in different groups. The results showed that the levels of IL-6 and IL-8 in the senCAFs group were significantly higher than those in the CAFs group; the levels of IL-6 and IL-8 in the YWJP + senCAFs group were significantly lower than those in the senCAFs group; meanwhile, compared with BS, the YWJP-containing serum group had significantly lower levels of IL-6 and IL-8 (Figure 2C). The results of quantitative PCR (qPCR) also confirmed the above conclusions (Figure 2D).

ELISA was used to detect the levels of HA and HAase in different groups. The results showed that HAase levels in the senCAFs group were significantly higher than those in the CAFs group, while HA levels were significantly lower than those in the CAFs group; the YWJP + senCAFs group had significantly lower HAase levels and higher HA content than the senCAFs group; compared with BS, the YWJP-containing serum group had significantly lower HAase levels, while the opposite was true for HA levels (Figure 2C). The results of qPCR also confirmed the above conclusions (Figure 2D). The above results support our previous finding that senCAFs secrete more HAase than CAFs, and YWJP can effectively reduce HAase secretion by senCAFs.

In summary, we confirmed that senCAFs can secrete not only more IL-6 and IL-8 but also HAase related to matrix remodeling compared with CAFs. YWJP can effectively inhibit the expression of β-galactosidase in senCAFs, and simultaneously suppress the secretion of IL-6, IL-8, and HAase by senCAFs, and this effect is not caused by the serum itself.

Mechanism study on YWJP promoting disulfidptosis in MKN45 cells by intervening in the HAase/GLUT1 pathway

Based on the conclusions in ELISA, MKN45 cells were treated with 45 pg/mL HAase. Results of the wound healing assay showed that the 48 hours migration rate and colony-forming ability of cells in the HAase group were significantly higher than those in the control group, indicating that HAase can promote the migration and proliferation of MKN45 cells (Figure 3). YWJP could effectively reduce the HAase-induced increase in migration rate and colony-forming ability of MKN45 cells, thereby inhibiting the malignant behaviors of MKN45 cells.

Figure 3 Effect of Yiqi Wenyang Jiedu prescription on malignant behaviors of cells under HAase action. A: Effect of Yiqi Wenyang Jiedu prescription (YWJP) on migration of MKN45 cells in HAase environment; B: Effect of YWJP on colony-forming ability of MKN45 cells in HAase environment. ^bP < 0.01. Error bars represent the standard error of the data. YWJP: Yiqi Wenyang Jiedu prescription; BS: Blank serum.

To further explore the mechanism by which YWJP interferes with the malignant behaviors of MKN45 cells under HAase action, combined with the results of previous mass spectrometry analysis, WB was used to detect the expression of disulfidptosis-related key protein SLC7A11 and GLUT1. The results showed that there was no significant difference in the expression level of SLC7A11 among all groups, indicating that YWJP does not act on SLC7A11 but instead exerts its effects upstream of GLUT1. Compared with the Control group, the expression of GLUT1 in the HAase group was significantly upregulated; the expression of GLUT1 in the YWJP + HAase group was significantly lower than that in the HAase group and BS + HAase group, suggesting that YWJP significantly reversed HAase-mediated GLUT1 upregulation; there was no statistically significant difference between the YWJP + HAase group and the GLUT1 Inhibitor + HAase group (Figure 4A).

Figure 4 Mechanism study on Yiqi Wenyang Jiedu prescription intervening in disulfidptosis of MKN45 gastric cancer cells. A: Effect of Yiqi Wenyang Jiedu prescription (YWJP) on the expression of SLC7A11 and GLUT1 in MKN45 cells in HAase environment; B: Effect of YWJP on the NADP⁺/NADPH ratio in MKN45 cells in HAase environment; C: Effect of YWJP on the expression of CYFIP1, Rac1, ARP2, and ARP3 in MKN45 cells in HAase environment; D: Effect of YWJP on the disulfide bond level in MKN45 cells in HAase environment; E: Effect of YWJP on PI staining of MKN45 cells. ^aP < 0.05, ^bP < 0.01, NS: Not significant. Error bars represent the standard error of the data. CAF: Cancer-associated fibroblasts; YWJP: Yiqi Wenyang Jiedu prescription; BS: Blank serum.

The NADP⁺/NADPH ratio was detected by ELISA. The results showed that compared with the HAase group and BS + HAase group, the NADP⁺/NADPH ratio in the YWJP + HAase group was higher, indicating that YWJP promoted the depletion of NADPH by downregulating the expression of GLUT1. Meanwhile, there was no statistically significant difference in the NADP⁺/NADPH ratio between the YWJP + HAase group and the GLUT1 Inhibitor + HAase group (Figure 4B).

Further studies were conducted on the expression of proteins related to the Rac1-CYFIP1-ARP2/3 axis downstream of GLUT1. Consistent with the expression pattern of GLUT1, YWJP also significantly reversed the upregulation of Rac1, CYFIP1, ARP2, and ARP3 protein expression. There was no statistically significant difference between the YWJP + HAase group and the GLUT1 Inhibitor + HAase group (Figure 4C).

We further detected the disulfide bond levels in each group using ELISA. The results showed that the disulfide bond level in the YWJP + HAase group was significantly higher than that in the HAase group and BS + HAase group; there was no statistically significant difference between the YWJP + HAase group and the GLUT1 Inhibitor + HAase group (Figure 4D). Similarly, PI staining showed that the proportion of dead cells in the YWJP + HAase group was significantly higher than that in the SASP group and BS + HAase group. There was no statistically significant difference between the YWJP + HAase group and the GLUT1 Inhibitor + HAase group (Figure 4E).

DISCUSSION

This study aimed to elucidate the novel molecular mechanism underlying the anti-gastric cancer effect of YWJP, with a specific focus on whether it influences the disulfidptosis process of MKN45 cells by regulating the functional state of in the tumor microenvironment. The results demonstrated that YWJP can effectively reverse the senescence of senCAFs, inhibit the secretion of their key effector molecule HAase, and thereby weaken the resistance of MKN45 cells to disulfidptosis through this pathway. This finding links the therapeutic effects of traditional Chinese medicine (TCM) compound prescriptions, the regulation of the tumor microenvironment, and cutting-edge cell death mechanisms, providing a new theoretical basis for gastric cancer treatment strategies.

The core role of the tumor microenvironment in tumor progression has been widely recognized[18-21]; this study further investigated the characteristics of senCAFs and their secreted SASP. Previous studies have demonstrated that CAFs can respond to TGF-β1 regulation, exhibiting a high capacity for HAase production[22,23]. Within SASP, increased TGF-β1 secretion further promotes the secretion of HAase. Currently, there are no specific studies on tumor diseases for this mechanism. This study confirmed that senCAFs not only secrete the inflammatory factors IL-6 and IL-8 but also upregulate HAase secretion. HAase can degrade HA on the cell surface, upregulate the expression of GLUT1, and promote glucose uptake by tumor cells.

Disulfidptosis, a novel form of cancer cell death discovered in 2023, mainly occurs in cancer cells with high SLC7A11 expression[24]. Tumor cells with high SLC7A11 expression have a high demand for NADPH and are strictly dependent on glucose uptake and NADPH supply[25-27]. This dependence makes them susceptible to disulfide stress when glucose uptake is insufficient, leading to the formation of abnormal intracellular disulfide bonds, cytoskeletal contraction, and ultimately disulfidptosis[28,29]. This study found that HAase derived from senCAFs can significantly enhance NADPH supply capacity by upregulating GLUT1 expression in MKN45 cells. This allows MKN45 cells to maintain sufficient NADPH levels to reduce abnormally accumulated disulfide bonds, thereby evading disulfidptosis. Mechanistically, this explains why MKN45 cells with high SLC7A11 expression exhibit higher malignancy in an HAase-rich microenvironment compared to those in regular culture medium.

The core finding of this study is the confirmation that the target of YWJP is not simply MKN45 cells; its active components also act on the senescent microenvironment and related stromal cells. YWJP mainly consists of eight core medicinal herbs: Astragalus membranaceus, Codonopsis pilosula, Curcuma zedoaria, Angelica dahurica, Nardostachys jatamansi, Paris polyphylla, Reynoutria japonica, and Actinidia chinensis root. Mass spectrometry analysis showed that its active components exerting anti-gastric cancer effects are mainly associated with cell cycle regulation, secondary metabolic processes, and extracellular matrix organization (Supplementary material). The component quercetin in YWJP reduces miR-155-5p expression and restores SIRT1 Levels, thereby inhibiting aging signals[30-32]. Quercetin suppresses reactive oxygen species (ROS) production and inflammatory cytokine secretion, directly alleviating DNA damage and inhibiting the secretion of IL-1, IL-6, IL-8 and TGF-βin SASP, thereby reducing the driving force for cellular senescence[33-35]. By Downregulating p65 expression or inhibiting its acetylation significantly attenuates NF-κB activity, reduces NF-κB-mediated inflammatory gene transcription, further diminishes SASP, and blocks the aging positive feedback loop[36-38]. Flavonoids enter the enzyme cavity via hydrophobic interactions, competitively bind to key residues at the active site within the HAase cavity, and alter the conformational environment of the HAase active center. This weakens the catalytic efficiency of HAase, leading to a decrease in its activity[39]. Resveratrol directly binds to the inner surface site of GLUT1, inhibiting GLUT1 expression and thereby reducing glucose uptake and metabolism in cancer cells[17,40]. Additionally, both resveratrol and curcumin can reduces intracellular ROS, inhibits the stability and transcriptional activity of HIF-1α, thereby diminishing HIF-1α's driving effect on the GLUT1 gene promoter[41-44]. Based on the above, the active components of YWJP can promote disulfidptosis in gastric cancer cells through the HAase/GLUT1 pathway.

The innovation of this study lies in being the first to link the mechanism of action of the traditional TCM compound YWJP in intervening gastric cancer to the cutting-edge concept of disulfidptosis, and clearly defining its effect as the regulation of senCAFs in the tumor microenvironment. However, this study still has limitations. First, as a TCM compound, YWJP has a complex composition; the specific active components and related targets involved in reversing senCAFs require further exploration. Second, the current conclusions are based on in vitro experiments; the effectiveness of YWJP in the complex physiological environment of living animals and whether it exerts synergistic effects such as immunomodulation need to be further verified using in vivo models. Additionally, the conclusions were only drawn using the MKN45 cell line; whether this mechanism is universally applicable to gastric cancer cells with other genetic backgrounds also requires further investigation.

CONCLUSION

In summary, this study reveals that YWJP promotes disulfidptosis in gastric cancer cells by reversing the proportion of senCAFs, inhibiting their HAase secretion, reducing glucose supply, inducing NADPH depletion and disulfide stress.