Fu Zheng Xiao Yu San Jie Decoction affects the proliferation of renal cell carcinoma via regulating E2F5 gene

Introduction

Renal carcinoma (RC) is a common and highly lethal malignant tumor in the urinary system. According to “The Global Cancer Statistics 2022”, RC ranks as the 14th most common cancer worldwide, with around 430,000 new cases each year. It has a mortality rate that places it 16th among all cancers, leading to approximately 160,000 deaths annually (1-3), and these figures are increasing. Histologically, renal cell carcinoma (RCC) is the most prevalent type of RC, accounting for about 90% of cases (4). Among these, clear cell RCC (ccRCC) constitutes around 70% (5). Currently, the main challenges in diagnosing and treating renal cancer are early detection and managing advanced stages. Patients with early-stage renal cancer often show no obvious symptoms. The classic triad of symptoms—hematuria, flank pain, and a palpable mass—typically appears only in advanced stages. Surgery is currently the only curative treatment for renal cancer, and it is most effective when performed in the early stages. For localized renal cancer without metastasis, the 5-year survival rate can be as high as 90% (6). Despite this, approximately 74% of patients who undergo surgical treatment experience recurrence or metastasis within 5 years (6). For these patients, as well as those with advanced renal cancer, targeted therapy combined with immunotherapy is the preferred treatment. However, issues such as targeted drug resistance and uncertainties surrounding immunotherapy remain significant challenges in renal cancer treatment.

Fu Zheng Xiao Yu San Jie Decoction (FZXYSJD), developed by the Department of Urology at the First Affiliated Hospital of Guangzhou University of Chinese Medicine, is a herbal formula designed to treat kidney cancer based on years of clinical experience. The formula consists of Huangqi (Hedysarum Multijugum Maxim) 30 g, Xiacao (Prunellae Spica) 30 g, Shancigu (Pseudobulbus Cremastrae Seu Pleiones) 10 g, Ezhu (Curcumae Rhizoma) 15 g, Sanqi [Panax Notoginseng (Burk.) F. H. Chen Ex C. Chow] 10 g, Huzhang (Polygoni Cuspidati Rhizoma Et Radix) 20 g, Feng Fang (Nidus Vespae) 10 g, and Gancao (licorice) 9 g. The key active ingredients, including astragaloside Ⅱ (7), rosmarinic acid (8), and notoginsenoside R1 (9), have been confirmed to effectively inhibit the proliferation of renal cancer cells. In China, the patient’s overall condition was typically considered in the use of traditional Chinese medicine (TCM) decoction. Various Chinese medicines are mixed and boiled together to achieve a harmonious balance among different organs in the body. Thus, while some ingredients in the decoction demonstrate therapeutic effects, further research is needed to explore the overall mechanism of action.

E2F transcription factor 5 (E2F5) belongs to the E2 promoter binding factor (E2F) family of transcription factors, which are crucial for regulating cell proliferation (10). Interestingly, E2F5 exhibits contrasting effects in cancer studies. For instance, research on oral cancer (11), gallbladder cancer (12), and prostate cancer (13) indicates that reducing E2F5 expression effectively decreases cancer cell proliferation. In contrast, research on breast cancer (14) and liver cancer (15) suggests that reducing E2F5 expression may promote cancer cell proliferation. However, current studies on E2F5 in RCC have primarily focused on bioinformatics and tissue expression. E2F5 expression in ccRCC tissue is lower than that in normal tissue (16,17), and some research shows that RCC proliferation may be associated with E2F5 (18,19).

In this study, we found that FZXYSJD drug serum inhibited the growth of ccRCC cell lines. Additionally, FZXYSJD promoted E2F5 expression. Silencing E2F5 promoted the proliferation of ccRCC cells. This finding indicated that FZXYSJD might inhibit ccRCC through E2F5 gene. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-aw-739/rc).

Methods

Materials and reagents

The herbs in FZXYSJD were all procured from the Department of Urology at First Affiliated Hospital of Guangzhou University of Chinese Medicine. According to the original formula, Huangqi 30 g, Xiacao 30 g, Shancigu 10 g, Ezhu 15 g, Sanqi 10 g, Huzhang 20 g, Feng Fang 10 g, and Gancao 9 g, add 600 mL of water and boil for 1 hour. Afterwards, prepare the decoction at concentrations of 0.71, 1.42, and 2.84 g/mL using by rotary evaporator.

Animals and grouping

Eighty adult male Sprague-Dawley (SD) rats, aged six weeks and weighing 180±10 g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (certification number: No. SCXK (liao) 2020---0001). All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of The First Affiliated Hospital of Guangzhou University of Chinese Medicine (Ethics number: GZTCMF1-20230083), in compliance with institutional guidelines for the care and use of animals. A protocol was prepared before the study without registration.

The rats were randomly divided into four groups: a blank control group (control) and low-dose (LD), medium-dose (MD), and high-dose (HD) FZXYSJD groups (LD, MD, and HD), with each group containing 20 rats. They were housed in a controlled room with a 12-hour light/dark cycle, temperature maintained at 22–25 °C, and relative humidity of 45–60%. Rats had free access to food and water. The blank control group was treated with 1 mL of 0.9% sodium chloride solution (Sichuan Kelun Pharmaceutical Co., Ltd., Sichuan, China) per 100 g body weight once daily, while the FZXYSJD groups received 7.1, 14.2, and 28.4 g/kg FZXYSJD once daily via intragastric administration. After seven days of treatment, the rats were sacrificed using an intraperitoneal injection of tribromoethanol (6 mL/kg, Nanjing Aibei Biotechnology Co., Ltd., Nanjing, China). Blood samples (5 mL) were collected from the abdominal aorta, centrifuged at 4 ℃, 3,000 rmp 30 min, and serum samples were stored at −80 °C.

Cell culture and transfection

Human ACHN, 786-O, and 293T cell lines were obtained from the American Type Culture Collection (ATCC). RCC cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% serum from either the control or FZXYSJD groups, along with 1% penicillin-streptomycin (PS) (100 µg/mL penicillin and 100 mg/mL streptomycin sulfates; Gibco). Cells were incubated at 37 °C in a 5% CO₂ atmosphere. Transfections of small interfering RNA (siRNA), microRNA (miRNA), inhibitors, and their respective negative controls (NC, mimic.nc, inhibtior.nc) were performed in RCC and 293T cell lines using Opti-MEM^® I Reduced Serum Medium (Gibco) mixed with Lipofectamine 2000 (Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot (WB) analysis were performed to validate transfection efficiency. The sequences for siRNA targeting E2F5 (siE2F5) are listed in Table 1.

Cell Counting Kit-8 (CCK-8) assay

The CCK-8 assay was used to evaluate the proliferation capacity of RCC cells. RCC cells were seeded into 96-well plates at a density of 1,000 cells per well. The cells were treated with the corresponding serum culture medium and transfected with siE2F5 or NCs. At 0, 24, 48, and 72 hours post-treatment, 10 µL of CCK-8 reagent (Beyotime Institute of Biotechnology, Shanghai, China) was added to each well and incubated for 30 minutes. The optical density (OD) of each well was measured at a wavelength of 450 nm by an ELISA microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Colony formation assay

ACHN and 786-O cells (600–1,000 cells per well) were inoculated into 6-well plates. After 24 hours of incubation, cells were treated with the corresponding serum culture medium and transfected with siE2F5 or NCs. The medium was changed every two days. After seven days of cultivation, cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet at room temperature for 20 minutes. The image was captured using an IPHONE X PRO MAX at a magnification of 1X. The number of cell colonies was counted using ImageJ software.

Bioinformatics analysis and target gene prediction

Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/index.html) was used for E2F family expression detection and survival analysis in RCC.

WB

WB was used to determine the expression level of E2F5 in renal cancer cells. First, RCC cell proteins were extracted with a lysis buffer. Polyacrylamide gel (SDS-PAGE) was prepared and electrophoresed 30 μg of protein until the bromophenol blue dye front appeared. After protein transfer onto a 0.22 μm polyvinylidene fluoride (PVDF) membrane, the membrane was washed with tris buffered saline with tween 20 (TBST) solution and blocked with Fast Blocking (Catalog: RK0501, Shenzhen Aipunuo Biotechnology Co., Ltd., Shenzhen, China). It was then incubated overnight at 4 °C with the primary antibodies: E2F5 (1:1,000, Catalog: D197169, BBI, Shanghai, China) and β-Actin (1:1,000, Catalog: ab8227, Abcam, Shanghai, China). Finally, after incubation with secondary antibodies: goat anti-mouse IgG (1:5,000, Catalog: D110087, BBI) or goat anti-rabbit IgG (1:5,000, Catalog: AS014, ABclonal, Wuhan, China), luminescence detection was performed. WB results were analyzed using Image Lab.

RNA extraction and complementary DNA (cDNA) synthesis

TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., California, USA) was used to isolate total RNA from RCC cells. The extracted RNA was reverse transcribed to synthesize cDNA using the miScript Reverse Transcription kit (Qiagen GmbH, Duesseldorf, Germany) and a Roche LightCycler 480 Real-Time PCR system (Roche Diagnostics, Basel, Switzerland).

RT-qPCR

Following RNA extraction and cDNA synthesis, the relative mRNA expression levels of E2F5 in cells were detected by Roche LightCycler 480 Real-Time PCR system (Roche Diagnostics). The reaction conditions were as follows: 95 °C for 15 minutes, followed by 40 cycles of 94 °C for 15 seconds, 55 °C for 30 seconds, and 70 °C for 30 seconds. Data analysis was performed using the 2^−ΔΔCq method. The primer sequences are provided in Table 2.

High-performance liquid chromatography-mass spectrometry (LC-MS)

In this study, LC-MS was used to analyze the composition of FXYJSD. The FXYJSD was procured from The First Affiliated Hospital of Guangzhou University of Chinese Medicine and prepared in decoction concentrations of 1.42 g/mL. The LC analysis was performed on a Vanquish UHPLC System (Thermo Fisher Scientific). Chromatography was carried out with an ACQUITY UPLC^® HSS T3 (2.1 mm × 100 mm, 1.8 µm) (Waters, Milford, MA, USA). The MS analysis was performed on a Mass spectrometric detection of metabolites was performed on Orbitrap Exploris 120 (Thermo Fisher Scientific) with an electrospray ionisation (ESI) ion source.

In-depth analysis of E2F5 by molecular docking with herb-identified components of FZXYSJD in Chinese Pharmacopoeia

Download the molecular structure (E2F5:5TV) from the PDB database (https://www.rsb.org/) and use Pymol software to remove water and residues, followed by Autodock software hydrogenation treatment. Search the identified components of FZXYSJD through the Chinese Pharmacopoeia 2022 (https://ydz.chp.org.cn). Download the molecular structure of the identified components from the PubChem database. Autodock Vina software to analyze the molecular docking binding energy between protein receptors and select the lowest binding energy. Biovia Discovery Studio software visualized optimized conformations, providing insights into molecular interactions.

Statistical analysis

Each experiment was repeated at least three times. Statistical comparisons between two groups were performed using Student’s t-test, while one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test was used for multiple groups. All statistical analyses were performed using GraphPad Prism software (version 8, GraphPad Inc., USA), and differences were considered statistically significant when P<0.05.

Results

FXYJSD inhibited cell growth of ccRCC

To determine whether FXYJSD could affect cell proliferation, ACHN and 786-O cells were cultured in DMEM containing 10% serum from the blank control group (control) or serum from the LD, MD, and HD FXYJSD groups, and cell growth was measured. CCK-8 assay results showed that, compared with the control, FXYJSD treatment led to a decrease in cell number at 72 hours. In ACHN cells, the results were as follows—LD: 30% (24 h, P=0.003), 29% (48 h, P<0.001), 32% (72 h, P<0.001); MD: 31% (24 h, P=0.01), 33% (48 h, P<0.001), 43% (72 h, P<0.001); HD: 31% (24 h, P=0.002), 39% (48 h, P<0.001), 50% (72 h, P<0.001). In 786-O cells, the results were as follows—LD: no significance (24 h, P>0.99), no significance (48 h, P=0.53), 13% (72 h, P=0.003); MD: 19% (24 h, P=0.02), 31% (48 h, P<0.001), 31% (72 h, P<0.001); HD: 20% (24 h, P=0.01), 33% (48 h, P<0.001), 39% (72 h, P<0.001) (Figure 1A). Furthermore, a colony formation assay was performed on cells cultured with serum from the FXYJSD groups, which suppressed the number of colonies. Compared with the control, in ACHN cells, the results were as follows—LD: 50% (P<0.001); MD: 90% (P<0.001); HD: 94% (P<0.001). In 786-O cells, the results were as follows—LD: 23% (P=0.04); MD: 74% (P<0.001); HD: 86% (P<0.001) (Figure 1B,1C).

Figure 1 FZXYJD suppressed the cell growth rate in ccRCC cell line. (A) CCK-8 assay. (B,C) Cell colony formation assay [IPHONE X PRO MAX] and date visualization (magnification: 1× crystal violet stained). *, P<0.05; **, P<0.01; ***, P<0.001. Control, cultured in SD rat serum. LD, cultured in FZXYJD lower dose group serum. MD, cultured in FZXYJD medium dose group. HD, cultured in FZXYJD high-dose group serum. CCK-8, Cell Counting Kit-8; ccRCC, clear cell renal cell carcinoma; FZXYSJD, Fu Zheng Xiao Yu San Jie Decoction; HD, high-dose; LD, low-dose; MD, medium-dose; OD, optical density.

E2F5 was down-regulated in ccRCC

The GEPIA was used for expression detection and survival analysis. The results indicated that E2F5 was the only down-regulated member of the E2F family in RCC (Figure 2A). Furthermore, down-regulation of E2F5 was associated with poor overall survival in RCC patients (Figure 2B).

Figure 2 Potential target genes of FZXYJD in E2F family were predicted. (A) E2F5 has low expression in ccRCC. (B) Patients had poorer survival with low expression of E2F5 in ccRCC. (C-F) FZXYJD can increase the expression level of E2F5 in ccRCC cell line. *, P<0.05; **, P<0.01; ***, P<0.001. Control, cultured in SD rat serum. LD, cultured in FZXYJD lower dose group serum. MD, cultured in FZXYJD medium dose group serum. HD, cultured in FZXYJD high-dose group serum. ccRCC, clear cell renal cell carcinoma; FZXYSJD, Fu Zheng Xiao Yu San Jie Decoction; HD, high-dose; HR, hazard ratio; KIRC, kidney renal clear cell carcinoma; LD, low-dose; MD, medium-dose; N, normal; SD, Sprague-Dawley; T, tumor; TPM, transcripts per million.

FXYJSD promoted E2F5 protein expression

To elucidate the suppression mechanism of FXYJSD, we verified that E2F5 protein expression was promoted in ccRCC cells after culturing with the corresponding FXYJSD serum groups for 72 hours (Figure 2C-2F).

siE2F5 promoted cell growth of ccRCC

Subsequently, ccRCC cells were treated with siE2F5 (Figure 3A) and then examined for cell growth. The results from the CCK-8 assay showed that gene silencing of E2F5 promoted the proliferation of ccRCC cells (Figure 3B). The colony formation assay results indicated that siE2F5 increased the number of colonies (Figure 3C).

Figure 3 E2F5 was involved in FZXYJD suppression of ccRCC cells proliferation. (A) Western blot and RT-qPCR were performed to analyze E2F5 expression in siE2F5-transfected ccRCC cell line. β-actin was used as an internal quantitative control. (B) CCK-8 assay. (C) Cell colony formation assay [IPHONE X PRO MAX] (magnification: 1×; crystal violet stained). *, P<0.05; **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; ccRCC, clear cell renal cell carcinoma; FZXYSJD, Fu Zheng Xiao Yu San Jie Decoction; NC, negative control; OD, optical density; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; siE2F5, small interfering RNA targeting E2F5.

Identification of components in FXYJSD decoction

The Chinese Pharmacopoeia 2022 found that the main components of FXYJSD are astragaloside IV, calycosin-7-O-β-D-glucoside, rosmarinic acid, ginsenoside Rg1, ginsenoside Rbl, notoginsenoside Rl, liquiritin, glycyrrhizic acid, emodin and polydatin (Table 3). Further combined with LC-MS analysis, only astragaloside IV, ginsenoside Rb1, notoginsenoside Rl cannot be detected among the above 10 compounds (Figure 4). These compounds were not detected, possibly because the Chinese Pharmacopoeia provided their substance identification components, or because polysaccharides/tannins in the pre-treatment compound may co-precipitate saponins, or the saponin dissolution rate is low during water extraction.

Figure 4 LC-MS analysis and molecular structures of active components in the Fuzheng Xiaoyu Sanjie Formula. (A) The base peak chromatogram obtained in negative ion mode displays two characteristic peaks (labeled as 4 and 7), along with their retention times and relative peak intensities. (B) The corresponding chromatogram in positive ion mode reveals complementary ionization profiles of the same compounds, showing five distinct characteristic peaks (labeled as 1, 2, 3, 5, and 6), together with their retention times and relative peak intensities. (C) Molecular structures of the bioactive compounds corresponding to these characteristic peaks: 1, calycosin-7-O-β-D-glucoside; 2, polydatin; 3, glycyrrhizic acid; 4, liquiritin; 5, ginsenoside Rg1; 6, rosmarinic acid; 7, emodin. FZXY, Fu Zheng Xiao Yu; LC-MS, liquid chromatography-mass spectrometry

Molecular docking of the E2F5 protein with FZXYSJD identified components

As shown in Figure 5, the binding free energies of E2F5 protein with different compounds were significantly lower than −6 kcal/mol and indicating strong binding affinity. The regulation of E2F5 may be related to these components.

Figure 5 Three-dimensional and two-dimensional docking patterns and interactions of FZXYSJD identified components with the E2F5 protein. FZXYSJD, Fu Zheng Xiao Yu San Jie Decoction.

Discussion

RCC is the most common malignant tumor of the urinary system and poses a significant health risk (20). Due to the limited efficacy of radiotherapy and chemotherapy in treating RCC, early surgical resection remains a primary and effective therapeutic strategy (21). However, many patients with RCC are diagnosed at an advanced stage, for which early diagnosis is difficult (22) and often results in missing the optimal window for surgery. Furthermore, the absence of tumor-specific biomarkers necessitates reliance on imaging findings for monitoring RCC recurrence, which makes it difficult to predict the prognosis of cancer patients (23). Consequently, the absence of effective recurrence monitoring and prognostic indicators leads to suboptimal disease management for many cancer patients post-surgery, resulting in decreased quality of life and reduced survival rates (24). Furthermore, challenges such as high recurrence rates post-surgery (6), resistance to targeted therapies (25), and the ineffectiveness of immunotherapy for RCC require further attention (26). Therefore, this study aims to examine the mechanisms of action of clinical TCM, identify its therapeutic targets, and aid in the discovery of tumor markers and drug interventions for the management of RCC.

In recent years, TCM adjuvant therapy has gained attention in cancer treatment. TCM is typically used for patients who have completed standard treatments such as surgery, chemotherapy, radiotherapy, and targeted immunotherapy, serving as either maintenance therapy or a method of “replacement and supplementation” (27,28). Lin et al. (29) discovered that rational use of TCM can effectively extend the median survival time of cancer patients while reducing adverse drug reactions. However, TCM practice in China relies more on physicians’ experience than on uniform standards, and the mechanisms of drug action remain unclear. Consequently, TCM faces limitations in anti-tumor treatment, highlighting the need for further basic and clinical research to improve its applications in cancer.

TCM views RCC as a disease caused by prolonged stagnation of qi and blood stasis due to the deficiency of positive qi. Treatment should follow the principles of nourishing positive qi, removing toxins, and dispersing nodules. FZXYSJD is a prescription based on these principles and has been used as a supplementary treatment for kidney cancer in clinical practice. However, there are currently no reports on the effects of FZXYSJD on ccRCC, and the underlying mechanisms remain unclear, indicating a need for further research. Therefore, we aim to investigate the effect of FZXYSJD on ccRCC cells. By preparing FZXYSJD drug-containing serum for intervention in ccRCC cells, the results of the CCK-8 assay and monoclonal formation showed that FZXYSJD could inhibit the proliferation and tumorigenicity of ccRCC cells.

Recently, the E2F family was localization in the nucleus (30), which has garnered significant attention for its role in regulating cell proliferation and the cell cycle (31). A study has shown that E2F5 is significantly reduced in ccRCC tissue (17). Similarly, bioinformatics analysis of ccRCC indicates that E2F5 is the only gene in the E2F family that is downregulated, and low E2F5 expression is associated with worse prognosis in ccRCC patients. At present, there is limited research on E2F5, with most studies focusing on miRNA regulation of E2F5. However, Ha et al. (32) demonstrated that Euxanthone (a compound extracted from the roots of Polygala tenuifolia Willd) can significantly upregulate the expression of E2F5, indicating the possibility that compounds can also combine with E2F5. Thus, we hypothesize that FZXYSJD may play a role in ccRCC cell proliferation by affecting E2F5. Subsequently, we confirmed that treatment with siE2F5 promoted ccRCC cell proliferation. In this study, we used Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS) to perform quality control of FZXYSJD according to the herb appraisal ingredient with Chinese Pharmacopeia 2022, and used molecular docking to predict the binding potential of ingredients with E2F5. But this is not enough to confirm that these ingredients can indeed bind to E2F5, which requires further experiments, such as using surface plasmon resonance (SPR). Finally, to confirm the effect of E2F5 on renal clear cells, we confirmed that siE2F5 treatment can promote ccRCC cell proliferation.

Limitations

Due to budget constraints, insufficient equipment, and long experimental time periods, this paper has some shortcomings, such as: not selecting 1–2 core components identified by LC-MS and verifying whether this single component can also upregulate E2F5 protein expression in a cell model; not conducting in vivo animal experiments; the concentration of active ingredients in drug serum is unknown, and it cannot be determined whether it is a single component or a multi-component synergistic regulation of E2F5. In the future, we will continue to conduct in-depth research.

Conclusions

In conclusion, the present study identified that FZXYSJD drug serum inhibited the proliferation of ccRCC cells by promoting E2F5 expression. Our clinical treatment experience suggests that FZXYSJD could serve as a complementary treatment to delay the progression of ccRCC. This study offers new insights into the TCM and molecular regulation. In the future, we will continue our research on treatments, molecular markers, diagnoses, and other related fields.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-aw-739/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-aw-739/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-aw-739/prf

Funding: This study was supported by the National Natural Science Foundation of China (No. 82205126), the Scientific Research Project of Guangdong Provincial Administration of Traditional Chinese Medicine (No. 20251104), the Guangdong Famous Traditional Chinese Medicine Studio (to J.X.), the fourth batch of famous traditional Chinese medicine master-apprentice program in Guangdong Province in 2024 (to J.X.), and the Young and Middle-aged Key Talent Training Project of The First Affiliated Hospital of Guangzhou University of Chinese Medicine (No. 09005650043).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-aw-739/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. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of The First Affiliated Hospital of Guangzhou University of Chinese Medicine (Ethics No. GZTCMF1-20230083), in compliance with institutional guidelines for the care 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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