Fig. 4: The intracellular pathway of adenosine production in response to 40 Hz flickering primarily involves energy metabolism.

a Description of metabolomic screening of the V1 tissue. b–g The effects of 30-min 40 Hz flickering on the intracellular metabolic changes, including major adenosine-generating, adenosine-degrading, and efflux pathways in V1 assessed 30 min after flickering cessation by the targeted UHPLC-MS/MS analysis. Adenosine (Ado, b), ATP (c), ADP (d), AMP (e), cAMP (f), and NAD⁺ (g). *P < 0.05 for the 40 Hz-treated vs normal light groups at 30 min; **P < 0.01 when comparing 40 Hz vs normal light groups at 240 min. h The effect of 40 Hz light flickering on the level of IMP, a main component in the salvage/de novo pathway, in V1. **P < 0.01 when comparing 40 Hz vs normal light groups at 240 min. i–l The effect of 40 Hz light flickering on metabolites involved in SAM-mediated transmethylation, including SAM (i), SAH (j), Hcy (k), and Met (l) in V1. *P < 0.05 when comparing 40 Hz with normal light groups at 240 min in (l), **P < 0.01 when comparing 40 Hz group at 30 min or 40 Hz group at 240 min with the groups exposed to normal light (l). m–o The effect of 40 Hz light flickering on the adenosine degradation product, inosine (by UHPLC-MS/MS analysis; m) and on mRNA expression of adenosine-degrading enzymes ADK (n) and ADA (o) in V1. *P < 0.05 when comparing the 40 Hz group with the normal light group at 240 min. p–r The effects of 40 Hz light flickering on the density and mRNA expression of AMPK-α1 (p) and AMPK-α2 (q) (by qPCR analysis) and on AMPK phosphorylation (by western blot analysis; r) in V1. *P < 0.05, Student’s t-test; comparing the 40 Hz group with the normal light group at 240 min after flickering cessation. s Schematic showing that 40 Hz flickering is mainly associated with the stepwise ATP dephosphorylation for energy metabolism, rather than SAM-mediated transmethylation or S-adenosylhomocysteine hydrolysis, or the salvage/de novo purine synthesis pathways.