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
Liu J, Mo Q, Lei G, Jia Y, Li A, Jia C, Pan L. · Journal of Traditional Chinese Medicine. 2025.
Study at a glance
Preclinical
Forty six-week-old female BALB/c mice; 32 tumor-bearing mice were distributed among model and intervention groups after a separate non-tumor control group was selected.
At each session, 0.05 mL of 1.6 ppm H₂-rich water was injected into each bilateral ST36 point within 30 minutes of production, every other day for three weeks. · H₂-rich solution was produced by passing hydrogen from an AMS-H-03 generator into 250 mL of 0.9% sodium chloride; concentration was measured as 1.6 ppm. Total per session was 0.10 mL across both points.
Every-other-day treatment for 21 days; tumors were collected on day 21.
H₂-rich-water ST36 injection was associated with lower tumor volume/mass and greater apoptosis versus the untreated model. However, acupuncture and saline ST36 injection also affected several outcomes. Body weight did not differ among some groups, MAPK10 immunofluorescence did not differ significantly across groups, and some sodium-chloride comparisons were non-significant. Omics findings were exploratory and only selected pathways were experimentally followed up.
Small preclinical study (n=8/group; 3-5 replicates in some assays), multiple active comparator effects, exploratory non-targeted omics, no independent validation of key metabolites and no human data. The work used a named commercial H₂ generator and outside commercial omics laboratories. The acknowledgements identify Chinese public/provincial/institutional grants; no explicit conflict declaration was identified, so absence is not inferred.
What kind of evidence is this?
Preclinical
Randomized five-group 4T1 breast-cancer xenograft mouse experiment with exploratory proteomics and metabolomics
Cancer research
H₂-rich water
Information not yet classified
Some editorial classification fields are still pending. The source-reported outcomes and result are shown below; Hydrogenology does not infer a positive or negative signal from prose automatically.
Methods
Forty six-week-old female BALB/c mice; 32 tumor-bearing mice were distributed among model and intervention groups after a separate non-tumor control group was selected.
40 mice total, 8 per group: non-tumor control, untreated tumor model, acupuncture at ST36, H₂-rich-water injection at ST36, and saline injection at ST36. Some downstream assays used 3-5 biological replicates per group.
Every-other-day treatment for 21 days; tumors were collected on day 21.
At each session, 0.05 mL of 1.6 ppm H₂-rich water was injected into each bilateral ST36 point within 30 minutes of production, every other day for three weeks.
H₂ dissolved in 0.9% saline and injected at acupuncture points — H₂ only, not Brown's gas.
H₂-rich solution was produced by passing hydrogen from an AMS-H-03 generator into 250 mL of 0.9% sodium chloride; concentration was measured as 1.6 ppm. Total per session was 0.10 mL across both points.
Untreated tumor model, non-tumor control, acupuncture-only ST36 and saline-injection ST36 groups.
Outcomes and reported result
Body mass, tumor volume and mass, histology, TUNEL apoptosis, proteomics, serum metabolomics, immunofluorescence, Western blot and qRT-PCR pathway validation.
H₂-rich-water ST36 injection was associated with lower tumor volume/mass and greater apoptosis versus the untreated model. However, acupuncture and saline ST36 injection also affected several outcomes. Body weight did not differ among some groups, MAPK10 immunofluorescence did not differ significantly across groups, and some sodium-chloride comparisons were non-significant. Omics findings were exploratory and only selected pathways were experimentally followed up.
The complete free PMC article, all five groups, H₂ preparation and dose, result sections including reported null findings, limitations and acknowledgements were checked.
A reported association, difference or mechanism is not automatically a clinical benefit.
Limitations and applicability
Small preclinical study (n=8/group; 3-5 replicates in some assays), multiple active comparator effects, exploratory non-targeted omics, no independent validation of key metabolites and no human data. The work used a named commercial H₂ generator and outside commercial omics laboratories. The acknowledgements identify Chinese public/provincial/institutional grants; no explicit conflict declaration was identified, so absence is not inferred.
4T1 mouse breast-cancer xenografts and an acupoint-injection protocol; it does not establish an anticancer treatment in people.
No single-study GRADE certainty rating is assigned. Read how records and evidence assessments are prepared.
Sources and record status
PMID: 41376217 · DOI: 10.19852/j.cnki.jtcm.20250929.002
Free full article in PubMed Central.
Complete PMC article and PubMed metadata; full-text extraction checked 9 August 2026.
10 August 2026
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