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. 2023 Jan-Dec:19:17448069231216234.
doi: 10.1177/17448069231216234.

Dehydrocorydaline alleviates sleep deprivation-induced persistent postoperative pain in adolescent mice through inhibiting microglial P2Y12 receptor expression in the spinal cord

Haikou Yang  1   2 , Yufeng Zhang  1   2 , Qingling Duan  1 , Kun Ni  3 , Yang Jiao  3 , Jixiang Zhu  3 , Jian Sun  2 , Wei Zhang  3 , Zhengliang Ma  1
Affiliations

Dehydrocorydaline alleviates sleep deprivation-induced persistent postoperative pain in adolescent mice through inhibiting microglial P2Y12 receptor expression in the spinal cord

Haikou Yang et al. Mol Pain. 2023 Jan-Dec.

Abstract

During adolescence, a second period of central nervous system (CNS) plasticity that follows the fetal period, which involves sleep deprivation (SD), becomes apparent. SD during adolescence may result in abnormal development of neural circuits, causing imbalance in neuronal excitation and inhibition, which not only results in pain, but increases the chances of developing emotion disorders in adulthood, such as anxiety and depression. The quantity of surgeries during adolescence is also consistently on the rise, yet the impact and underlying mechanism of preoperative SD on postoperative pain remain unexplored. This study demonstrates that preoperative SD induces upregulation of the P2Y12 receptor, which is exclusively expressed on spinal microglia, and phosphorylation of its downstream signaling pathway p38Mitogen-activated protein/Nuclear transcription factor-κB (p38MAPK/NF-κB)in spinal microglia, thereby promoting microglia activation and microglial transformation into the proinflammatory M1 phenotype, resulting in increased expression of proinflammatory cytokines that exacerbate persisting postoperative incisional pain in adolescent mice. Both intrathecal minocycline (a microglia activation inhibitor) and MRS2395 (a P2Y12 receptor blocker) effectively suppressed microglial activation and proinflammatory cytokine expression. Interestingly, supplementation with dehydrocorydaline (DHC), an extract of Rhizoma Corydalis, inhibited the P2Y12/p38MAPK/NF-κB signaling pathway, microglia activation, and expression of pro-inflammatory cytokines in the model mice. Taken together, the results indicate that the P2Y12 receptor and microglial activation are important factors in persistent postoperative pain caused by preoperative SD in adolescent mice and that DHC has analgesic effects by acting on these targets.

Keywords: P2Y12 receptor; dehydrocorydaline; incisional pain; inflammation; microglia; sleep deprivation.

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Conflict of interest statement

Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Time-dependent changes in the PWMT after continuous 4.5-day-SD in mice was tested by Von Frey. n = 8 per group. Group C vs group I, &&&P < 0.001; group C vs group SI, ###P < 0.001; group I vs group SI, $$P < 0.01, $$$P < 0.001.
Figure 2.
Figure 2.
The expression of microglia in the spinal dorsal horn was measured by immunochemical staining with IBA1 antibody on postoperative day 1 and 7. The number and body area of microglia in each group. Scale bar = 50 μm, n = 3 per group. (a) Microglial activation on postoperative day 1. (b) Microglial activation on postoperative day 7. (c, d) The number and body area of microglia in each group on postoperative day 1 and 7, ###P < 0.001.
Figure 3.
Figure 3.
The protein levels in the spinal cord were detected with western blotting, n = 4 per group. The results were considered significant at #p < 0.05, ##p < 0.01, and ###p < 0.001. (a) Protein expression in the spinal cord on postoperative day 1. (b) The protein expression in spinal cord on postoperative day 7. C-F iNOS, Arg1, IL-1β, and IL-6 levels in the spinal cord of each group postoperative day 1 and day 7.
Figure 4.
Figure 4.
Time-dependent changes in the PWMT after intrathecal injection of minocycline in mice was tested by Von Frey, n = 8 per group, #P < 0.05, ##P < 0.01, ###P < 0.001.
Figure 5.
Figure 5.
Effects of minocycline on microglia activation in mice on postoperative day 7, n = 3 per group,#p < 0.05, ###p < 0.001. (a) microglial activation detected by immunofluorescence. (b,c) The number and body area of microglia in each group on postoperative day 7.
Figure 6.
Figure 6.
The protein levels in the spinal cord after minocycline injection, n = 4 per group. (a) The protein expression in the spinal cord on postoperative day 7. (B-E) iNOS, Arg1, IL-1β, and IL-6 levels in the spinal cord of each group on postoperative day 7, ##P < 0.01, and ###P < 0.001.
Figure 7.
Figure 7.
The effects of SD on the P2Y12/p38MAPK/NF-κB signaling pathway in each group, n = 4 per group. (a-d) The protein expression in the spinal cord on postoperative days 1 and 7. 7. (e-h) p38MAPK, p-p38MAPK, P65, and p-P65 levels in the spinal cord of each group on postoperative day 1 and day 7, #P < 0.05, ##P < 0.01, and ###P < 0.001.
Figure 8.
Figure 8.
The P2Y12 receptor expression in spinal cord in each group and pain behavior after intrathecal injection of MRS2395. (a) The P2Y12 receptor expression on Day1 and Day7, n = 4. (b) PWMT was tested by Von Frey after intrathecal injection of MRS2395, n = 8. #P < 0.05, ##P < 0.01, and ###P < 0.001.
Figure 9.
Figure 9.
Effects of MRS2395 on microglia activation on Day7, n = 3 per group,#P < 0.05. (a) microglia activation detected by immunofluorescence. (b, c) The number and body area of microglia in each group on Day7.
Figure 10.
Figure 10.
The inhibitory effects of MRS2395 on P2Y12/p38MAPK/NF-κB signaling pathway examined by immunoblotting, n = 4 per group,#P < 0.05. (a,d) The protein levels in spinal cord on Day 7. (b-c,e-g) p38MAPK, p-p38MAPK, P65, p-P65 and P2Y12 receptor levels in spinal cord of Day7.
Figure 11.
Figure 11.
The inflammation related proteins expression after MRS2395 intervention on Day7, n = 4 per group,#P < 0.05,##P < 0.05. (a) The proteins expression after MRS2395 injection.(b-d) iNOS, Arg1, IL-1β and IL-6 levels in spinal cord on Day7.
Figure 12.
Figure 12.
Pain behavior was exhibited by PWMT after injection of DHC, n = 8. ###P < 0.001.
Figure 13.
Figure 13.
The effect of DHC on microglial activation on Day7, n = 3 per group,##P < 0.01, ###P < 0.001. (a) microglia activation detected by immunofluorescence with IBA1. (b, c) The number and body area of microglia in each group on Day7.
Figure 14.
Figure 14.
The suppression of DHC on P2Y12/p38MAPK/NF-κB signaling pathway detected by immunoblotting, n = 4 per group,#P < 0.05. (a,d) The protein levels in spinal cord on Day 7. (b-c,e-g) p38MAPK, p-p38MAPK, P65, p-P65 and P2Y12 receptor levels in spinal cord of Day7.
Figure 15.
Figure 15.
The inflammation related proteins expression after DHC injection on Day7, n = 4 per group, #P < 0.05,##P < 0.05. (a) The proteins expression after DHC injection. (b-d) iNOS, Arg1, IL-1β and IL-6 levels in spinal cord on Day7.
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