In this research, we established an inflammatory pain model by injection CFA into the right hind paw of mouse. One week after CFA injection, the mouse exhibited thermal hyperalgesia [18], and P2RY14 was elevated in the bilateral S1HL, mainly observed in neurons. We found that EA, not sham EA, alleviates thermal hyperalgesia and decreases P2RY14 expression. Furthermore, EA-induced analgesic effects were reversed by UDPG, a specific P2RY14 agonist. However, the PPTN, a P2RY14 antagonist, did not impact the EA analgesic effects. These findings suggested that the upregulated P2RY14 in the S1HL may mediate pain hypersensitivities and EA-induced analgesic may be achieved by suppressing the P2RY14 expression.
In the inflammatory pain model, S1HL exhibits a number of structural and functional changes including: sustained enhancement of synaptic transmission, dendritic spine formation, increased intrinsic cellular excitability and ion channel rearrangement, which together contribute to central nociceptive sensitization [19]. EA of the ST36, but not non-acupoint, effectively activates S1HL neurons and astrocytes [20]. In addition, the activation of both local neuronal inhibitory microcircuits and astrocytes in S1HL were involved in the mechanism of EA analgesia [21]. Purinergic receptors play an important role in EA analgesia and are broadly distributed throughout the nervous system [6]. However, the role of cerebral purinergic receptors in mediating EA analgesia remains to be elucidated. Here, we investigated for the first time the alterations of P2RY14 expression in S1HL in CFA-induced inflammatory pain and its possible mechanisms involved in EA analgesia.
Numerous studies have shown that P2RY14, in the periphery nerves and spinal cord, is a mediator of nociceptive sensitization and inflammation [22, 23]. However, the role of P2RY14 in the brain in pain modulation and EA-induced analgesia remains incompletely understood. Previous study has shown that peripheral injection of CFA rapidly increases P2RY14 expression on trigeminal ganglion astrocytes [24]. Another study reported that microglial P2RY14 expression in the trigeminal cervical complex (TCC) was significantly increased in a rat model of migraine induced by repeated inflammatory stimulation of the dura mater [25]. Our findings suggested that the elevated P2RY14 colocalized with neurons, but not astrocytes (supplementary figure), in the S1HL of a CFA-induced inflammatory pain model. However, there were some limitations in this research. First, P2RY14 expression in S1HL was not continuously observed after CFA injection, and temporal variability in P2RY14 expression cannot be excluded. Second, we did not co-label P2RY14 with microglia. Therefore, we could not rule out the possibility that S1HL microglia express P2RY14 and are involved in EA-induced pain relief. Finally, we chose the doses of agonist and antagonist based on the available literature. We did not perform concentration gradients or electrophysiological validation of the drugs' efficacy.
In summary, we have shown that P2RY14 in S1HL mediates CFA-induced hypersensitivity. We demonstrated that EA-induced analgesia may be achieved by downregulating P2RY14 on neurons in S1HL. For the first time, we investigated the role of P2RY14 in S1HL in EA-induced analgesia and enriched the purinergic signaling mechanism of EA analgesia.