Advanced Search

Journal of Traditional Chinese Medicine >

2025 , Vol. 45 >Issue 6: 1238 - 1253

DOI: https://doi.org/10.19852/j.cnki.jtcm.20250929.002

Original Articles

Four-dimensional data independent acquisition proteomics and metabolomics reveal mechanisms of hydrogen-rich water at Zusanli (ST36) point against triple-negative breast cancer in mice

  • Jingxuan LIU ,
  • Qian MO ,
  • Guowu LEI ,
  • Yejuan JIA ,
  • Aiying LI ,
  • Chunsheng JIA ,
  • Lijia PAN
Expand
  • 1 College of Acupuncture and moxibustion and Massage, Hebei University of Chinese Medicine, Shijiazhuang 050200, China
    2 College of Basic Medicine, Hebei University of Chinese Medicine, Shijiazhuang 050200, China

Received date: 2025-06-03

  Accepted date: 2025-09-09

  Online published: 2025-09-29

Supported by

Exploring the Advantages of Micro-needle System Therapy for Specific Diseases and the Patterns of Acupoint Selection through Data Mining Techniques(81473773);Exploring the Molecular Mechanism behind How Hydrogen Mitigates the Aging of Vascular Endothelial Cells Induced by Chronic Intermittent Hypoxia, Utilizing the Keap1-Nuclear factor erythroid 2-related factor 2 Signaling Pathway(ZD2020142);Inhibitory Effect and Mechanism of Hydrogen-rich Water Acupoint Injection on Tumors in Mice with Triple-negative Breast Cancer(CXZZBS2024154)

Fold

Abstract

OBJECTIVE: To develop a safe and effective green therapy for triple-negative breast cancer, this study combines hydrogen-rich water with acupuncture point injection, and finds that it can prevent tumor growth and minimize cancer metastasis.

METHODS: After 21 d of hydrogen rich water injection treatment on 4T1 (mouse breast cancer cells) xenograft mice, in order to systematically identify differentially expressed proteins in tumor samples between the model group and the Zusanli (ST36) group injected with hydrogen rich water at acupoints, with a focus on functional proteins or signaling pathways related to tumor occurrence and development, researchers conducted four-dimensional data independent acquisition (4D-DIA) proteomic analysis on tumor tissues. In order to further investigate the dynamic changes of metabolites after therapeutic intervention, researchers conducted liquid chromatography-tandem mass spectrometry untargeted metabolomics identification and analysis on mouse serum. The results of the joint proteomics-metabolomics analysis were validated using experimental methods such as immunofluorescence, Western blotting, and quantitative reverse transcription polymerase chain reaction detection.

RESULTS: Injecting hydrogen-rich water into acupoints significantly inhibited tumor growth (P < 0.05). 4D-DIA proteomics and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses uncovered pathways such as T helper 1 cell (Th1) and T helper 2 cell (Th2) cell differentiation. The KEGG metabolic pathways identified in the metabolomics analysis included galactose metabolism along with fructose and mannose metabolism. Based on the combined proteomics and metabolomics analysis, the key pathways included the C-type lectin receptor signaling pathway. The major cancer-related differential proteins detected in Th1 and Th2 cell differentiation [interleukin 6 signal transducer, nuclear factor of activated T cells 4, recombinant mitogen activated protein kinase 10 (MAPK10), and MAPK11] were upregulated after the injection of hydrogen-rich water into the Zusanli (ST36) acupoint, whereas Linker for activation of T cells (Lat), signal transducer and activator of transcription 1, and protein kinase C, theta were downregulated.

CONCLUSION: The injection of hydrogen-rich water into the Zusanli (ST36) acupoint effectively inhibited the hyperplasia of 4T1 BC cells and enhanced their apoptosis, potentially exerting a therapeutic effect through multiple pathways and targeting various sites.

Key words： acupuncture; acupoint injection; hydrogen-rich water; omics; triple-negative breast cancer

Cite this article

Jingxuan LIU , Qian MO , Guowu LEI , Yejuan JIA , Aiying LI , Chunsheng JIA , Lijia PAN . Four-dimensional data independent acquisition proteomics and metabolomics reveal mechanisms of hydrogen-rich water at Zusanli (ST36) point against triple-negative breast cancer in mice[J]. Journal of Traditional Chinese Medicine, 2025 , 45(6) : 1238 -1253 . DOI: 10.19852/j.cnki.jtcm.20250929.002

References

1. Bray F, Laversanne M, Sung H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA 2024; 74: 229-63.
2. Katsika L, Boureka E, Kalogiannidis I, et al. Screening for breast cancer: a comparative review of guidelines. Life 2024; 14: 777.
3. Zheng RS, Zhang SW, Sun KX, et al. Cancer statistics in China, 2016. Zhong Hua Zhong Liu Za Zhi 2023; 45: 212-20.
4. Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA 2023; 73: 17-48.
5. Park JH, Ahn JH, Kim SB. How shall we treat early triple-negative breast cancer (TNBC): from the current standard to upcoming immuno-molecular strategies. Esmo open 2018; 3: e000357.
6. Stevens KN, Vachon CM, Couch FJ. Genetic susceptibility to triple-negative breast cancer. Cancer Res 2013; 73: 2025-30.
7. Xie S, Sun Y, Zhao X, et al. An update of the molecular mechanisms underlying anthracycline induced cardiotoxicity. Front Pharmacol? 2024; 15: 1406247.
8. Aghili M, Taherioun M, Jafari F, et al. Duloxetine to prevent neuropathy in breast cancer patients under paclitaxel chemotherapy (a double-blind randomized trial). Support Care Cancer 2024; 32: 493.
9. Ma W, Wang X, Zhang D, Mu X. Research progress of disulfide bond based tumor microenvironment targeted drug delivery system. Int J Nanomed? 2024; 19: 7547-66.
10. Zhao D, Li Z, Ji DK, Xia Q. Recent progress of multifunctional molecular probes for triple-negative breast cancer theranostics. Pharmaceutics 2024; 16: 803.
11. Greenlee H, DuPont-Reyes MJ, Balneaves LG, et al. Clinical practice guidelines on the evidence-based use of integrative therapies during and after breast cancer treatment. CA Cancer J Clin 2017; 67: 194-232.
12. Zhang J, Qin Z, So TH, et al. Acupuncture for chemotherapy-associated insomnia in breast cancer patients: an assessor-participant blinded, randomized, sham-controlled trial. Breast Cancer Res 2023; 25: 49.
13. Xu Y, Yu J, Shen R, Shan X, Zhou W, Wang J. Comparison efficacy and safety of acupuncture and moxibustion therapies in breast cancer-related lymphedema: a systematic review and network Meta-analysis. PLoS One 2024; 19: e0303513.
14. Hershman DL, Unger JM, Greenlee H, et al. Comparison of acupuncture vs sham acupuncture or waiting list control in the treatment of aromatase inhibitor-related joint pain: a randomized clinical trial. JAMA Netw Open 2022; 5: e2241720.
15. Gosain R, Gage-Bouchard E, Ambrosone C, Repasky E, Gandhi S. Stress reduction strategies in breast cancer: review of pharmacologic and non-pharmacologic based strategies. Semin Immunopathol 2020; 42: 719-34.
16. Tran S, Hickey M, Saunders C, Ramage L, Cohen PA. Nonpharmacological therapies for the management of menopausal vasomotor symptoms in breast cancer survivors. Support Care Cancer 2021; 29: 1183-93.
17. Zhang ZJ, Man SC, Yam LL, et al. Electroacupuncture trigeminal nerve stimulation plus body acupuncture for chemotherapy-induced cognitive impairment in breast cancer patients: an assessor-participant blinded, randomized controlled trial. Brain Behav Immun 2020; 88: 88-96.
18. H?xtermann MD, Haller H, Aboudamaah S, et al. Safety of acupuncture in oncology: a systematic review and Meta-analysis of randomized controlled trials. Cancer 2022; 128: 2159-73.
19. Epstein AS, Liou KT, Romero SAD, et al. Acupuncture vs massage for pain in patients living with advanced cancer: the IMPACT randomized clinical trial. JAMA Netw Open 2023; 6: e2342482.
20. Li A, Cao T, Feng L, Hu Y, Zhou Y, Yang P. Recent advances in metal-hydride-based disease treatment. ACS Appl Mater Interfaces? 2024; 16: 5355-67.
21. Hirano SI, Ichikawa Y, Sato B, Yamamoto H, Takefuji Y, Satoh F. Molecular hydrogen as a potential clinically applicable radioprotective agent. Int J Mol Sci 2021; 22: 4566.
22. Li Y, Li G, Suo L, Zhang J. Recent advances in studies of molecular hydrogen in the treatment of pancreatitis. Life Sci 2021; 264: 118641.
23. Qiu P, Liu Y, Zhang J. Recent advances in studies of molecular hydrogen against sepsis. Int J Biol Sci 2019; 15: 1261-75.
24. Ji P, Yang K, Xu Q, et al. Mechanisms and application of gas-based anticancer therapies. Pharmaceuticals 2023; 16: 1394.
25. Zhou W, Zhang J, Chen W, Miao C. Prospects of molecular hydrogen in cancer prevention and treatment. J Cancer Res Clin 2024; 150: 170.
26. Hirano SI, Yamamoto H, Ichikawa Y, Sato B, Takefuji Y, Satoh F. Molecular hydrogen as a novel antitumor agent: possible mechanisms underlying gene expression. Int J Mol Sci 2021; 22: 8724.
27. Chen JB, Kong XF, Qian W, et al. Two weeks of hydrogen inhalation can significantly reverse adaptive and innate immune system senescence patients with advanced non-small cell lung cancer: a self-controlled study. Med Gas Res 2020; 10: 149-54.
28. Li S, Liao R, Sheng X, et al. Hydrogen gas in cancer treatment. Front Oncol 2019; 9: 696.
29. Shih YW, Yang SF, Chien MH, Chang CW, Chang VHS, Tsai HT. Significant effect of acupressure in elevating blood stem cell factor during chemotherapy in patients with gynecologic cancer. J Nurs Res 2018; 26: 411-19.
30. Zhang B, Xu F, Hu P, et al. Needleless transcutaneous electrical acustimulation: a pilot study evaluating improvement in post-operative recovery. Am J Gastroenterol 2018; 113: 1026-35.
31. Mao H, Mao JJ, Guo M, et al. Effects of infrared laser moxibustion on cancer-related fatigue: a randomized, double-blind, placebo-controlled trial. Cancer 2016; 122: 3667-72.
32. Hsiung WT, Chang YC, Yeh ML, Chang YH. Acupressure improves the postoperative comfort of gastric cancer patients: a randomised controlled trial. Complement Ther Med 2015; 23: 339-46.
33. Zhang FC, Gao TY, Zhang CX, et al. Effect of moxibustion combined with chemotherapy on immune checkpoints in tumor tissue of breast cancer-bearing mice. Zhen Ci Yan Jiu 2025; 50: 319-26.
34. Liu S, Wang Z, Su Y, et al. A neuroanatomical basis for electroacupuncture to drive the vagal-adrenal axis. Nature 2021; 598: 641-45.
35. Liu F, Wang Y, Lyu K, et al. Acupuncture and its ability to restore and maintain immune homeostasis. QJM 2024; 117: 167-76.
36. Wang N, Zhao L, Zhang D, Kong F. Research progress on the immunomodulatory mechanism of acupuncture in tumor immune microenvironment. Front Immunol 2023; 14: 1092402.
37. Gao S, Hsu TW, Li MO. Immunity beyond cancer cells: perspective from tumor tissue. Trends cancer 2021; 7: 1010-19.
38. Basu A, Ramamoorthi G, Albert G, et al. Differentiation and regulation of TH Cells: a balancing act for cancer immunotherapy. Front immunol 2021; 12: 669474.
39. Martínez-Pérez C, Leung J, Kay C, et al. The signal transducer IL6ST (gp130) as a predictive and prognostic biomarker in breast cancer. J Pers medicine 2021; 11: 618.
40. Martínez-Pérez C, Kay C, Meehan J, Gray M, Dixon JM, Turnbull AK. The IL6-like cytokine family: role and biomarker potential in breast cancer. J Pers Med 2021; 11: 1073.
41. Mathe A, Wong-Brown M, Morten B, et al. Novel genes associated with lymph node metastasis in triple negative breast cancer. Sci Rep 2015; 5: 15832.
42. Zhong QH, Zha SW, Lau ATY, Xu YM. Recent knowledge of NFATc4 in oncogenesis and cancer prognosis. Cancer Cell Int 2022; 22: 212.
43. Fougère M, Gaudineau B, Barbier J, et al. NFAT 3 transcription factor inhibits breast cancer cell motility by targeting the lipocalin 2 gene. Oncogene 2010; 29: 2292-301.
44. Guaddachi F, Bergerat D, et al. Extracellular vesicles produced by NFAT3-expressing cells hinder tumor growth and metastatic dissemination. Sci Rep 2020; 10: 8964.
45. Barollo S, Bertazza L, Watutantrige-Fernando S, et al. Overexpression of L-Type Amino Acid Transporter 1 (LAT1) and 2 (LAT2): novel markers of neuroendocrine tumors. PLoS One 2016; 11: e0156044.
46. Furuya M, Horiguchi J, Nakajima H, Kanai Y, Oyama T. Correlation of L-type amino acid transporter 1 and CD98 expression with triple negative breast cancer prognosis. Cancer Sci 2012; 103: 382-9.
47. Du D, Liu C, Qin M, et al. Metabolic dysregulation and emerging therapeutical targets for hepatocellular carcinoma. Acta Pharm Sin B 2022; 12: 558-80.
48. Zhang C, Xu J, Xue S, Ye J. Prognostic value of L-Type Amino Acid Transporter 1 (LAT1) in various cancers: a Meta-analysis. Mol Diagn Ther 2020; 24: 523-36.
49. H?fliger P, Charles RP. The L-Type Amino Acid Transporter LAT1-an emerging target in cancer. Int J Mol Sci 2019; 20: 2428.
50. Cormerais Y, Giuliano S, LeFloch R, et al. Genetic disruption of the multifunctional CD98/LAT1 complex demonstrates the key role of essential amino acid transport in the control of mTORC1 and tumor growth. Cancer Res 2016; 76: 4481-92.
51. Kurozumi S, Kaira K, Matsumoto H, et al. Association of L-type amino acid transporter 1 (LAT1) with the immune system and prognosis in invasive breast cancer. Sci Rep 2022; 12: 2742.
52. Li YJ, Zhang C, Martincuks A, Herrmann A, Yu H. STAT proteins in cancer: orchestration of metabolism. Nat Rev Cancer 2023; 23: 115-34.
53. Erdogan F, Radu TB, Orlova A, et al. JAK-STAT core cancer pathway: an integrative cancer interactome analysis. J Cell Mol Med 2022; 26: 2049-62.
54. Wong GL, Manore SG, Doheny DL, Lo HW. STAT family of transcription factors in breast cancer: pathogenesis and therapeutic opportunities and challenges. Semin Cancer Biol 2022; 86: 84-106.
55. Banik S, Rakshit S, Sarkar K. The role of STAT1 in T Helper Cell Differentiation during breast cancer progression. J Breast Cancer 2021; 24: 253-65.
56. Serrano López J, Jiménez-Jiménez C, Chutipongtanate S, et al. High-throughput RNA sequencing transcriptome analysis of ABC-DLBCL reveals several tumor evasion strategies. Leukemia Lymphoma 2022; 63: 1861-70.
57. Li H, Li Y, Zhang Y, et al. MAPK 10 expression as a prognostic marker of the immunosuppressive tumor microenvironment in human hepatocellular carcinoma. Front Oncol 2021; 11: 687371.
58. Atyah M, Zhou C, Zhou Q, et al. The age-specific features and clinical significance of NRF2 and MAPK10 expression in HCC patients. Int J Gen Med 2022; 15: 737-48.
59. Sun R, Xiang T, Tang J, et al. 19q13 KRAB zinc-finger protein ZNF471 activates MAPK10/JNK3 signaling but is frequently silenced by promoter CpG methylation in esophageal cancer. Theranostics 2020; 10: 2243-59.
60. Tsai YC, Huang CY, Hsueh YM, et al. Genetic variants in MAPK 10 modify renal cell carcinoma susceptibility and clinical outcomes. Life Sci 2021; 275: 119396.
61. Rachwa? K, Nied?wied? I, Wa?ko A, et al. Red Kale (Brassica oleracea L. ssp. acephala L. var. sabellica) induces apoptosis in human colorectal cancer cells in vitro. Molecules 2023; 28: 6938.
62. Wang Z, Liu W, Chen C, Yang X, Luo Y, Zhang B. Low mutation and neoantigen burden and fewer effector tumor infiltrating lymphocytes correlate with breast cancer metastasization to lymph nodes. Sci Rep 2019; 9: 253.
63. Roche O, Fernández-Aroca DM, Arconada-Luque E, et al. p38β and cancer: the beginning of the road. Int J Mol Sci 2020; 21: 7524.
64. Katopodis P, Kerslake R, Zikopoulos A, Beri N, Anikin V. p38β-MAPK11 and its role in female cancers. J Ovarian Res 2021; 14: 84.
65. Gao Y, Hu S, Li R, et al. Hyperprogression of cutaneous T cell lymphoma after anti-PD-1 treatment. JCI Insight insight 2023; 8: e164793.
66. Belguise K, Sonenshein GE. PKCtheta promotes c-Rel-driven mammary tumorigenesis in mice and humans by repressing estrogen receptor alpha synthesis. JCI Insight 2007; 117: 4009-21.
67. Byerly JH, Port ER, Irie HY. PRKCQ inhibition enhances chemosensitivity of triple-negative breast cancer by regulating Bim. Breast Cancer Res 2020; 22: 72.
68. Byerly J, Halstead-Nussloch G, Ito K, Katsyv I, Irie HY. PRKCQ promotes oncogenic growth and anoikis resistance of a subset of triple-negative breast cancer cells. Breast Cancer Res 2016; 18: 95.
69. Nicolle A, Zhang Y, Belguise K. The emerging function of PKCtheta in cancer. Biomolecules 2021; 11: 221.
70. Zhu J, Thompson CB. Metabolic regulation of cell growth and proliferation. Nat Rev Mol Cell Biol 2019; 20: 436.
71. Xu J, Zhao Y, Tyler Mertens R, Ding Y, Xiao P. Sweet regulation-the emerging immunoregulatory roles of hexoses. J Adv Res 2025; 69: 361-79.
72. Dougan J, Hawsawi O, Burton LJ, et al. Proteomics-metabolomics combined approach identifies peroxidasin as a protector against metabolic and oxidative stress in prostate cancer. Int J Mol Sci 2019; 20: 3046.
73. Alwehaibi MA, Al-Ansari MM, Alfadda AA, et al. Proteomics investigation of the impact of the enterococcus faecalis secretome on MCF-7 tumor cells. Int J Mol Sci 2023; 24: 14937.
74. Majtan T, Olsen T, Sokolova J, et al. Deciphering pathophysiological mechanisms underlying cystathionine beta-synthase-deficient homocystinuria using targeted metabolomics, liver proteomics, sphingolipidomics and analysis of mitochondrial function. Redox Biol 2024; 73: 103222.
75. Lu L, Kotowska AM, Kern S, et al. Metabolomic and proteomic analysis of ApoE4-carrying H4 neuroglioma cells in Alzheimer's disease using OrbiSIMS and LC-MS/MS. Anal Chem 2024; 96: 11760-70.
76. Wettersten HI, Hakimi AA, Morin D, et al. Grade-dependent metabolic reprogramming in kidney cancer revealed by combined proteomics and metabolomics analysis. Cancer Res 2015; 75: 2541-52.
77. Bai T, Wan Q, Yue C, et al. Combined spatial metabolomics and 4D-DIA quantitative proteomics approaches to explore the relationship between lung cancer and the heart. Sci Rep 2025; 15: 14878.
Options
Outlines

/

〈 〉