The Roles of Extracellular Purinergic Signaling in Local Acupoints In ChronoAcupuncture Analgesia

Abstract

Objective: To explore the peripheral mechanism of chrono-acupuncture with respect to purinergic signaling in local acupoints. Methods:The Sprague Dawley (SD) rats were randomly divided into two groups. One group was injected 0.1ml saline as control group. Another group was established by subcutaneous injecting 0.1ml complete Freund's adjuvant (CFA). After modeling successfully, model rats were randomly divided into model group and acupuncture group based on the basic pain threshold. The basic pain threshold was tested by tailflick method. The extracellular ATP concentration in local acupoints was detected by high-performance liquid chromatography (HPLC) after acupunctured at different times, which were zeitgeber time 0(ZT0) (7:00), ZT4(11:00), ZT8(15:00), ZT12(19:00), ZT16(23:00), and ZT20(3:00) respectively. Based on the characteristics of the variation of pain threshold and extracellular ATP concentration, the peak phase and valley phase were selected for the further experiment. The protein expression of P2X3 receptors in skin of "Zusanli" (ST36), dorsal root ganglion (DRG) and spinal dorsal horn (SDH) were separately detected by immunohistochemistry.Results: Time had an evident influence on pain threshold (P=0.047, P<0.05) which indicated the pain threshold at different time points was different. The variation of pain threshold among control group, model group and acupuncture group were significantly different (P<0.01). In acupuncture group, the of peak value and valley value appeared at ZT8 and ZT16 respectively. Pain threshold showed complex interactions with extracellular ATP, time factor and acupuncture stimuli. The extracellular ATP concentrations were significantly different in comparison among control group, model group and acupuncture group respectively. (P<0.01). As for protein expression of P2X3 receptors in skin of "Zusanli" (ST36), compared with control group, it obviously increased in model group at ZT8 and ZT16. And there was significant difference between control group and model group (P<0.01). In addition, compared with model group, it obviously decreased after acupunctured at ZT8. And there was significant difference between model group and acupuncture group at ZT8(P<0.01), but not at ZT16 (P>0.05). Interestingly, the protein expression of P2X3 receptors in both DRG and SDH was consistent with that in skin of "Zusanli" (ST36). Conclusion:Temporal variation of purinergic signaling participated in the initial mechanism of chrono-acupuncture analgesia in local acpoints. Also, that might be the base of dynamic variation of acupoints reactivity.