Hua-Zhuo-Jie-Du Decoction Combined with Cisplatin Inhibits the Development of Gastric Cancer Cells by Regulating Immune and Autophagy Signaling

Abstract

Host immunity and autophagy of cancer cells markedly impact the development of gastric cancer. Hua-Zhuo-Jie-Du decoction (TDP) has been used in gastritis clinically. This study aimed to evaluate the effects of TDP combined with cisplatin (DDP) on gastric cancer and explore the molecular mechanism. A total of 16 BALB/c nude mice were used to model the SGC7901 cells xenograft and treated with TDP and DDP or both, with the model group as the control. Hematoxylin–Eosin (H&E) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining were performed, and the expression levels of CD31 and Ki-67 were quantified by immunohistochemistry staining. Additionally, cyclooxygenase (COX)-2, matrix metalloproteinas (MMP)-2, and MMP-9 expression levels were quantified using quantitative real-time PCR (qRT-PCR) and Western blotting (WB). WB was used to determine Cleaved-caspase3, Beclin-1, LC3B, and p-p62 levels. Lastly, flow cytometry was employed to evaluate immune responses in mice. TDP and DDP significantly decreased tumor weight and nuclear division, resulting in loosely distributed cells. Besides, TDP and DDP down-regulated the protein expression levels of Ki-67, CD31, COX-2, MMP-2, and MMP-9, as well as decreased the number of CD4+ IL-17+ cells. Conversely, TDP and DDP up-regulated Cleaved-caspase3 expression and the proportion of CD3+/CD4+ and CD8+/CD3+ cells. Notably, optimal effects were achieved using the combination of DDP and TDP. Furthermore, DDP increased the LCII/LCI ratio and the Beclin-1 levels while down-regulating p62 levels. However, TDP alleviated these effects. These results collectively indicated that the combination of TDP with DDP can inhibit the development of gastric cancer cells by mediating the immune and autophagy signaling pathways.

INTRODUCTION

As is well documented, gastric cancer (GC) is the fourth most prevalent cancer globally and ranks second in terms of cancer-related mortality.¹⁾ During the early stages, gastric cancer cells may invade the gastric mucosa or submucosa.²⁾ At present, laparoscopic gastrectomy is typically performed for the treatment of early-stage GC owing to its safety profile, convenience, affordability, and shorter recovery time.^3,4) Despite these benefits, the efficacy of laparoscopic treatment remains suboptimal. After the surgical excision of the tumor, adjunctive anti-tumor drugs are generally required. Cisplatin (DDP) is commonly administered to improve 5-year (yr) and 10-yr survival rates for GC patients.³⁾ However, its efficacy is limited after prolonged use. Therefore, there is an urgent need to investigate and develop effective anti-gastric cancer drugs in combination with DDP to improve the clinical outcomes of GC patients.

Hua-Zhuo-Jie-Du decoction (TDP) is a Traditional Chinese Medicine (TCM) formula that comprises Yin Chen (Oriental wormwood, YC), Huang Qin (Scutellaria baicalensis, HQ), Huang Lian (Coptis chinensis, HL), Huo Xiang (Agastache rugosus, HX), Pei Lan (herba eupatorii, PL), Baihua Shelan Cao (Oldenlandia diffusa, BSC), Ban Bian Lian (Chinese lobelia, BBL), Ban Zhi Lian (Sculellaria barbata, BZL), Ban Lan Gen (radix isatidis, BLG), Ku Sen (radix sophorae flavescentis, KS), and Jiao Gu Lan (gynostemma pentaphylla, JGL). As early as 2011, Li et al. described that it can delay chronic atrophic gastritic precancerosis (CAGP) progression by decreasing the levels of lactic acid, nitrites, and tumor markers, including CEA, CA19-9, CA72-4, and CA125.⁵⁾ In addition, TDP is an effective treatment for precancerous lesions of gastric cancer (PLGC). Notably, TDP treatment significantly improves the integrity of the gastric mucosa, inhibits PLGC cell proliferation, and promotes apoptosis.⁶⁾ Two recent studies pointed out that it can prevent CAGP in mice by regulating the intestinal flora.^7,8) Therefore, we postulate that TDP possesses anti-gastric cancer properties.

T-cell immunity is vital for cancer treatment, with CD8+ and CD4+ T cell activation recognizing and destroying cancer cells.⁹⁾ Meanwhile, autophagy is a key factor in the cancer cell survival pathway. In chemotherapy-treated tumor cells, autophagy is activated, conferring anti-tumor drug resistance.¹⁰⁾ The combination of TDP and DDP has been reported to exert synergistic effects. In the present study, the combination of TDP and DDP exerted significant therapeutic effects, thereby contributing to the regulation of immunity and autophagy of GC cells in mice.

MATERIALS AND METHODS

Xenograft Experiments in Vivo

A total of 16 BALB/c nude male mice (4–6 weeks old) procured from Chengdu Dasuo Experimental Animal Co., Ltd. (Chengdu, China) were used. They were housed with ad libitum access to water and food and with a 12-h light/dark cycle. The gastric cancer cell line SGC7901 (Cell Bank of the Chinese Academy of Sciences, Shanghai) was cultured to the log-growth phase, and its concentration was adjusted to 4 × 10⁶ cells/mL in phosphate-buffered saline (PBS). Next, mice were subcutaneously injected with 100 µL cell suspension into the right axillary region. Initially, a tissue homogenizer was utilized to generate a single-cell suspension of the spleen, followed by the implementation of Magnetic Cell Separation technique (Dynabeads™ FlowComp™, Thermo Fisher, Waltham, MA, U.S.A.) for the isolation of Pan T cells. T cells were cultured in GT-T551 supplemented with 10% fetal bovine serum (FBS), interleukin-2 (1L-2) (200U/mL) and 100 mg/mL of PSG. T cell activation and expansion were performed using cluster of differentiation 3/cluster of differentiation 28 beads (CD3/CD28) and IL-2. The amplifyed T cells were collected, washed, and re-suspended with PBS to adjust the cell concentration to 1 × 10⁷ cells/100 µL. When the tumor reaches a certain size (100–200 mm³), T cell (1 × 10⁶ cells per mouse) suspension is drawn and injected into the tail vein. In this study, all animal experiments were conducted in accordance with the ethical standards of experimental animals (Ethics Committee of Hebei Medical University, No: IACUC-Hebmu-2021018).

Compound Preparation of Chinese Herbal Medicine Preparation and Mice Treatment

Turbid and detoxifying prescription (TDP) was prepared by decocting traditional Chinese medicinal materials, namely 15 g YC, 12 g HQ, 12 g HL, 9 g HX, 9 g PL, 15 g BSC, 15 g BBL, 15 g BZL, 15 g BLG, 10 g KS, and 15 g JGL, in 1000 mL of water for 2 h. Next, 10 mL of TDP solution was administered to mice. After 5 d, the mice were randomly assigned to four groups: Model, Model + DDP, Model + TDP, and Model + DDP + TDP. Model + DDP group was given 5 mg/kg DDP (Hefei BASF Biotechnology Co., Ltd., Hefei, China) via intraperitoneal injection for 7 d, and the Model+ TDP group was administered 20.2 mg/g TDP by gastric gavage for 3 weeks. Mice in the Model + DDP + TDP group received DDP and TDP. The administration of TDP and DDP both commenced via gavage on the 6th day post-modeling, with TDP being administered continuously for 3 weeks, and DDP for 7 d. The drug was administered after 6 d of molding. The model group served as a control and was injected or fed with an equivalent volume of PBS.

Hematoxylin–Eosin (H&E) and Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate Nick-End Labeling (TUNEL) Staining

Tumor samples were fixed in 4% formaldehyde and dehydrated in a graded ethanol/xylene series after washing. The tissues were subsequently paraffin-embedded and sectioned into 5 µm thick slices. After that, the slices were dewaxed, followed by H&E and TUNEL staining. H&E staining was performed using H&E, and TUNEL staining was performed using a TUNEL kit (Roche, Switzerland) according to the manufacturer’s protocol. Lastly, the stained sections were visualized under a light microscope.

IHC Staining Assay

IHC staining assay was performed to quantify the expression levels of CD31 and Ki-67. The tumor slices were prepared as described in 1.3 and blocked using Tris-buffered saline (TBS) containing 5% goat serum for 1 h. Thereafter, the sections were incubated with antibodies against CD31 (1 : 25, A20228, Abclonal) and Ki-67 (1 : 100, A16919, Abclonal). Finally, the sections were observed under a light microscope (40 × lens).

Quantitative Real-Time PCR (qRT-PCR)

Total RNA was extracted from tumor samples using TRIzol reagent following the manufacturer’s instructions (Invitrogen, U.S.A.). The expression levels of matrix metalloproteinas (MMP)-2, MMP-9, and cyclooxygenase (COX)-2 were quantified using a SYBR Green assay (Vazyme, China) using the 2^−ΔΔCt method, with β-actin acting as the control. The primers are listed in Table 1. qRT-PCR was conducted under the following conditions: initial denaturation at 94 °C for 5 min; 35 cycles of denaturation at 94 °C for 2 s; annealing at 59 °C for 30 s, and a melting curve stage at 95 °C for 30 s, 59 °C for 30 min, and 95 °C for 30 s.

Table 1. Primers for qRT-PCR

Gene name	Forward primer 5′–3′	Reverse primer 5′–3′
MMP-2	gca agg atg gag gca cga ttg gtc tg	ccg cat ggt ctc gat ggt gtt ctg g
MMP-9	cca cca ccg cca act atg acc agg at	gta ctg ctt gcc cag gaa agc gaa gg
COX-2	tgt atc ccg ccc tgc tgg tgg aaa	ctt gcg ttg atg gtg gct gtc ttg gt
β-Actin	acc gtg aaa aga tga ccc aga t	agc ctg gat ggc tac gta cat g

Flow Cytometry

Blood samples were collected from mice, and T lymphocyte subpopulations were identified using flow cytometry. mAbs against CD3, CD4, and IL-17, purchased from eBioscience, were used in this study. Flow cytometry was carried out using a Cyan ADP Color flow cytometer (Beckman Coulter, Brea, CA, U.S.A.), and data analysis was performed using Flow Jo 8 software (Tree Star, Woodburn, OR, U.S.A.).

Western Blotting

The expression of cleaved-caspase3, COX-2, MMP-2, MMP-9, Beclin-1, LC3B, and p-p62 was quantified via WB, with β-actin serving as the reference protein. Tumors from each group were lysed using RIPA buffer (Beyotime, China) and then centrifuged at 12000 rpm for 10 min at 4 °C, following which the supernatant was collected. Total protein concentrations were quantified using a BCA kit (Beyotime) and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes, and the membranes were blocked with 5% non-fat milk for 1 h at room temperature. Then, the membranes were incubated with primary antibodies (Beclin-1: 1 : 2000, A7353, abclonal; Cleaved-caspase3: 1 : 500, ab32040, Abcam; COX-2: 1 : 4000, A1253, abclonal; LC3B: 1 : 2000, A19665, abclonal; MMP-2: 1 : 2000 A19080, abclonal; MMP-9: 1 : 2000, A2095, abclonal; p-p62: 1 : 2000, A19250, abclonal) 4 °C overnight. Afterward, they were incubated with the secondary antibody (β-actin: 1 : 100000, AC026, abclonal) for 1 h. Protein expression levels were measured using a high-sensitivity ECL chemiluminescence detection kit (Proteintech, China) and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, U.S.A.).

Statistical Analysis

Statistical analyses were performed using GraphPad Prism 9.1.2. Data were expressed as the mean ± standard deviation (S.D.). One-way ANOVA or two-way ANOVA was used to compare differences between groups. Furthermore, the student t-test was used to compare two groups. p < 0.05 was considered statistically significant. All experiments were performed in triplicate.

RESULTS

Effects of the Combination of TDP and DDP on Tumor Growth in SGC7901 Xenograft-Bearing Nude Mice

Nude mice were transplanted with SGC7901 cells to construct a subcutaneous tumor model for gastric. Then, mice were treated with DDP, TDP, or both. As illustrated in Fig. 1A, DDP and TDP treatment both decreased tumor size. As anticipated, the combination of DDP with TDP yielded optimal results (Figs. 1A, B). As displayed in Fig. 1C, H&E staining displayed pathological nuclear divisions and an uneven cell arrangement. However, DDP and TDP treatment ameliorated nuclear divisions and resulted in a looser cell arrangement. Notably, drug-treated groups exhibited significant neutrophil infiltration in the tumors. Furthermore, the most significant reduction in nuclear division and the loosest cell arrangement were observed in the DDP + TDP group.

Fig. 1. Effects of the Combination of TDP and DDP on Tumor Growth in SGC7901 Xenograft-Bearing Nude Mice

(A) Tumor growth curve. (B) Tumor weight. (C) H&E staining. Data were expressed as the mean ± S.D. Compared with the Model group, * p < 0.05, ** p < 0.01, *** p < 0.001; Compared with the Model + DDP + TDP group, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001.

Effects of the Combination of TDP and DDP on SGC7901 Cell Apoptosis in Xenograft Tumors of Nude Mice

A tunnel assay was carried out to explore the effects of the combination of TDP and DDP on apoptosis in xenograft tumors (Fig. 2A). As depicted in Fig. 2B, DDP outperformed TDP in terms of apoptotic effect. However, the combination of DDP and TDP resulted in the most pronounced apoptotic effect (Fig. 2B). In addition, WB was performed to determine the expression of Cleaved-caspase 3 in tumors (Fig. 2C). As delineated in Fig. 2D, the protein expression of Cleaved-caspase 3 exhibited a consistent trend across groups. The combination of TDP and DDP demonstrated the highest effects on SGC7901 cell apoptosis in xenograft tumors of nude mice.

Fig. 2. Effects of the Combination of TDP and DDP on SGC7901 Cell Proliferation and Apoptosis in Xenograft Tumors of Nude Mice

(A) TUNEL staining. (B) Quantification of cell apoptosis by TUNEL assay. (C, D) WB was performed to detect the expression of Cleaved-caspase 3. (E) Ki-67 immunohistochemical staining. (F) Ki-67 expression (OD) Statistical analysis. Data are expressed as the mean ± S.D. Compared with the Model group, * p < 0.05, *** p < 0.001; compared with the Model + DDP + TDP group, ^#p < 0.05, ^###p < 0.001.

Effects of the Combination of TDP and DDP on SGC7901 Cell Proliferation in Xenograft Tumors of Nude Mice

Cell proliferation was evaluated by quantifying Ki-67 expression levels (Figs. 2E, F). As presented in Fig. 2F, DDP and TDP significantly down-regulated the expression of Ki-67. Moreover, the expression levels MMP-2, MMP-9, and COX-2 were determined by WB (Fig. 3A). MMPs are a class of enzymes capable of degrading the extracellular matrix. Thus, the level of MMPs reflects the aggressiveness and metastatic potential of tumors. COX-2 is a marker of tumorigenesis and is up-regulated in various cancers. Thus, detecting the level of COX-2 can assist in tumor diagnosis, prognostic assessment, and treatment. The results indicated that DDP and TDP down-regulated the expression of the aforementioned proteins (Fig. 3B), with their combination showing the most pronounced effects. CD31 serves as a marker for vascular endothelial cells and reflects the proliferative and migratory properties of cancer cells. Immunohistochemical staining and statistical analysis of CD31 are displayed in Figs. 3C and D. DDP (** p < 0.01), TDP (* p < 0.05), and DDP + TDP (*** p < 0.001) decreased the expression level of CD31 in xenograft tumors of nude mice.

Fig. 3. Effects of the Combination of TDP and DDP on SGC7901 Cell Proliferation and Apoptosis in Xenograft Tumors of Nude Mice

(A, B) WB analysis of MMP-2, MMP-9, and COX-2 proteins. (C) CD31 immunohistochemical staining. (D) Statistical analysis of CD31 expression. Data are expressed as the mean ± S.D. Compared with the Model group, * p < 0.05, ** p < 0.01, *** p < 0.001; compared with the Model + DDP + TDP group, ^#p < 0.05.

Effects of the Combination of TDP and DDP on the Immunity of Nude Mice

The detected T cells in this experiment originated from C57BL/6 mice. To assess the impact of SGC7901 cell injection on the immunity of nude mice, flow cytometry was performed to quantify the levels of immune cells (Fig. 4A), CD8+/CD3+ (Fig. 4B), and CD4+ IL-17+ (Fig. 4C) cells. As portrayed in Figs. 4D and E, the percentages of CD3+/CD4+ and CD8+/CD3+ cells were consistent. The combination of DDP and TDP markedly up-regulated the levels of CD3+/CD4+ (Fig. 4D) and CD8+/CD3+ (Fig. 4E). Conversely, CD4+ IL-17+ cells showed an opposite trend compared to CD3+/CD4+ and CD8+/CD3+ cells (Fig. 4F). Lastly, DDP decreased the percentage of CD3+/CD4+ cells and increased that of CD4+ IL-17+ cells, whereas TDP treatment did not significantly affect these levels.

Fig. 4. Effects of the Combination of TDP and DDP on the Immune Response of Nude Mice

The detected T cells in this experiment originated from C57BL/6 mice. (A) Flow cytometry analysis of CD3+/CD4+ cells. (B) Flow cytometry analysis of CD8+/CD3+ cells. (C) Flow cytometry analysis of CD4+/IL-17+ cells. (D) Percentage of CD3+ CD4+ cells (%). (E) Percentage of CD3+ CD8+ cells (%). (F) Percentage of CD4+ IL-17+ cells (%). Data are expressed as the mean ± S.D. Compared with the Model group, * p < 0.05, ** p < 0.01; compared with the Model + DDP + TDP group, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001.

Effects of the Combination of TDP and DDP on Tumor Autophagy

WB was conducted to quantify the protein levels of p62, Beclin-1, LCII, and LCI, to assess the effects of the combination of TDP and DDP on tumor autophagy in the nude mouse xenograft model (Fig. 5A). The results suggested that TDP treatment did not significantly alter the LCII/LCI ratio (Fig. 5B), p62 levels (Fig. 5C), and Beclin-1 (Fig. 5D) levels. However, DDP increased the LCII/LCI ratio, up-regulated the expression of Beclin-1, and down-regulated the expression of p62. However, TDP alleviated the changes induced by DDP.

Fig. 5. Effects of the Combination of TDP and DDP on Tumor Autophagy in the Nude Mouse Xenograft Model

(A) WB analysis of autophagy-related proteins, including p-p62, Beclin-1, LCII, and LCI. (B) LCII/LCI ratio. (C) p-p62 protein expression. (D) Beclin-1 protein expression. Data are expressed as the mean ± S.D. Compared with the Model group, * p < 0.05, *** p < 0.001; compared with the Model + DDP + TDP group, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001.

DISCUSSION

To increase patient survival rate and QOL, it is crucial to develop novel drugs. Compared with synthetic drugs, TCM anticancer drugs have fewer side effects and are less susceptible to drug resistance.¹¹⁾ The combination of DDP with TCM has been explored for the treatment of GC. Of note, the TDP formula has been extensively used for the treatment of chronic atrophic gastritis (CAG), which is considered a precursor of GC.¹²⁾ According to earlier studies, the TDP formula mediates cell proliferation and apoptosis,^13,14) consistent with the results of the current study. More importantly, the combination of TDP and DDT suppressed the growth of GC tumors and concomitantly promoted SGC7901 cell apoptosis.

Loss of intercellular junctions in the primary tumor drives cell migration by secreting various proteases to degrade the extracellular matrix (ECM), which is composed of collagen, fibronectin, elastin, laminin, actin, and proteoglycan. The balance between degradation and synthesis of ECM plays a decisive role in maintaining tissue homeostasis.¹⁵⁾ It is worthwhile emphasizing that matrix metalloproteinases (MMPs) are integral components that participate in the regulation of the ECM degradation process.¹⁶⁾ Indeed, previous studies concluded that inhibiting MMP2 and MMP9 suppressed GC cell proliferation.^17,18) Herein, the combination of TDP and DDP effectively down-regulated the expression of MMP2 and MMP9. Likewise, it down-regulated the expression of COX-2, an enzyme that promotes tumorigenesis in numerous types of cancers.

Ma et al. identified a correlation between Ki-67 up-regulation and immunosuppression.¹⁹⁾ Specifically, Ki-67 overexpression could serve as a predictive biomarker for poor prognosis in gastric cancer patients. At the same time, Ki-67 overexpression reflects a high proliferation and migration of cancer cells.²⁰⁾ Moreover, CD31, also referred to as PECAM-1, has been identified as a candidate marker for vascular endothelial cells and tumor micro-vessel density, which indirectly reflects the proliferative and migratory properties of cancer cells.²¹⁾ IHC staining assay was performed to measure the levels of Ki-67 and CD31, uncovering that the combination of TDP and DDP down-regulated the expression level of CD31 and Ki-67 and suggesting a significant inhibition of the proliferation and migration of SGC7901 cells. The experimental results indicate that TDP&DDP treatment did not inhibit the expression of CD31 and Ki-67 in tumor layer samples; instead, it led to a decrease in their expression. This phenomenon may be attributed to the increased apoptosis (programmed cell death) induced by TDP&DDP treatment, which consequently reduced the expression levels of these two indicators. Apoptotic cells are no longer involved in the cell cycle, hence the decrease in Ki-67 expression. Additionally, apoptosis may also affect the function of endothelial cells, leading to a reduction in CD31 expression. Taken together, we posit that TDP is a promising new drug for the treatment of gastric cancer.

In a previous study, cyclophosphamide (CTX), an immunosuppressant, decreased the number of CD3+ CD4+ and CD3+ CD8+ T lymphocytes in mice.²²⁾ Herein, flow cytometry results signaled that DDP exerted comparable immunosuppressive effects to CTX. However, TDP reversed the decrease in CD3+ CD4+ and CD3+ CD8+ T cell numbers. CD8, as mentioned, is indeed a key marker of cytotoxic T lymphocytes (CTLs), which play a vital role in the immune system’s ability to recognize and eliminate infected or abnormal cells. When CD8 counts are elevated, it generally implies a robust immune response to a pathogen or cancer. If the number of CD8 cells increases under the treatment of “Model + DDP + TDP,” this may indicate an enhanced response of the immune system. Excessive activation of CD8 cells may lead to an enhanced inflammatory response, which can cause damage to normal tissue. Meanwhile, compared with DDP treatment, the number of CD4+ IL-17+ cells was significantly decreased by TDP. Interleukin-17 (IL-17) is a proinflammatory cytokine synthesized by CD4 T cells, with low IL-17 levels indicating a poor prognosis in gastric adenocarcinoma patients.²³⁾ This finding is in line with the findings of previous research, which suggested that the upregulation of IL-17 may be implicated in the positive feedback loop in GC occurrence and development.²⁴⁾ Therefore, the TDP formula may contribute to improving the immune responses of mice, thereby preventing the development of GC.

A previous study documented that P62 can be selectively bound to autophagosomes by direct binding to microtubule-associated protein 1 light chain 3 (LC3) and is efficiently degraded by autophagy. The LC3II/LC3I ratio and the expression of P62 reflect the degree of autophagy. In 2016, research demonstrated that tumors positive for LC3 and Beclin-1 were undergoing authophagy.²⁵⁾ In the present study, DDP significantly increased the LC3II/LC3I ratio and concurrently up-regulated Beclin-1 expression and down-regulated p62 expression, collectively implying that it promotes the autophagy of SGC7901 cells. On the other hand, TDP reversed DDP-induced autophagy despite not exerting effects on autophagy when used alone. Autophagy plays a dual protective role, and in some cases, can promote cell survival by degrading damaged organelles and proteins, especially in response to chemotherapy-induced stress. Besides, autophagy exerts pro-apoptotic effects, and overactivity of autophagy can lead to cell death. In this study, cisplatin-induced autophagy may be a protective mechanism for cells, potentially contributing to subsequent cisplatin resistance following prolonged exposure.^26,27) Notwithstanding, TDP inhibited autophagy, which may increase cisplatin sensitivity and thus enhance its tumoricidal effects.

CONCLUSION

To the best of our knowledge, this is the first study to provide evidence of the anti-GC effects of the combination of TDP and DDP. In summary, TDP combined with DDP suppressed the growth of GC tumors by promoting cell apoptosis, enhancing host immunity, and inhibiting cell proliferation and autophagy. Overall, our research lays a theoretical reference for the clinical drug application of GC treatments. Nevertheless, the effect of the TDP formula on GC in vivo warrants further exploration.

Funding

This work was supported by the Science and technology capability improvement project of Hebei University of traditional Chinese medicine (No. KTY2019022) and Provincial Medical Talents Project funded by the Government in 2021 (2100199).

Author Contributions

Conceptualization: Chun-xia Sun and Cui-huan Yan. Formal analysis and investigation: Chun-xia Sun and De-hui Li. Methodology: Ya-pei Xu and Zhu-feng Yang. Writing-original draft preparation: Chun-xia Sun. Writing-review and editing: Chun-xia Sun and Li-ying Wei. Supervision: Yue-ming Gao and Yi Liu. Yong-zhang Li played a crucial role in the initial experimental planning and provided invaluable guidance during the writing of our manuscript. All authors gave final approval, agreed to be accountable for all aspects of work ensuring integrity and accuracy.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability

The data sets used and analyzed during the current study are available from the corresponding author on reasonable request.

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