Effects of intrathecal injection P2Y12 receptor inhibitor on interleukin-1 beta and interleukin-6 in spinal cord of rat bone cancer pain model

Abstract

To explore the roles of P2Y12 receptor (P2Y12R) in bone cancer pain by observing the changes of inflammatory cytokines (IL-1β, IL-6) after intrathecal injection (i.t.) of P2Y12R antagonist MRS2395. Thirty-two female SD rats were randomly divided into 4 groups (n = 8 each): sham group (group S), MRS2395 group (group M), cancer group (group A) and cancer + MRS2395 group (group MA).Groups S and M received an injection of 10 µl Hank's solution into left tibia medullar cavity while groups A and MA had an injection of Walker 256 mammary cancer cells (10 µl, 2×10(7) cells/ml) into the same place. At Day 9-12 post-inoculation, groups S and A received an injection of saline (0.9%, 15 µl, i.t.) while groups M and MA had MRS2395 (400 pmol/µl, 15 µl, i.t.). Intrathecal catheterization between L3 and L4 was performed immediately after inoculating tumor cells by inserting a small tube into vertebral space. Mechanical withdrawal thresholds were measured on left hind paws before and during 10-minute intervals after dosing. Spinal cords (L4-L6 segments) were removed for determining the expressions of IL-1β and IL-6 by enzyme-linked immunosorbent assay (ELISA) at Day 12 after drug delivery. At 20 min post-injection, mechanical withdrawal thresholds of groups S, M, A and MA were (34.2 ± 5.8), (34.4 ± 5.7), (21.0 ± 2.0) and (25.4 ± 2.3) g respectively at Day 9 post-inoculation (F = 18.679, P < 0.01); mechanical withdrawal thresholds of group A obviously decreased versus groups S and M; mechanical withdrawal thresholds in group MA increased obviously versus group A. The expressions of IL-1β in groups S, M, A and MA were (74.0 ± 18.6), (98.4 ± 17.3), (253.5 ± 66.4) and (146.3 ± 22.3) pg/ml at Day 12 post-inoculation (F = 18.221, P < 0.01); compared with groups S and M, the expression of IL-1β in group A showed a significant up-regulation. Likewise, the expressions of IL-6 were (377.4 ± 65.8), (331.6 ± 67.9), (856.1 ± 53.4) and (596.1 ± 34.9) pg/ml (F = 70.880, P < 0.01) respectively in groups S, M, A and MA; compared with groups S and M, the expression of IL-6 increased obviously in group A. There were significant decreases of IL-1β and IL-6 in group MA versus group A. An intrathecal injection of MRS2395 may alleviate hyperalgesia and inhibit the up-regulated expression of spinal cord inflammatory cytokines in bone cancer rats. And P2Y12 receptor may be involved in the formation of bone cancer pain through regulating the expressions of IL-1β and IL-6.