Yiqi Chutan Method Alleviates Tumor-Related Skeletal Muscle Injury by Reducing Inflammatory Infiltration

YINGCHAO WU^{1,2,3,4,5}, ZIQIAN LUO^{2,4}, ZHONGJIA YI^{6}, DAJIN PI^{6}, JIAQI CUI^{6}, LIZHU LIN^{1}, MINGZI OUYANG^{6}, QIANJUN CHEN^{1,2,3,4,5}

^{1}Guangdong Provincial Hospital of Chinese Medicine, Guangzhou 510120, Guangdong, China
^{2}The Second Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong, China
^{3}State Key Laboratory of Traditional Chinese Medicine Syndrome, Guangzhou 510120, Guangdong, China
^{4}Department of Breast Oncology, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou 510120, Guangdong, China
^{5}Guangdong Academy of Traditional Chinese Medicine, Guangzhou 510120, Guangdong, China
^{6}College of Traditional Chinese Medicine, Jinan University, Guangzhou 510632, Guangdong, China

Corresponding authors: MINGZI OUYANG, Associate Professor/Associate Chief Physician of Traditional Chinese Medicine; E-mail: mingzioy@jnu.edu.cn
QIANJUN CHEN, Chief Physician of Traditional Chinese Medicine/Doctoral Supervisor; E-mail: cqj55@163.com

Abstract

Background: Cancer patients frequently experience muscle wasting and other skeletal muscle impairments, yet the underlying pathological mechanisms remain incompletely understood. Objective: This study investigates the pathological mechanisms of tumor-related skeletal muscle injury and evaluates the therapeutic effects of the Yiqi Chutan method on this condition. Methods: Between October 2020 and May 2021, thirty 8-week-old female C57BL/6 mice were enrolled. Twenty mice were randomly selected for tumor model establishment via subcutaneous injection of LLC cells. Successful models were divided into a tumor group (n=10) and a Yiqi Chutan group (YQCT group, n=10), with an additional control group (n=10). The YQCT group received Yifei Sanjie pills via gavage (3 g·kg⁻¹·d⁻¹) for 21 days, while control and tumor groups received equal volumes of normal saline. Twenty-four hours after the final administration, behavioral assessments were conducted using open field and elevated plus maze tests. Gastrocnemius muscle tissues were harvested for histopathological analysis to evaluate pathological changes. Transcriptomic analysis was performed to identify differentially expressed genes, and enzyme-linked immunosorbent assay (ELISA) was used to measure inflammatory cytokine levels. RAW264.7 cells in logarithmic growth phase were randomly divided into mouse serum control group (RAW264.7 group), mouse tumor serum group (RAW264.7-LPS group), and mouse Yiqi Chutan serum group (YQCT group). These groups were treated for 24 hours with media containing 10% serum from control mice, 10% serum from tumor-bearing mice + 100 ng/mL LPS, or 10% serum from YQCT-treated mice + 100 μg/L LPS, respectively. ELISA was then used to detect inflammatory cytokine levels in RAW264.7 cells. After co-culturing these treated RAW264.7 cells with mouse C2C12 cells for 48 hours, lysosomal red fluorescent probe kits were used to assess autophagolysosome levels in C2C12 cells.

Results: Significant differences were observed among the three groups in total movement distance in both open field and elevated plus maze tests (P<0.001). The tumor group exhibited reduced movement compared to controls, while the YQCT group showed increased movement relative to the tumor group (P<0.001). Histopathological results revealed significant muscle cell damage in the tumor group compared to controls, with reduced damage severity in the YQCT group. Transcriptomic analysis demonstrated similar gene expression patterns between control and YQCT groups, contrasting with the opposite trend observed in the tumor group. Immunofluorescence staining showed increased infiltration of M1 macrophages, neutrophils, T lymphocytes, and B lymphocytes in tumor group gastrocnemius muscles compared to controls. In contrast, YQCT group exhibited increased M2 macrophage infiltration and decreased pro-inflammatory cell infiltration compared to the tumor group. ELISA results demonstrated elevated IL-1β, IL-6, and TNF-α levels in the tumor group versus controls, with significantly lower levels in the YQCT group compared to the tumor group (P<0.001). Cell experiments revealed that RAW264.7-LPS group had higher IL-1β, IL-6, and TNF-α levels than controls, while YQCT group showed lower levels than RAW264.7-LPS group (P<0.001). Activated RAW264.7 cells induced increased autophagolysosome formation in C2C12 cells.

Conclusion: The Yiqi Chutan method alleviates tumor-induced skeletal muscle inflammatory cell infiltration, thereby reducing inflammatory injury, preserving skeletal muscle function, and protecting motor function in tumor-bearing mice.

Keywords: Yiqi Chutan method; transcriptomics; tumor; inflammation; skeletal muscle

Funding: National Natural Science Foundation of China (82474504); Guangdong Provincial Natural Science Foundation (2023A1515011115, 2025A1515011760); Traditional Chinese Medicine Guangdong Laboratory (Hengqin Laboratory) Cultivation Project (HQL2024PZ023); Guangzhou Science and Technology Program (SL2024A03J0850); Guangdong Provincial Key Laboratory of Traditional Chinese Medicine Syndrome Clinical Research (YN2023ZH10); State Key Laboratory of Traditional Chinese Medicine Syndrome (QZ2023ZZ13); Guangzhou University of Chinese Medicine and Zhongshan Hospital of Traditional Chinese Medicine High-level Hospital Co-construction Project Discipline Construction Special Fund (GZYZS2024XKG05)

Introduction

According to the 2022 global cancer statistics report from the World Health Organization's International Agency for Research on Cancer [1], there were 19.965 million new cancer cases and 9.737 million cancer-related deaths worldwide that year. The epidemiological analysis identified lung cancer, female breast cancer, colorectal cancer, prostate cancer, and gastric cancer as the five most common malignancies, while lung cancer, colorectal cancer, liver cancer, female breast cancer, and gastric cancer accounted for the highest mortality. Projections based on current demographic trends estimate that global cancer incidence will exceed 35 million new cases annually by 2050. Historically, due to the severe health threats posed by malignant tumors, their poor prognosis, and limited research resources, oncology research has long focused primarily on the tumor itself, leading to the development of targeted interventions against tumor cell proliferation pathways [2,3]. However, with economic development and increased life expectancy in China, both cancer incidence and prevalence continue to rise [4,5]. The advent of effective treatments has enabled more patients to survive with tumors, substantially increasing the population of cancer survivors [6]. Consequently, tumor-related adverse effects have gradually gained attention during both pathological progression and clinical intervention phases [7].

Reports indicate that 30% of cancer patients experience tumor-related skeletal muscle injury symptoms including fatigue, weight loss, and muscle mass reduction [8,9], with muscle mass loss affecting 23% of men and 10% of women [9], particularly common in lung cancer patients [10]. Previous research inadequately addressed these tumor-related adverse effects, causing studies on tumor-induced skeletal muscle injury to lag significantly behind tumor-centric research and directly limiting effective clinical solutions. Shifting or expanding research focus to encompass the entire spectrum of tumor occurrence, development, and treatment, and comprehensively addressing emerging clinical problems from basic research to clinical application has become an urgent priority. Therefore, investigating the pathological mechanisms of tumor-related skeletal muscle injury and developing effective therapeutic interventions holds significant clinical importance.

The Yiqi Chutan method is a classical therapeutic approach summarized by national TCM master Daihan Zhou based on extensive clinical experience for treating non-small cell lung cancer [11]. The classical formulation consists of eight traditional Chinese medicinals: Maozhaocao (Ranunculi Ternati Radix), fried Jiangcan (Bombyx Batryticatus), Zhongjiefeng (Sarcandrae Herba), Fabanxia (Pinelliae Rhizoma Praeparatum), Shancigu (Cremastrae Pseudobulbus Pleiones Pseudobulbus), Zhebeimu (Fritillariae Thunbergii Bulbus), Lingzhi (Ganoderma), and Xiyangshen (Panacis Quinquefolii Radix), which has been manufactured as a hospital preparation for long-term clinical use. Clinical studies have confirmed that the Yiqi Chutan method achieves favorable efficacy in lung cancer treatment, effectively prolonging progression-free survival, median survival, and overall survival [11-13]. Additionally, clinical research demonstrates that this method improves quality of life and ameliorates tumor-related adverse effects such as fatigue and weight loss during survival with tumor [14-16], though its mechanisms of action remain inadequately elucidated. Preliminary basic research has confirmed that Yiqi Chutan method can effectively prevent muscle injury during tumor progression and chemotherapy [17-19], yet its precise biological mechanisms require further investigation and reporting. Therefore, systematically studying the pathways through which Yiqi Chutan method intervenes in tumor-related skeletal muscle injury holds important theoretical value for expanding the application of traditional Chinese medicine in cancer supportive care.

Materials and Methods

1.1.2 Cell Lines and Experimental Animals

Mouse LLC lung cancer cells, mouse RAW264.7 mononuclear macrophages, and mouse C2C12 myoblasts were maintained in the Ouyang Mingzi research group at the College of Traditional Chinese Medicine, Jinan University. For animal experiments, thirty 8-week-old SPF-grade female C57BL/6 mice (body weight 18±1 g) were provided by Beijing Huafukang Biotechnology Co., Ltd. [License No.: SCXK (Beijing) 2019-0008]. Animals were housed in controlled animal rooms (5 mice/cage) under standard conditions: temperature 23±2°C, humidity 60±10%, and pressure differential 25±2 Pa, with free access to food and water and a 12-hour light/dark cycle (light period 6:00-18:00). This study was approved by the Laboratory Animal Ethics Committee of Jinan University (Approval No.: IACUC-20200923-06).

1.1.3 Experimental Equipment

Cell culture incubator [Thermo Fisher Scientific (USA), Model 371]; mouse behavioral analysis platform with EthoVision XT 14 software [Noldus (Beijing) Information Technology Co., Ltd.]; fluorescence microscope [Olympus (Japan), Model IX71]; transmission electron microscope [Hitachi (Japan), Model HT7800/HT7700]; ultrasonic disruptor (Ningbo Xinzhi Biotechnology Co., Ltd., Model JY92-IIN); low-speed centrifuge [Eppendorf (Germany), Model 5702R].

1.1.4 Experimental Reagents

DMEM high-glucose medium [Gibco (USA), Cat# 11965092]; fetal bovine serum [Gibco (USA), Cat# 10270106]; penicillin/streptomycin dual antibiotic [Gibco (USA), Cat# 10378016]; Yifei Sanjie pills (First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangdong Pharmaceutical Preparation Record Z20190015000); Hematoxylin-Eosin staining kit (Shanghai Jizhi Biochemical Technology Co., Ltd., Cat# AG1120); One-step TUNEL apoptosis detection kit (Shanghai Beyotime Biotechnology Co., Ltd., Cat# C1086); rabbit anti-inducible nitric oxide synthase (iNOS) monoclonal antibody [Cell Signaling Technology (USA), Cat# 13120T]; rabbit anti-mannose receptor (CD206) monoclonal antibody [Cell Signaling Technology (USA), Cat# 24595T]; rabbit anti-integrin αM chain (CD11b) monoclonal antibody [Cell Signaling Technology (USA), Cat# 17800T]; rabbit anti-T lymphocyte surface antigen (CD3) monoclonal antibody [Cell Signaling Technology (USA), Cat# 78588T]; anti-rabbit leukocyte differentiation antigen 20 (CD20) monoclonal antibody [Cell Signaling Technology (USA), Cat# 70168T]; goat anti-rabbit IgG antibody [Cell Signaling Technology (USA), Cat# 4412S]; mouse interleukin (IL)-1β ELISA kit (Hangzhou Lianke Biotechnology Co., Ltd., Cat# EK201B-48), mouse IL-6 ELISA kit (Hangzhou Lianke Biotechnology Co., Ltd., Cat# EK206/3-48), and mouse tumor necrosis factor (TNF)-α ELISA kit (Hangzhou Lianke Biotechnology Co., Ltd., Cat# EK282HS-48); lipopolysaccharide (LPS, Shanghai Jizhi Biochemical Technology Co., Ltd., Cat# AC12037); lysosomal red fluorescent probe kit (Shanghai Beyotime Biotechnology Co., Ltd., Cat# C1046).

1.2.1 Cell Culture

Mouse LLC lung cancer cells, RAW264.7 mononuclear macrophages, and C2C12 myoblasts were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum and dual antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL). Cultures were maintained at 37°C in a 5% CO₂ humidified incubator with fresh medium replacement every 48 hours. Cells were passaged upon reaching 90% confluence, and all subsequent experiments utilized cells in logarithmic growth phase.

1.2.2 Tumor Mouse Model Establishment and Grouping

Following our established tumor-bearing animal model protocol [19], thirty female C57BL/6 mice were acclimated for one week. Ten mice were randomly assigned to the control group, while the remaining twenty received subcutaneous injection of 1×10⁵ LLC cells in the right axillary region to establish tumor-bearing models. Seven days post-injection, the twenty tumor-bearing mice were randomly divided into tumor and Yiqi Chutan (YQCT) groups (n=10 each). The YQCT group received Yifei Sanjie pills via gavage (3 g·kg⁻¹·d⁻¹) for 21 days, while control and tumor groups received equal volumes of normal saline. Behavioral assessments were performed and samples were collected 24 hours after the final administration.

1.2.3 Cell Model Establishment, Grouping, and Intervention

RAW264.7 mononuclear macrophages in logarithmic growth phase were randomly divided into three groups: mouse serum control group (RAW264.7 group), mouse tumor serum group (RAW264.7-LPS group), and mouse Yiqi Chutan serum group (YQCT group). Cells were treated for 24 hours with media containing 10% serum from control mice, 10% serum from tumor-bearing mice + 100 ng/mL LPS, or 10% serum from YQCT-treated mice + 100 μg/L LPS, respectively. ELISA was then used to detect inflammatory cytokine levels in RAW264.7 cells. Additionally, after co-culturing these treated RAW264.7 cells with mouse C2C12 cells for 48 hours, lysosomal red fluorescent probe kits were used to assess autophagolysosome levels in C2C12 cells.

1.2.4 Behavioral Testing

(1) Open Field Test: Following established protocols [20], mice were placed in a 40 cm × 40 cm × 30 cm open field apparatus and recorded for 5 minutes under low-light conditions. Total movement distance was analyzed using Noldus EthoVision XT 14 software.

(2) Elevated Plus Maze Test: Using a standard elevated plus maze paradigm [21] with a central platform (5 cm × 5 cm) connecting two open arms (30 cm × 5 cm × 0.5 cm) and two closed arms (30 cm × 5 cm × 15 cm), mouse behavior was recorded for 5 minutes. Movement trajectory data were quantified using EthoVision XT 14 software to determine total movement distance.

1.2.5 Histopathological Analysis

At the experimental endpoint, mice were euthanized via intraperitoneal injection of sodium pentobarbital (50 mg/kg). Whole blood was collected simultaneously, and gastrocnemius muscle tissues were harvested and preserved according to standard protocols.

(1) Hematoxylin-Eosin (HE) Staining: Following our previous research [22], fresh gastrocnemius muscle tissues were fixed in 4% paraformaldehyde for 24 hours, then processed through tissue trimming, graded dehydration, and paraffin embedding. Continuous 4 μm sections were prepared, dewaxed in xylene, rehydrated through graded alcohols, and stained with hematoxylin (5 seconds), differentiated in hydrochloric acid alcohol (25 seconds), and counterstained with eosin (5 seconds). After dehydration, clearing, and mounting, histological observation was performed at consistent anatomical levels under light microscopy.

(2) Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL): Paraffin sections were prepared as described above. Following our previous protocol [23], sections were incubated with permeabilization solution at 37°C for 8 minutes, washed three times with phosphate-buffered saline (PBS) for 3 minutes each, then incubated with proteinase K at 37°C for 20 minutes. After PBS washing, terminal deoxynucleotidyl transferase (TDT) and dUTP mixture (1:9) was applied at 37°C for 1 hour. Following PBS washes, fluorescent mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) was applied. Apoptotic cells were observed under fluorescence microscopy at consistent tissue levels.

(3) Transmission Electron Microscopy (TEM): According to established protocols [24], fresh gastrocnemius muscle tissues were initially fixed in 2.5% glutaraldehyde for 4 hours, then rinsed three times with 0.01 M PBS for 15 minutes each. Post-fixation was performed with 1% osmium tetroxide for 2 hours. After thorough PBS washing, samples underwent graded acetone dehydration, Epon812 resin infiltration, and programmed polymerization (37°C for 6 hours → 45°C for 12 hours → 63°C for 24 hours). Ultrathin 70 nm sections were prepared, double-stained with uranyl acetate and lead citrate, and examined via transmission electron microscopy to observe ultrastructural features of muscle fibers.

(4) Immunofluorescence Staining (IF): Paraffin sections were prepared as described above. Following our previous protocol [25], after antigen retrieval, sections were sequentially treated with peroxidase blocking (room temperature, 20 minutes) and serum blocking (10% goat serum, room temperature, 20 minutes). Primary antibodies against iNOS, CD206, CD11b, CD3, and CD20 were applied and incubated overnight at 4°C. After washing, fluorescent secondary antibodies were applied (room temperature, 2 hours, protected from light) followed by mounting with DAPI-containing fluorescent medium. Structural changes were observed under fluorescence microscopy at consistent tissue levels.

1.2.6 Transcriptomic Analysis of Mouse Gastrocnemius Muscle

Fresh gastrocnemius muscle samples were obtained and preprocessed. Total RNA was extracted using the RNAprep pure Tissue kit (Cat# DP431, Tiangen Biotech). RNA integrity and quantity were assessed using the Agilent 2100 bioanalyzer system. Qualified total RNA was used for library construction to ensure library quality. Sequencing was performed using qualified libraries to obtain sequence information. Reference sequences and gene annotations were obtained from genomic databases. The reference genome index was constructed using HISAT2 v2.0.5 and aligned with paired-end clean reads. Feature Counts (1.5.0-p3) was used to calculate reads mapped to each gene. DESeq2 (1.20.0) was employed for differential expression analysis between two groups to identify differentially expressed transcripts (DETs), with P<0.05 set as the significance threshold. Transcriptomic data were analyzed for differential expression and enrichment using the OmicShare platform [26].

1.2.7 ELISA Detection of Inflammatory Cytokines in Tissues and Cells

Mouse IL-1β, IL-6, and TNF-α ELISA kits were used according to manufacturer instructions to detect inflammatory cytokine levels in gastrocnemius muscle tissues and RAW264.7 cells.

1.2.8 Detection of Autophagolysosome Levels in Cells

Lysosomal red fluorescent probe kits were used according to manufacturer instructions to detect autophagolysosome levels in C2C12 cells.

1.3 Statistical Analysis

SPSS 13.0 statistical software was used for data analysis. Normally distributed measurement data are expressed as mean ± standard deviation (x̄±s). One-way ANOVA was used for multi-group comparisons, with LSD-t test for pairwise comparisons. GraphPad Prism 9 software was used for data visualization. P<0.05 was considered statistically significant.

Results

2.1 Behavioral Comparisons Among Three Groups of Mice

Significant differences were observed among the three groups in total movement distance in both open field and elevated plus maze tests (P<0.001). The tumor group exhibited reduced movement compared to the control group, while the YQCT group showed increased movement relative to the tumor group (P<0.001) [FIGURE:1, TABLE:1].

TABLE 1 Comparison of Total Movement Distances Among Three Groups of Mice (x̄±s, mm)

Group n Open Field Test Elevated Plus Maze Test Control 10 21,919.10 ± 2,325.29 8,914.00 ± 968.61 Tumor 10 3,018.06 ± 1,189.86^a 1,135.94 ± 222.16^a YQCT 10 20,172.66 ± 3,261.84^b 8,165.38 ± 872.03^b

Note: YQCT = Yiqi Chutan method; ^a indicates P<0.05 vs. control group; ^b indicates P<0.05 vs. tumor group.

2.2 Comparison of Gastrocnemius Muscle Structure Among Three Groups of Mice

HE staining revealed intact muscle structure with compact, orderly arranged muscle fibers in the control group. In contrast, tumor group mice exhibited loose muscle structure with disorganized fiber arrangement. The YQCT group showed alleviated muscle damage compared to the tumor group, with intact and orderly fiber structure and significantly reduced inter-fiber spaces [FIGURE:2]A. TUNEL staining demonstrated significant muscle cell damage in the tumor group compared to controls, with reduced damage in the YQCT group [FIGURE:2]B. TEM analysis revealed marked morphological changes in tumor group skeletal muscle, including disorganized fiber arrangement, widened fiber gaps, and increased mitochondrial autophagosomes. YQCT group showed significant improvement compared to the tumor group, with substantially reduced mitochondrial autophagosomes [FIGURE:2]C. These findings suggest that Yiqi Chutan method can improve skeletal muscle injury, enhance muscle strength and energy reserve capacity, and improve energy metabolism in tumor-bearing mice.

2.3 Comparison of Differentially Expressed Genes in Gastrocnemius Muscle Transcriptome Among Three Groups

Transcriptomic analysis of gastrocnemius muscle tissues showed intra-group homogeneity and inter-group heterogeneity in sample distribution between control vs. tumor and tumor vs. YQCT groups [FIGURE:3]A, 3C. Compared with controls, the tumor group exhibited 642 upregulated and 480 downregulated genes [FIGURE:3]B. Compared with the tumor group, YQCT group showed 1,054 upregulated and 768 downregulated genes [FIGURE:3]D. Notably, 217 differentially expressed genes in the tumor group were reversed by Yiqi Chutan intervention [FIGURE:3]E. The bidirectional clustering heatmap of these 217 genes [FIGURE:3]F revealed similar expression trends between control and YQCT groups, contrasting with the opposite trend in the tumor group.

2.4 Enrichment Analysis of Differentially Expressed Genes in Gastrocnemius Muscle

GO enrichment analysis of the 217 differentially expressed genes revealed enrichment in biological processes and molecular functions including regulation of leukocyte proliferation, immune system processes, regulation of lymphocyte proliferation, regulation of mononuclear cell proliferation, positive regulation of cytokine production, mitochondrial inner membrane protein complexes, and cytokine binding [FIGURE:4]A. KEGG pathway enrichment analysis showed enrichment in oxidative phosphorylation, complement and coagulation cascades, and notably, the immune system [FIGURE:4]B. Reactome pathway enrichment analysis demonstrated enrichment in innate immune system, neutrophil degranulation, respiratory electron transport, and exocytosis of secretory granule membrane proteins [FIGURE:4]C.

2.5 Immunofluorescence Staining Analysis of Gastrocnemius Muscle

Immunofluorescence staining revealed significantly increased infiltration of M1 (pro-inflammatory) macrophages, neutrophils, T lymphocytes, and B lymphocytes in tumor group gastrocnemius muscle compared to controls. In contrast, YQCT group showed significantly increased M2 (anti-inflammatory) macrophage infiltration and decreased pro-inflammatory cell infiltration compared to the tumor group [FIGURE:5].

2.6 Comparison of IL-1β, IL-6, and TNF-α Levels in Gastrocnemius Muscle Among Three Groups

ELISA results demonstrated significant differences in IL-1β, IL-6, and TNF-α levels among the three groups (P<0.001). Tumor group exhibited higher levels of these cytokines compared to controls, while YQCT group showed significantly lower levels compared to the tumor group (P<0.001) [TABLE:2].

TABLE 2 Comparison of IL-1β, IL-6, and TNF-α Levels Among Three Groups of Mice (x̄±s, ng/L)

Group IL-1β IL-6 TNF-α Control 2.17 ± 0.42 11.10 ± 1.45 17.07 ± 1.22 Tumor 11.93 ± 2.36^a 29.30 ± 5.31^a 33.23 ± 2.98^a YQCT 4.17 ± 1.30^b 12.40 ± 1.60^b 16.37 ± 3.31^b

Note: IL = interleukin, TNF = tumor necrosis factor; ^a indicates P<0.05 vs. control group; ^b indicates P<0.05 vs. tumor group.

2.7 Comparison of IL-1β, IL-6, and TNF-α Levels in Cell Models

Using LPS to activate RAW264.7 cells, we established an inflammatory cell activation model. Significant differences in IL-1β, IL-6, and TNF-α levels were observed among the three cell groups (P<0.001). RAW264.7-LPS group exhibited higher cytokine levels than controls, while YQCT group showed significantly lower levels than RAW264.7-LPS group (P<0.05) [TABLE:3].

TABLE 3 Comparison of IL-1β, IL-6, and TNF-α Levels Among Three Cell Models (x̄±s, ng/L)

Group IL-1β IL-6 TNF-α RAW264.7 36.87 ± 3.92 44.92 ± 8.22 83.50 ± 9.75 RAW264.7-LPS 62.00 ± 3.83^a 1,249.20 ± 107.96^a 328.80 ± 35.60^a YQCT 36.93 ± 3.07^b 132.37 ± 63.47^b 129.57 ± 28.25^b

Note: ^a indicates P<0.05 vs. RAW264.7 group; ^b indicates P<0.05 vs. RAW264.7-LPS group.

2.8 Comparison of Autophagolysosome Levels in Cell Models

Co-culture experiments with RAW264.7 and C2C12 cells demonstrated that activated RAW264.7 cells induced increased autophagolysosome formation in C2C12 cells, indicating that pro-inflammatory cell infiltration directly causes muscle cell injury. After Yiqi Chutan intervention to modulate RAW264.7 cell activation, C2C12 cell damage was significantly attenuated [FIGURE:6].

Discussion

From the perspective of traditional Chinese medicine theory, lung cancer pathogenesis manifests as deficiency in the root and excess in the branch, with lung-spleen qi deficiency constituting the pathological foundation that influences the entire disease course, while the branch excess is characterized by "phlegm-toxin." Phlegm-turbidity accumulation can promote tumor formation, and tumor progression further exacerbates phlegm-turbidity generation, creating a dual pathological attribute as both causative factor and pathological result that forms a vicious cycle. Phlegm evil obstructing the viscera and blocking qi flow can induce various secondary pathological changes [27]. Long-standing cancer with phlegm evil invading the muscle exterior leads to impaired qi flow and abnormal distribution of essential substances, damaging the muscular striae. Therefore, an etiological association exists between tumor progression and tumor-related skeletal muscle injury. The eight-medicinal combination in Yiqi Chutan method, dominated by the "cultivating earth to generate metal" approach, restores transportation and transformation functions through spleen-strengthening and qi-tonifying, promotes qi-blood generation, and possesses the effects of diffusing lung qi, resolving phlegm, and detoxifying and dispersing nodules, precisely addressing the etiology and pathogenesis of tumor-related skeletal muscle injury. Elucidating the therapeutic mechanism of Yiqi Chutan method for this condition holds significant importance for guiding clinical medication.

Modern medical research indicates that tumor growth and treatment generate substantial inflammatory factors and mediators [28], which enter systemic circulation and create a hyper-inflammatory state that may be an important trigger for tumor-related adverse effects [29]. Previous studies have confirmed that tumor-induced chronic inflammation leads to symptoms such as fatigue [30]. Furthermore, research demonstrates that prolonged muscle exposure to high inflammatory infiltration environments causes abnormal energy metabolism in muscle cells, ultimately damaging myocytes [17]. Normal energy metabolic function is fundamental for maintaining motor function. This study found that during tumor progression, skeletal muscle pro-inflammatory cell infiltration and inflammatory cytokine levels were significantly higher than in non-tumor-bearing states, causing inflammatory injury to skeletal muscle cells and ultimately impairing motor function in tumor-bearing mice.

Under the guidance of TCM Yiqi Chutan theory, this study confirmed that the Yifei Sanjie pill formulation can promote M2 macrophage polarization in skeletal muscle of tumor-bearing mice, reduce pro-inflammatory cell infiltration and pro-inflammatory cytokine levels, effectively alleviate tumor-related skeletal muscle injury caused by chronic hyper-inflammatory states, and preserve normal muscle function. Currently, clinical treatment for severe skeletal muscle injury with intense pain typically involves symptomatic analgesics such as ibuprofen sustained-release capsules or diclofenac sodium sustained-release tablets. However, these medications can reduce muscle growth rate and slow muscle injury recovery [31]. Similar to these reports, our study found that Yiqi Chutan method also alleviates tumor-related skeletal muscle injury by reducing inflammatory infiltration, suggesting potential synergistic and toxicity-reducing effects with conventional clinical treatments. Based on these findings, this study not only preliminarily reveals the pathological mechanisms of tumor-related skeletal muscle injury but also proposes an effective and safe therapeutic strategy that may address current treatment gaps and provide a potential approach for comprehensive cancer patient management.

Notably, due to intestinal barrier limitations, most components of Yifei Sanjie pills cannot enter the bloodstream to exert direct therapeutic effects, and the formulation may possess multiple pharmacological mechanisms involving multiple components and targets. Therefore, future studies should employ advanced big data artificial intelligence methods such as artificial neural networks, DeepSeek, and ChatGPT to screen for high-potential lead compounds from Yifei Sanjie pills to support subsequent pharmacological validation and drug development. Additionally, while Yifei Sanjie pills have been primarily used clinically for lung cancer treatment, their application in relieving tumor-related side effects lacks extensive clinical evidence. Thus, future clinical studies are necessary to further clarify the clinical potential of Yifei Sanjie pills.

In summary, Yiqi Chutan method may reduce tumor burden-induced skeletal muscle tissue inflammatory infiltration by promoting M2 macrophage polarization, thereby alleviating inflammatory injury to skeletal muscle cells, preserving normal muscle function, and ultimately relieving tumor-induced fatigue and other adverse symptoms. This study clarifies the efficacy of Yifei Sanjie pills in alleviating tumor-related skeletal muscle injury and confirms the effectiveness of treatment guided by TCM Yiqi Chutan theory. These findings provide basic research evidence for expanding the clinical application of Yifei Sanjie pills.

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Acknowledgments: The authors thank the funding agencies for their support.

Author Contributions: Yingchao Wu designed the study and wrote the manuscript; Ziqian Luo performed animal experiments; Zhongjia Yi conducted cell experiments; Dajin Pi collected and organized data; Jiaqi Cui performed statistical analysis; Lizhu Lin provided TCM theoretical guidance; Mingzi Ouyang revised the manuscript; Qianjun Chen was responsible for quality control and overall manuscript supervision.

Conflict of Interest: The authors declare no conflicts of interest.

Received: April 8, 2025; Revised: May 29, 2025; Accepted: [Epub ahead of print]

Correspondence to: MINGZI OUYANG, E-mail: mingzioy@jnu.edu.cn; QIANJUN CHEN, E-mail: cqj55@163.com