Construction of Lung Adenocarcinoma Xenograft Model in Mice and Evaluation of the Efficiency of XY Decoction through MAPK Pathway

This study demonstrates that XY decoction exerts therapeutic effects against lung adenocarcinoma by regulating the MAPK pathway.

Lung adenocarcinoma is increasing worldwide, prompting the exploration and development of effective treatment methods. In recent years, the therapeutic potential of traditional Chinese medicine for lung adenocarcinoma, such as the XiaoYi (XY) decoction, has been increasingly recognized. The mechanism of action of traditional Chinese medicine in treating lung adenocarcinoma continues to be elucidated. In this study, animal models were used to investigate the therapeutic effects of XY decoction in the treatment of lung adenocarcinoma. The blood-entry chemical components of XY decoction in mice were separated and analyzed in both positive and negative ion modes using UHPLC-QE-MS. The findings were then validated through in vivo experiments, which demonstrated reduced tumor size, improved pathological changes, and increased apoptosis. As a result, fifteen components absorbed into the blood following XY decoction administration were identified: Stachyose, Quercetin-3-O-beta-glucopyranosyl-7-O-alpha-rhamnopyranoside; Imperialine; Spinosin B; Peimine; Flavone base + 3O, 2MeO, O-Hex; Picropodophyllotoxin; (2S,3R,4S,5S,6R)-2-[(2R,3R,4S,5S,6R)-4,5-dihydroxy-2 [[(3S,5R,8R,10R,12R,13R,14R,17S)-12-hydroxy-17-(2-hydroxy-6-methylhept-5-en-2-yl) 4,4,8,10,14-pentamethyl-2,3,5,6,7,9,11,12,13,15,16,17-dodecahydro-1H cyclopenta[a]phenanthren-3-yl]oxy]-6-(hydroxymethyl)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol; Tricin; Ginsenoside Rg1; (20R)-Ginsenoside Rh1; Jujuboside B; Aucubin; Ginsenoside Rg3; and beta-Elemonic acid. Animal experiments demonstrated that XY decoction inhibited tumor growth, improved pathological changes, and promoted tumor cell apoptosis in a dose-dependent manner. The mechanisms involved may be related to the upregulation of p-JNK and p-P38 protein expression in the MAPK pathway and the downregulation of p-ERK protein expression in the MAPK pathway. In conclusion, XY decoction has the potential to regulate the MAPK pathway and exert therapeutic effects against lung adenocarcinoma.

Lung cancer incidence is rising worldwide. It accounts for 18% of all malignancies and is responsible for a significant number of cancer-related deaths^1,2. Non-small cell lung cancer (NSCLC) is a prevalent form of lung cancer, with adenocarcinoma as the main pathological type³. Due to its insidious symptoms, NSCLC is often diagnosed at an advanced stage and has a high mortality rate^4,5. Stage IV NSCLC patients have a five-year survival rate of only 5%⁶, while even surgically resectable early-stage NSCLC has a five-year survival rate of just 50%⁷.

Currently, the treatment of lung adenocarcinoma includes surgery, radiotherapy, chemotherapy, immunotherapy, and targeted therapy. Additionally, traditional Chinese medicine (TCM) for lung adenocarcinoma is increasingly being recognized and valued⁸. TCM has a long history and has been used in the treatment of various oncological diseases with effective therapeutic outcomes^9,10. It regards the human body as a whole entity, with the therapeutic principle focused on restoring the movement of Qi (Qi refers to the vital energy that promotes the circulation of blood and body fluids, contributing to enhanced immunity and reduced inflammation in NSCLC patients) in internal organs to restore overall balance and inhibit cancer progression.

TCM not only treats oncological diseases with low toxicity, multiple targets, and high efficiency¹¹, but it also facilitates the repair of damage caused by radiotherapy and chemotherapy, thereby prolonging survival¹².

Liquid chromatography-mass spectrometry (LC-MS) technology integrates liquid chromatography with mass spectrometry to achieve the separation and detection of components in complex compound mixtures¹³. LC-MS offers high resolution, high sensitivity, and high specificity, allowing for the separation and identification of various chemical components in traditional Chinese medicine (TCM) decoctions. When combined with serum medicinal chemistry, which separates and detects blood-entry active components after oral administration^14,15, LC-MS enables the prediction and analysis of potential drug action mechanisms. This approach helps in understanding the chemical composition and pharmacological components of TCM decoctions, providing essential data for in-depth research on their pharmacological effects and clinical applications, making it highly applicable in TCM research^16,17.

The MAPK signaling pathway plays a crucial role in the genesis, progression, metastasis, and invasion of lung cancer, with Erk, p38, and JNK as important subfamilies. TCM decoctions have been shown to influence the expression of proteins in the MAPK pathway¹⁸. XiaoYi (XY) decoction is derived from YiGuan decoction and is used to treat lung adenocarcinoma at the First Affiliated Hospital of Changchun University of Chinese Medicine (Figure 1), demonstrating precise clinical efficacy. However, its therapeutic mechanism remains unclear.

In this study, ultra-high-performance liquid chromatography coupled with Q-Exactive Orbitrap mass spectrometry (UHPLC-QE-MS) was employed to elucidate the possible therapeutic mechanism of XY decoction against lung adenocarcinoma, and its effects were validated through in vivo studies.

All animal procedures were approved by the Animal Ethics Committee of Changchun University of Traditional Chinese Medicine (Ethics No. 2024008). Six-week-old C57BL/6 male mice (20 ± 2 g), bred under specific pathogen-free (SPF) conditions, exhibited typical physical activity patterns in a controlled environment with constant temperature and humidity. They had ad libitum access to food and water and were maintained under a 12-h light/dark cycle. Details of the reagents and equipment used in this study are provided in the Table of Materials.

1. Preparation of XY decoction aqueous extract

1. Place Panax ginseng C.A. Mey (Renshen, 15 g), Chinemys reevesii (Gray) (Guiban, 30 g), and Trionyx sinensis Wiegmann (Biejia, 30 g) into a decoction pot. Add 1000 mL of deionized water and soak for 30 min at room temperature.
2. Boil the three herbs from step 1.1 for 1 h. Simultaneously, soak Rehmannia glutinosa Libosch (Dihuang, 30 g), Trichosanthes kirilowii Maxim. (Tianhuafen, 30 g), Coix lacryma-jobi L. var. mayuen (Roman.) Stapf (Yiyiren, 20 g), Paeonia lactiflora Pall. (Baishao, 30 g), Boswellia carterii Birdw. (Ruxiang, 7 g), Commiphora myrrha Engl. (Moyao, 7 g), Agrimonia pilosa Ledeb. (Xianhecao, 20 g), Alpinia oxyphylla Miq. (Yizhiren, 20 g), Ziziphus jujuba Mill. var. spinosa (Bunge) (Suanzaoren, 30 g), Melia toosendan Sieb. et Zucc. (Chuanlianzi, 5 g), Fritillaria thunbergii Miq. (Zhebeimu, 15 g), Anemarrhena asphodeloides Bge. (Zhimu, 10 g), Platycodon grandiflorum (Jacq.) (Jiegeng, 10 g), Polygala tenuifolia Willd. (Yuanzhi, 15 g), and Phragmites communis Trin. (Lugen, 30 g) in another container for 30 min. After soaking, transfer them to the decoction pot from step 1.1.
3. Boil all 18 herbs from steps 1.1 and step 1.2 for 30 min. Pour out the herbal liquid, add 1000 mL of deionized water, and boil for another 30 min. Repeat this boiling process three times to obtain three portions of herbal liquid, then combine and mix them thoroughly.
4. Filter the mixed herbal liquid through a 400-mesh cloth. Concentrate the filtrate under reduced pressure, then transfer the concentrated herbal liquid to cooling equipment for further processing.
5. Pour the concentrated herbal liquid into a lyophilization tray and place it in the pre-freezing chamber of a vacuum lyophilizer. Once pre-freezing is complete, transfer the tray to the sublimation drying chamber to remove water.
6. Remove any residual moisture at a high temperature. Pulverize the freeze-dried herbal extract using a pulverizer to obtain fine lyophilized XY decoction powder (Figure 2).

2. Drug-containing serum preparation

1. Randomly assign six-week-old C57BL/6 male mice (20 ± 2 g) into either the XiaoYi (XY) group or the control group.
   NOTE: The mouse dose was calculated using the human-to-mouse conversion factor:
   Equivalent experimental dose for mice (g/kg) = {Human dose (g/kg) / Body weight (70 kg)} x 9.1
   Based on this calculation, the dosage for mice was determined to be 2.6 g/kg.
2. Weigh 0.9152 g of lyophilized XY decoction powder and dissolve it in 3.2 mL of distilled water to prepare the drug solution.
   NOTE: Mice were fasted for 12 h before oral administration.
3. Administer the XY decoction to the mice in the XY group via gavage, while administering the control group with an equivalent volume of deionized water.
4. 2 h after oral administration, anesthetize the mice with pentobarbital sodium (following institutionally approved protocols). Enucleate the eyeballs using tweezers to collect blood samples, then euthanize the mice via CO₂ asphyxiation followed by cervical dislocation following ethical guidelines¹⁹.
5. Allow the blood samples to stabilize at room temperature for 90 min. Centrifuge at 3450 x g (4 °C) for 15 min to collect the supernatant.

3. UHPLC-QE-MS analysis

1. Add 100 mg of XY decoction lyophilized powder to 500 µL of extraction solution (methanol: water = 4:1). Mix thoroughly, sonicate, and incubate in an ice-water bath for 1 h.
2. Maintain the mixture at -40 °C for 1 h, then centrifuge at 13,800 x g (4 °C) for 15 min. Filter the supernatant through a 0.22 µm filter membrane, then combine 100 µL of each supernatant to create quality control (QC) samples.
3. After thawing the plasma samples, take 400 µL of the control group plasma sample and 400 µL of the XY group plasma samples. Add 40 µL of hydrochloric acid (2 mol/L) to each sample. Vortex for 1 min and let the mixture stand at 4 °C for 15 min. Repeat this step four times.
4. Add 1.6 mL of acetonitrile and vortex for 5 min. Centrifuge at 13,800 x g for 5 min at 4 °C. Subject 1,800 µL of the supernatant to nitrogen drying.
5. Reconstitute the dried samples in 150 µL of 80% methanol containing 10 µg/mL of the internal standard by vortexing for 5 min. Centrifuge at 13,800 × g for 5 min at 4 °C, then transfer 120 µL of the supernatant to a fresh glass vial for LC-MS analysis (Figure 3).

4. Cell culture and animal preparation

1. Seed 1 mL of Lewis lung carcinoma cells in a culture dish containing Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Incubate the cells under controlled conditions at 37 °C with 5% CO₂.
2. When the cells reach 80% confluence, passage them using 0.25% trypsin. Continue passaging until the cell state stabilizes, then adjust the cell concentration to 3 x 10⁶ cells/mL.
3. Inject 0.1 mL of the prepared cell suspension subcutaneously into the right axilla of male C57BL/6 mice to establish the lung adenocarcinoma model.
   NOTE: Tumor tissue can be palpated in the axilla of mice 5 days after inoculation.
4. After 5 days, randomly divide the mice into five groups (n = 6 per group): Model group (M) - No treatment; XY decoction low-dose group (XY-L) - 1.3 g/kg; XY decoction medium-dose group (XY-Z) - 2.6 g/kg; XY decoction high-dose group (XY-H) - 5.2 g/kg; Cisplatin group (DDP) - Treated with cisplatin²⁰.
5. Dissolve the XY decoction powder in 200 µL of distilled water at the corresponding dose and administer it via gavage. The M and DDP groups receive an equal volume of distilled water via gavage.
6. Administer cisplatin to the DDP group via intraperitoneal injection (3 mg/kg) once every 3 days. Inject the same volume of physiological saline into the other groups.

5. Pathological analysis of tumor tissue

1. After 21 days, euthanize all mice using CO₂ asphyxiation followed by cervical dislocation (following ethical guidelines) and excise the tumor tissue²¹. Measure the tumor volume and weight (Figure 4).
2. Fix the excised tumor tissues in 4% paraformaldehyde for 24 h). Dehydrate the tissue using graded ethanol solutions.
3. Clear the dehydrated tissue blocks using xylene, then embed them in melted paraffin wax. Allow the wax to solidify into a paraffin block. Section the tissue block into 5 µm-thick slices using a microtome.
4. Dewax the tissue sections in xylene, followed by rehydration in graded ethanol solutions. Stain the sections with hematoxylin for 5 min, then rinse with deionized water.
5. Counterstain the sections with 0.5% eosin solution for 1 min. Observe the stained sections under an optical microscope and capture images for analysis.

6. Immunohistochemical analysis of tumor tissue

1. Perform dewaxing and rehydration following the procedures described in steps 5.2-5.4.
2. Incubate the sections in an endogenous peroxidase blocker at room temperature for 15 min. Block non-specific binding by incubating the sections with goat serum.
3. Apply the primary antibodies: Ki-67 (1:600) and Caspase-3 (1:100). Incubate the sections overnight at 4 °C. Wash with PBS buffer), then apply a general-purpose secondary antibody (1:500) and incubate at 37 °C for 30 min.
4. Develop the color reaction by adding DAB solution. Counterstain the sections with hematoxylin solution.
5. Perform dehydration, clearing, and sealing of the stained sections. Observe protein expression under an optical microscope and capture images for analysis.

7. Western blot analysis

1. Homogenize the tumor tissues using a tissue lyser. Extract the total protein and determine its concentration using a bicinchoninic acid (BCA) assay kit (see Table of Materials).
2. Perform protein quantification, followed by SDS-PAGE electrophoresis and protein transfer onto a membrane.
3. Incubate the membrane overnight at 4 °C with primary antibodies: P38 (1:2000), p-P38 (1:1000), ERK (1:20000), p-ERK (1:500), JNK (1:2000), p-JNK (1:5000), β-actin (1:200) (see Table of Materials).
4. The next day, incubate the membrane with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1.5 h. Detect protein bands using an enhanced chemiluminescence (ECL) system.
   NOTE: Protein band intensities were quantified using ImageJ software.

XY powder solution and serum samples from the control group and the XY group were analyzed using UHPLC-QE-MS, revealing well-separated peaks. The total ionization diagrams for the positive and negative ion modes are shown in Figure 3. The composition of the XY decoction was identified based on retention time, acquisition mode, and mass spectrum fragments. Fifteen blood-entry components were identified by comparing the serum of the blank and XY decoction oral administration groups with the components of the XY powder solution (Table 1).

The tumor volume line graph indicates that after 21 days of gavage with XY decoction, the tumor volume in mice decreased compared to the M group and exhibited a dose-dependent effect (Figure 4). Tumor histopathology was observed using H&E staining (Figure 5). In the M group, tumor cells were densely arranged and exhibited active proliferation, with abundant pathological mitosis. As the XY decoction dose increased, the arrangement of tumor cells gradually became sparse, and intercellular space increased. Various degrees of necrosis were observed in tumor tissues, and pathological mitosis was rare²¹.

To verify the effects of XY decoction on the proliferation and apoptosis of tumor tissues in mice, immunohistochemistry (IHC) experiments were conducted. After staining with Ki-67 and Caspase 3 antibodies, scattered brownish-yellow particles were observed in the tumor tissue sections. Compared with the M group, Ki-67 levels in tumor tissues of the XY and DDP groups decreased sequentially, whereas Caspase 3 levels increased (Figure 6).

To determine whether XY decoction exerts therapeutic effects on lung adenocarcinoma through the MAPK pathway, Western blot analysis was performed on tumor tissues from mice. As shown in Figure 7, treatment with XY decoction led to a decrease in p-ERK expression, with the XY-H group showing a significant difference compared to the M group (p < 0.001). The levels of p-JNK and p-P38 increased in the XY-H group compared to the M group, with statistically significant differences observed in the XY-H group (p < 0.01) and the DDP group (p < 0.05) compared to the M group. Additionally, the difference between the DDP and XY-H groups was statistically significant.

Figure 1: Components of XY decoction. This figure presents the individual drugs contained in XY decoction. Please click here to view a larger version of this figure.

Figure 2: Preparation of XY decoction aqueous extract. This figure illustrates the preparation process of the aqueous extract of XY decoction. Please click here to view a larger version of this figure.

Figure 3: Analysis of XY decoction aqueous extract. (A) The extraction process of drug-containing serum. (B) Mass spectrometry analysis in positive ion mode. (C) Mass spectrometry analysis in negative ion mode. Please click here to view a larger version of this figure.

Figure 4: Effects of XY decoction on mouse tumor volume and morphology. (A) Cell culture, tumor implantation, and sampling procedures. (B) Line graph depicting tumor volume over time. (C) Tumor morphology after 21 days of gavage treatment. Please click here to view a larger version of this figure.

Figure 5: Histological analysis of mouse tumor tissues. (A) Tumor tissue arrangement becoming sparser with increasing doses of XY decoction, with signs of necrosis in a dose-dependent manner. (B) Pathological mitosis in tumor tissues, showing a decrease in pathological mitosis with increasing doses of XY decoction. Scale bars: 200 µm. Please click here to view a larger version of this figure.

Figure 6: Ki-67 and Caspase-3 expression levels. (A) Ki-67 expression levels. (B) Caspase-3 expression levels. Scale bars: 400 µm. (C) Quantification of Ki-67-positive areas, showing a decrease with increasing doses of XY decoction. (D) Quantification of Caspase-3-positive areas, showing an increase with increasing doses of XY decoction. ***p < 0.001, ****p < 0.0001; ns, not significant. Please click here to view a larger version of this figure.

Figure 7: Protein expression levels in the MAPK pathway. (A) Western blot analysis of protein expression. (B) ERK expression levels. (C) p-ERK expression levels. (D) JNK expression levels. (E) p-JNK expression levels. (F) P38 expression levels. (G) p-P38 expression levels. XY decoction upregulated the expression of p-JNK and p-P38 proteins while downregulating p-ERK expression. *p < 0.05, **p < 0.01, ***p < 0.001. Please click here to view a larger version of this figure.

Table 1: Components absorbed into the blood following XY decoction administration. The table lists the active components detected in the blood after administration of XY decoction: A: Rehmannia glutinosa Libosch (Dihuang), B: Fritillaria thunbergii Miq. (Zhebeimu), C: Trichosanthes kirilowii Maxim. (Tianhuafen), D: Panax ginseng C.A. Mey (Renshen), E: Boswellia carterii Birdw. (Ruxiang), F: Ziziphus jujuba Mill. var. spinosa (Bunge) (Suanzaoren), G: Phragmites communis Trin. (Lugen).

The incidence of cancer is rising annually, attracting significant clinical and research interest^22,23. Recent studies have demonstrated the effectiveness of traditional Chinese medicine (TCM) in treating lung cancer^24,25,26. For instance, Maimendong decoction has been shown to enhance the number and activity of NK cells, thereby inhibiting lung cancer metastasis²⁷. Similarly, Mahuang Fuzi Xixin decoction has achieved therapeutic effects on lung cancer by influencing multiple signaling pathways²⁸. This study utilizes LC-MS analysis and in vivo experiments to predict the possible mechanisms by which XY decoction exerts therapeutic effects on lung adenocarcinoma.

LC-MS analysis identified 15 blood-entry components. Among these, tricin and picropodophyllotoxin have been shown to inhibit drug resistance and reduce the expression levels of p-PRKCA, SPHK1, SPHK2, and EGFR, thereby suppressing lung cancer cell activity^29,30. Ginsenoside Rg1 and Ginsenoside Rg3, components of Panax ginseng C.A. Meyer, can block cell mitosis, inhibit mTOR pathway signaling, exert anti-angiogenic effects, enhance immune function, and ultimately inhibit tumor growth and metastasis while reducing drug resistance^31,32,33,34. Stachyose can induce apoptosis in intestinal cancer cells by modulating the expression of pro-apoptotic proteins³⁵. Imperialine, an alkaloid with anti-inflammatory and anti-proliferative properties, has potential as an early-stage antitumor agent³⁶. Peimine can arrest the cell cycle by inducing apoptosis, inhibit cell migration through endoplasmic reticulum stress, and regulate NF-κB pathway-related protein expression to suppress the growth of breast and gastric cancer cells^37,38. Aucubin and Jujuboside B have been reported to promote autophagy and apoptosis in tumor cells while inhibiting angiogenesis, thereby exerting antitumor effects^39,40,41.

The MAPK pathway is essential for various biological processes, including cell activation, proliferation, autophagy, and apoptosis. Several compounds detected in the blood-entry components of XY decoction, such as Ginsenoside Rg1, Ginsenoside Rg3, Peimine, and Jujuboside B, may exert antitumor effects through the MAPK pathway^42,43,44,45. The MAPK pathway involves the ERK, JNK, and P38 signaling cascades and is widely studied in cancer research due to its role in regulating physiological processes through the protein cascade activation of MAP3K-MAP2K-MAPK^46,47.

Activation of the ERK pathway stimulates cell proliferation and inhibits apoptosis by regulating the activity and expression of pro-apoptotic and anti-apoptotic proteins at the transcriptional and translational levels^48,49. Protein inhibitors targeting the ERK pathway have been extensively studied and are used in cancer treatment^50,51,52. Traditional Chinese medicine (TCM) monomers and formulations have also been shown to exert antitumor effects by modulating the ERK pathway. For example, the QYHJ formula inhibits the proliferation and promotes apoptosis of pancreatic ductal adenocarcinoma cells by regulating the expression of p-P38 and p-ERK1/2⁵³. Ferulic acid derivatives suppress proliferation, arrest the cell cycle, and induce apoptosis in the A549 human lung cancer cell line⁵⁴.

The JNK/P38 pathway plays distinct roles by regulating different downstream effector proteins through phosphorylation, thereby influencing the cell cycle, cell proliferation, and apoptosis^55,56. Studies have shown that triptolide modulates the expression of p-P38 and p-JNK and inhibits the proliferation of A549 cells, exerting antitumor effects⁵⁷. Additionally, baicalein induces apoptosis in HeLa cells by affecting p-P38 and p-JNK expression⁵⁸. In vivo and in vitro studies have demonstrated that Aidi injection influences the expression of p-JNK and p-P38 in HepG2 cells and hepatocellular carcinoma mouse models, thereby inhibiting tumor proliferation⁵⁹.

Ki-67 and Caspase 3 are key indicators of tumor proliferation and apoptosis. Ki-67 is a nuclear protein that is highly expressed during cell proliferation and is absent in quiescent cells (G0 phase)⁶⁰. Since tumor development is closely linked to cell proliferation, research on the correlation between Ki-67 and tumorigenesis has gained significant attention. Ki-67 expression is positively correlated with tumor malignancy, aggressiveness, and prognosis^61,62, making it a widely used biomarker for cancer proliferation⁶³. It has been extensively studied in the pathogenesis and prognosis of gastrointestinal, lung, breast, and cervical cancers^64,65,66,67.

The caspase family consists of proteolytic enzymes that regulate apoptosis and inflammation. Caspase 3, primarily found in the cytoplasm, is a key effector of apoptosis. It facilitates cell death by cleaving cytoskeletal proteins, inactivating apoptosis-inhibitory proteins, and degrading DNA repair enzymes^68,69,70. Alterations in Caspase 3 expression can influence tumor cell sensitivity to chemotherapy, thereby affecting invasion, metastasis, and the progression of various cancers^71,72,73,74.

This study demonstrated that the XY decoction inhibited tumor proliferation in LLC tumor-bearing mice in a dose-dependent manner. Pathological analysis of tumor tissues from the XY and DDP treatment groups revealed looser cell arrangements compared to the M group, along with reduced pathological mitosis. Additionally, necrosis and hemorrhage were observed in the tumor tissues of the treatment groups. Immunohistochemistry (IHC) results further confirmed that the XY decoction inhibited tumor cell proliferation and promoted apoptosis in a dose-dependent manner. Western blot analysis of tumor tissues indicated significant changes in the expression levels of key proteins in the MAPK pathway. Overall, the pathological and IHC findings suggest that the XY decoction exerts therapeutic effects against lung adenocarcinoma by modulating the expression of proteins in the MAPK signaling pathway.

The authors have nothing to disclose.

This work was supported by the Jilin Province Science and Technology Development Plan Item (YDZJ202301ZYTS459).