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  • Riepilogo
  • Abstract
  • Introduzione
  • Protocollo
  • Risultati
  • Discussione
  • Divulgazioni
  • Riconoscimenti
  • Materiali
  • Riferimenti
  • Ristampe e Autorizzazioni

Riepilogo

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

Abstract

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.

Introduzione

Lung cancer incidence is rising worldwide. It accounts for 18% of all malignancies and is responsible for a significant number of cancer-related deaths1,2. 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 rate4,5. 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 outcomes9,10. 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 administration14,15, 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 research16,17.

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.

Protocollo

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.

Risultati

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 ...

Discussione

The incidence of cancer is rising annually, attracting significant clinical and research interest22,23. Recent studies have demonstrated the effectiveness of traditional Chinese medicine (TCM) in treating lung cancer24,25,26. For instance, Maimendong decoction has been shown to enhance the number and activity of NK cells, thereby inhibiting lung cancer metastasis

Divulgazioni

The authors have nothing to disclose.

Riconoscimenti

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

Materiali

NameCompanyCatalog NumberComments
AcetonitrileCNW Technologies5/8/1975
2-Chloro-L phenylalanineShanghai Hengbo Biotechnology Co., Ltd.103616-89-3
Ammonium Hydroxide SolutionChina National Pharmaceutical Group Chemical Reagent Co., Ltd.#2028
Antibody-β-actinAbcamab8228
Anti-Erk1 (pT202/pY204) + Erk2 (pT185/pY187) AntibodyAbcamab4819
Anti-ERK1/ERK2 AntibodyAbcamab184699
Anti-JNK1 (pY185) + JNK2 (pY185) + JNK3 (pY223) AntibodyAbcamab76572
Anti-JNK1/JNK2/JNK3 AntibodyAbcamab208035
Anti-Ki67 AntibodyCell Signaling TechnologyGB121141
Anti-p38 (phospho T180) AntibodyAbcamab178867
Anti-p38 alpha/MAPK14 AntibodyAbcamab170099
BCA protein concentration assay kitBoster Biological Technology17E17B46
Bovine Serum Albumin (BSA)DAKOBIOFROXX
Broad-spectrum phosphatase inhibitor (100x)Boster Biological TechnologyAR1183
C57bl/6 miceLiaoning Changsheng Biotechnology Co., Ltd.SCXK2021-0006
Chromatographic columnWatersACQUITY UPLC BEH C18 1.7 μm2.1*100 mm
CisplatinChina National Pharmaceutical Group Chemical Reagent Co., Ltd.232120
Citrate (pH 6.0) Antigen Retrieval SolutionWuhan Baiqiandu Biotechnology Co., Ltd.B2010
Cover SlipJiangsu Shitai Laboratory Equipment Co., Ltd.10212432C
Cryogenic StageWuhan Junjie Electronics Co., Ltd.JB-L5
DAB Chromogenic KitJiangsu Shitai Laboratory Equipment Co., Ltd.2005289
Decolorization shakerBeijing Liu Yi Instrument FactoryWD-9405A
DehydratorWuhan Junjie Electronics Co., Ltd.JJ-12J
Drying OvenShanghai Huitai Instrument Manufacturing Co., Ltd.DHG-9140A
Dulbecco's modified eagle mediumGibco8121587
EDTA (pH 8.0) Antigen Retrieval SolutionWuhan Baiqiandu Biotechnology Co., Ltd.B2001
EDTA (pH 9.0) Antigen Retrieval SolutionWuhan Baiqiandu Biotechnology Co., Ltd.B2002
Electrophoresis bufferNCM Biotech20230801
Embedding machineWuhan Junjie Electronics Co., Ltd.JB-P5
EosinChina National Pharmaceutical Group Chemical Reagent Co., Ltd.212954
EthanolChina National Pharmaceutical Group Chemical Reagent Co., Ltd.493511
Fetal bovine serumGibco2166090RP
Formic acidSIGMA64-18-6
GraphPad Prism softwareGraphPad software, LLCVersion 9.0.0
HematoxylinChina National Pharmaceutical Group Chemical Reagent Co., Ltd.H3136
High performance liquid chromatographyAgilent1260 Infinity II Prime
High speed refrigerated centrifugeThermo Fisher ScientificLegend Micro 17R
High-Resolution Mass SpectrometryThermo Fisher ScientificQ Exactive Focus
HRP-conjugated Goat Anti-MouseSeraCare5220-0341
HRP-conjugated Goat Anti-RabbitSeraCare5220-0336
HRP-conjugated Goat Anti-Rabbit/Mouse Universal Secondary AntibodyDAKOK5007
HRP-conjugated Goat Anti-RatSeraCare5220-0364
HRP-conjugated Rabbit Anti-GoatSeraCare5220-0362
Hydrochloric acidChina National Pharmaceutical Group Chemical Reagent Co., Ltd.H9892
Hydrogen PeroxideChina National Pharmaceutical Group Chemical Reagent Co., Ltd.B12555
Hypersensitive ECL chemiluminescence kitNCM BiotechP10300
ImageJ softwareNational Institutes of Healthv1.8.0
Imaging SystemNikon (Japan)NIKON DS-U3
Lewis lung carcinoma cellsChina National Pharmaceutical Group Chemical Reagent Co., Ltd.90020104
Mesh clothBaijie100110779650
Metal heating temperature control instrumentBaiwan Electronictechnology Co., Ltd.HG221-X3
MethanolAladdinM116118
MicropipetteDlab Scientific Co., Ltd.KE0003087/KA0056573
Microscope SlideJiangsu Shitai Laboratory Equipment Co., Ltd.10212432C
MicrotomeShanghai Leica Instruments Co., Ltd.RM2016
Microwave OvenGalanz Microwave Electrical Appliances Co., Ltd.P70D20TL-P4
Neutral BalsamChina National Pharmaceutical Group Chemical Reagent Co., Ltd.10004160
Optical microscopeNikon (Japan)Nikon Eclipse CI
OvenShanghai Huitai Instrument Manufacturing Co., Ltd.DHG-9140A
PAGE gel rapid preparation kitBiosharpPL566B-5
ParaformaldehydeChina National Pharmaceutical Group Chemical Reagent Co., Ltd.8.18715
Phosphate buffered saline (1x)Gibco8120485
Pre-stained color protein marker (10-180 kDa)Cywin Innovation (Beijing) Biotechnology Co., Ltd.SW176-02
Protein loading buffer (5X)Boster Biological TechnologyAR1112
pulverizerShangzhiqiao Co., Ltd.DFT-100A
PVDF (0.45 μm)Cywin Innovation (Beijing) Biotechnology Co., Ltd.SW120-01
Rapid membrane transfer solutionCywin Innovation (Beijing) Biotechnology Co., Ltd.SW171-02
RIPA lysateBoster Biological Technology Co., Ltd.AR0105
TBS bufferNCM Biotech23HA0102
Tissue Flotation WorkstationJinhua Kedi Instrument Equipment Co., Ltd., Zhejiang Province.KD-P
Transmembrane bufferNCM Biotech23CA2000
Trypsin (0.25%, 1x)HyCloneJ210045
Ultrasonic cell disruption apparatusNingbo Xinyi ultrasonic equipment Co., Ltd.JY92-IIDN
Upright Optical MicroscopeNikon (Japan)Nikon Eclipse CI
vacuum lyophilizerLanjing Co., Ltd.HD-LG20D
Vortex mixerKylin-BellXW-80A
XyleneChina National Pharmaceutical Group Chemical Reagent Co., Ltd.214736

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