Relationship between P2X 7 receptors and NLRP3/IL-1β pathway in spinal neurons in development of inflammatory pain in rats / 中华麻醉学杂志

ABSTRACT

Objective:To evaluate the relationship between P2X 7 receptors and nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3)/interleukin-1beta (IL-1β) pathway in spinal neurons in the development of inflammatory pain (IP) in rats. Methods:SPF healthy adult male Wistar rats, weighing 180-220 g, were used in this study.Forty rats in which intrathecal catheters were successfully implanted were divided into 5 groups ( n=8 each) using a random number table method: control group (group CON), group IP, IP plus dimethyl sulfoxide (DMSO) group (group IP-DMSO), IP plus P2X 7 receptor antagonist A740003 group (group IP-A) and IP plus P2X 7 receptor agonist ATP group (group IP-ATP). Rats were anesthetized with pentobarbital sodium 40 mg/kg.IP was induced by injecting complete Freund′s adjuvant 50 μl into the right ankle joint cavity, while group CON was injected with the equal volume of normal saline instead.On 1 day before establishing the model, immediately after establishing the model, and on 1, 2 and 3 days after establishing the model, 1% DMSO 10 μl was intrathecally injected once a day in group IP-DMSO, A740003 0.1 nmol(dissolved in DMSO 10 μl) was intrathecally injected once a day in group IP-A, and ATP 150 nmol(dissolved in DMSO 10 μl) was injected intrathecally once a day in group IP-ATP.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured on 3 days after establishing the model.Enzyme-linked immunosorbent assay was used to determine the prostaglandin E2 (PGE2) concentrations in right ankle tissues and IL-1β concentrations in cerebrospinal fluid (CSF). Then rats were sacrificed, and the lumber segments (L 4-6) of the spinal cord were removed for determination of the expression of NLRP3, casepase-1, IL-1β (by Western blot) and co-expression of P2X 7 receptors with neuron-specific nucleoprotein (NeuN) and NLRP3 and with NeuN (by immunofluorescence). Results:Compared with group CON, PGE2 contents in ankle tissues were significantly increased in group IP, and the MWT was significantly decreased, the TWL was shortened, the concentrations of IL-1β in CSF were increased, and the expression of NLRP3, caspase-1 and IL-1β was up-regulated in the other four groups ( P<0.05). Compared with group IP, the MWT was significantly increased, the TWL was prolonged, the concentrations of IL-1β in CSF were decreased, and the expression of NLRP3, caspase-1 and IL-1β was down-regulated in group IP-A ( P<0.05), the MWT was significantly decreased, TWL was shortened, the concentrations of IL-1β in CSF were increased, and the expression of NLRP3, caspase-1 and IL-1β was up-regulated in group IP-ATP ( P<0.05), and no significant change was found in the parameters mentioned above in group IP-DMSO ( P>0.05). P2X 7 was co-expressed with NeuN, and NLRP3 was co-expressed with NeuN. Conclusion:P2X 7 receptors in spinal neurons are involved in the development of inflammatory pain by activating NLRP3/IL-1β signaling pathway in rats.