Role Of Spinal COX And PGD₂ In Fentanyl-induced Postoperative Hyperalgesia In Mice

Opioids are the main drugs for the treatment of moderate and severe pain in the clinic.By binding to different types of opioid receptors,they can participate in pain regulation and exert a strong analgesic effect.In addition to producing analgesic effects,opioids can also induce sensitization of nociceptive pathways in the peripheral and central nervous systems,causing opioid-induced hyperalgesia（OIH）,which greatly limits their clinical application.The research on remifentanil-induced hyperalgesia（RIH）is extensive,but the hyperalgesia caused by fentanyl is not clear.Elucidating the mechanism of fentanyl-induced hyperalgesia helps to implement effective treatment measures.Studies have shown that cyclooxygenase inhibitors have a preventive effect on OIH.Our research team has confirmed that flurbiprofen axetil,a non-selective COX inhibitor can relieve RIH,which can indirectly indicate that COX plays an important role in nociceptive signals.At the same time,PGD₂,one of the main products of COX,has recently been found to have anti-inflammatory effects in a variety of disease models.There are two PGD₂ synthetases,H-PGDS and L-PGDS on its upstream,and there are two receptors,DP1 and DP2 on its downstream,and research has found DP1 receptor plays a key role in neuroprotection.This study is to investigate the postoperative hyperalgesia induced by fentanyl in mice and the mechanism of COXs-PGDS-PGD₂-DP1 pathway in fentanyl-induced postoperative hyperalgesia in mice.Objective To detect the influence of COXs-PGDS-PGD₂-DP1 pathway in the mice spinal cord of fentanyl-induced postoperative hyperalgesia,and evaluate its effect.MethodsI:Male C57BL/6J mice（64 mice）,aged 6～8 weeks,weighing about 20～25 g,were divided into 8 groups by random number table method:In groups C or group I,0.1 ml of saline was injected through the tail vein,4 injections with a 15-min interval.In group F or group IF(50μg·kg^-1),0.1ml of fentanyl(12.5μg·kg^-1)was injected through the tail vein,4 injections with a 15-min interval.In group IF(40μg·kg^-1),IF(60μg·kg^-1),IF(80μg·kg^-1)or IF(100μg·kg^-1),0.1ml of fentanyl(10μg·kg^-1,15μg·kg^-1,20μg·kg^-1,25μg·kg^-1,respectively)was injected,4 injections with a15-min interval.An incision pain model was established 15 minutes after the first fentanyl injection.The PWT and TWL were measured at 1d before treatment and 3h,6h,1d,2d,3d,5d,and 7d after the last fentanyl injection.The mice in the group C and group IF（50μg/kg）were anesthetized and sacrificed after the tests,and lumbar spinal cords were collected.II:40 male mice were divided into 5 groups:group 6h,group 1d,group 2d,group3d,and group 5d.Fentanyl(50μg·kg^-1)incision pain models were prepared.The mice were anesthetized and sacrificed at 6h,1d,2d,3d,and 5d after injection.Collected the lumbar spinal cords,the PGE₂ and PGD₂ protein contents were measured by ELISA,and H-PGDS m RNA,L-PGDS m RNA,DP1 m RNA and DP2m RNA were measured by RT-PCR.The content and distribution of COX-1 and COX-2 in spinal cord were analyzed by immunofluorescence staining.III:80 male mice were divided into 10 groups:group C,group IF+Vehicle,group IF+BW245C(8μg·10μl^-1),group IF+BW245C(80μg·10μl^-1),group I+BW245C(80μg·10μl^-1),group F+BW245C(80μg·10μl^-1),group IF+BWA868C(4μg·10μl^-1),group IF+BWA868C(40μg·10μl^-1),group I+BWA868C(40μg·10μl^-1)and group F+BWA868C(40μg·10μl^-1).Injected intrathecally with the corresponding doses of DMSO,BW245C or BWA868C 1 hour after the preparation of fentanyl incision pain model.PWT and TWL were measured1d before the intravenous injection and 3h,6h,1d,2d,3d,5d,7d after the last intravenous injection.ResultsI:Compared with group C,the PWT and TWL of group I,group F,and group IF were decreased,and the reduction was most significant at 2d after injection（P<0.05）;compared with group I and group F,PWT and TWL of group IF were significantly reduced（P<0.05）,but there was no difference between group I and F（P>0.05）;compared with 1d before injection,TWL in each group and at all time points were significantly reduced（P<0.05）,and PWT in group I,group F,and group IF were significantly reduced at 1d,2d,and 3d（P<0.05）.And the dose of fentanyl increased,the results of PWT and TWL decreased significantly（P<0.05）.II:Compared with 1d before injection,PGE₂ increased from 1d to 3d（P<0.05）,PGD₂ increased from 1d to 7d,and PGD₂ increased most significantly at 5d and 7d（P<0.05）;H-PGDS m RNA increased most significantly at 5d（P<0.05）;DP1 m RNA increased significantly at 5d and 7d（P<0.05）,and there was no difference in L-PGDS m RNA and DP2 m RNA at all time points（P>0.05）.III:2 days after fentanyl injections,the levels of COX-1 and COX-2 in the spinal dorsal horn of mice in group IF increased significantly,and both COX-1 and COX-2co-localized with Neun.Compared with group IF,PWT and TWL in group IF+BW245C(8μg·10μl^-1)and group IF+BW245C(80μg·10μl^-1)were significantly higher（P<0.05）;compared with group I and group F,the PWT and TWL in group I+BW245C(80μg·10μl^-1)and group F+BW245C(80μg·10μl^-1)were significantly higher（P<0.05）.Compared with the IF group,PWT and TWL in group IF+BWA868C(4μg·10μl^-1)group and group IF+BWA868C(40μg·10μl^-1)were significantly reduced（P<0.05）;compared with group I and group F,PWT and TWL in group I+BWA868C(40μg·10μl^-1)and group F+BWA868C(40μg·10μl^-1)were significantly reduced（P<0.05）.Conclusion Fentanyl induced mechanical and thermal postoperative hyperalgesia in mice was dose-dependent.The COXs—H-PGDS—PGD₂—DP1 pathway may be involved in the mechanism of pain threshold elevation after fentanyl-induced hyperalgesia.Agitating DP1 receptor can alleviate hyperalgesia induced by fentanyl in mice,and inhibiting DP1 receptor can aggravate hyperalgesia induced by fentanyl in mice,all of these were dose-dependent.