To explore the molecular mechanism of cumulative analgesic effect induced by repeated electroacupuncture (EA) intervention via targeted regulation of adenosine monophosphate-activated protein kinase (AMPK) signaling pathway.

Mice were randomly divided into control group, model group, single EA group, repeated EA group and sham EA group; as well as vehicle + EA group and inhibitor + EA group, with 8 mice in each group. A mice inflammatory pain model was established by complete Freund’s adjuvant (CFA) injection. Repeated EA stimulation was applied to mice in repeated EA group, vehicle + EA group and inhibitor + EA group at left “Zusanli” (ST36) and “Shangjuxu” (ST37). The parameters were set as frequency 2 Hz, current 1 mA, 30 min per intervention, once a day for 6 consecutive days. The single EA group only received a single EA treatment on the second day after modeling. The sham EA group only received superficial subcutaneous puncture without electrical stimulation. Mice in inhibitor + EA group were intraperitoneally injected with Compound C at a dose of 20 mg/kg 30 minutes before daily EA. Mechanical pain threshold and thermal pain latency were detected to evaluate pain behaviors. Western blot was used to determine the protein expression levels of p-AMPK, AMPK and c-Fos in the spinal dorsal horn. Immunofluorescence staining was adopted to detect the number of c-Fos positive cells.

Compared with the control group, mechanical pain threshold and thermal pain latency were markedly decreased in the model group (P<0.05). Compared with the model group, single EA significantly elevated mechanical pain threshold and thermal pain latency at 1, 2 and 4 h after treatment (P<0.05); repeated EA increased pain thresholds after intervention (P<0.05). The ratio of p-AMPK/AMPK in lumbar spinal dorsal horn was significantly higher in repeated EA group than that in control and model groups (P<0.05), while this indicator was obviously lower in sham EA group than that in the EA group (P<0.05). Pain thresholds from day 2 to day 6 were higher in vehicle + EA group relative to the model group (P<0.05), and notably declined in inhibitor + EA group compared with vehicle + EA group (P<0.05). The expression level and positive neuron number of c-Fos were increased in the model group versus the control group (P<0.05), and reduced in vehicle + EA group (P<0.05). Such changes were reversed after Compound C intervention (P<0.05).

Repeated EA can effectively alleviate CFA-induced pain in mice. The cumulative analgesic effect may be mediated by persistent activation of AMPK in the lumbar spinal dorsal horn.