Mechanism of cumulative analgesic effect of electroacupuncture： targeted regulation based on AMPK signaling pathway

ObjectiveTo 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.MethodsMice 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 minutes 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.ResultsCompared 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 indicato was obviously lower in sham EA group than those 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）.ConclusionRepeated 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.