Methylene blue activated energy and cell-defence pathways together in cultured human fibroblasts
Mark Kemp · 9 October 2026
In cultured human fibroblasts, 100 nM methylene blue briefly raised NAD/NADH and AMPK, then induced PGC1α and SURF1, doubled complex IV activity and slowed telomere erosion from 0.49 to 0.03 kb per week. A plain summary of Atamna et al., Redox Biology 2015.
In cultured human IMR90 fibroblasts, 100 nM methylthioninium chloride (methylene blue) raised the NAD/NADH ratio by 63% within 15 minutes, followed by a transient rise in AMPK phosphorylation, then induction of PGC1α and SURF1 — two regulators of mitochondrial and complex IV biogenesis (Atamna et al., Redox Biology 2015). Complex IV activity more than doubled, cellular oxidants fell 28%, and telomere erosion slowed from about 0.49 kb per week to 0.03 kb per week. Treated cells gained 18 population doublings before senescence versus 2.1 for the chronic AMPK activator AICAR.
Citation: Atamna H, Atamna W, Al-Eyd G, Shanower G, Dhahbi JM. Redox Biol 2015;6:426–435. doi:10.1016/j.redox.2015.09.004. PMID 26386875.
What the study did
This is the follow-up to the same group's 2008 paper. That one showed methylene blue delayed senescence in IMR90 cells and raised complex IV activity; this one asks what connects the two.
The authors treated IMR90 human lung fibroblasts with 100 nM methylene blue and tracked a chain of signals over time: the NAD/NADH ratio, phosphorylation of AMPK, levels of PGC1α and SURF1, complex IV activity, cellular oxidant levels, telomere length and the number of divisions before senescence. They compared the result against AICAR, a drug that activates AMPK continuously rather than briefly.
The paper notes that at micromolar concentration methylene blue inhibits complex IV; all results below are at 100 nanomolar.
What it found
The sequence ran in order. NAD/NADH rose 63% at 15 minutes and returned toward baseline by 24 hours. AMPK phosphorylation rose gradually, peaked around 24 hours, and was back to control by 48 hours. PGC1α was induced after 24 hours; SURF1 rose over the following days. Both are known to drive mitochondrial and complex IV biogenesis.
By day five, complex IV activity had more than doubled — a larger change than the 30% reported in the group's 2008 paper, which measured it over a different treatment period. Cellular oxidants, measured by DCFH-DA staining, were 28% lower than control.
Telomeres shortened at about 0.49 kb per week in untreated cells and 0.03 kb per week in treated cells. Control cells reached senescence at week 11; treated cells at week 17, an extra 18 population doublings in this experiment (the 2008 paper reported more than 20 under its own conditions). AICAR, the continuous AMPK activator, gave 2.1.
The authors' interpretation: a brief activation of the energy-sensing pathway (AMPK), combined with the cell-defence pathway they had reported earlier (Keap1/Nrf2), explains the effect better than either alone. Methylene blue did not alter the cell cycle.
| Model | IMR90 human lung fibroblasts, in vitro |
| Compound | Methylthioninium chloride, 100 nM |
| Comparator | AICAR 100 µM (continuous AMPK activator) |
| Measured | NAD/NADH; pAMPK/AMPK; PGC1α; SURF1; complex IV activity; cellular oxidants; telomere length; population doublings |
| Result | NAD/NADH +63% at 15 min (transient); AMPK transient; PGC1α and SURF1 induced; complex IV >2×; oxidants −28%; telomere erosion 0.49 → 0.03 kb/week; +18 PDL vs +2.1 for AICAR |
| Not tested | Animals; humans; oral administration; any concentration other than 100 nM |
What it means, and what it doesn't
The finding is a sequence: a brief activation of AMPK, the cell's energy sensor, followed by induction of PGC1α and SURF1, the proteins that drive complex IV biogenesis. The comparison with AICAR is the interesting part — pressing the same switch continuously was far less effective than pressing it briefly.
None of this is evidence about ageing in a person. "Senescence" here is the number of times a cell divides in a flask. The cells are a standard laboratory line, the concentration is a bath concentration in culture medium, and nothing was given to an animal or a person. The authors describe the effect as anti-senescence activity in vitro; that is the limit of what was tested.
Read the paper
Atamna H, Atamna W, Al-Eyd G, Shanower G, Dhahbi JM. Combined activation of the energy and cellular-defense pathways may explain the potent anti-senescence activity of methylene blue. Redox Biology 2015;6:426–435.
This summarises a laboratory study on cultured cells. It is not evidence of an effect in humans and is not a claim about any product.