Nrf2 Regulates Incisional Pain-remifentanil Iduced Hyperalgesia By Autophagy In Rats

Objective:Opioids,widely used in the treatment of intraoperative analgesia and in various types of moderate and severe pain,have been one of the most widely used powerful analgesic drugs,having important clinical status.Opioids could cause some side effects.Over the years,the opioid-induced hyperalgesia?OIH?,one of the side effects,has been concerned by scholars.OIH that induced by contacting opioids in surgeries,often complicates patients situations by harder postoperative pain controling,increased dosage of analgesic drugs and even the development of an acute pain to chronic pain.Due to the undefined mechanism,there is still a short of effective solutions.Remifentanil is potent as the only ultra short-acting MOR agonist in opioids.Its unique pharmacokinetic properties meet the requirements of clinical anesthesia that being efficient,safe and controllable so that it have been frequently used in intravenous analgesia in general anesthesia.Meanwhile,OIH and OPH induced by remifentanil is more likely to occur than that by other opioids,affecting the clinical application of remifentanil.The study of its mechanism is of great meaning to the prevention and intervention of this clinical problem.Autophagy is a kind of cellular"self-feeding"phenomenon.In this process cells sequester excessive cytoplasmic molecules or organelles like lipids,long-lived proteins,mitochondria with double membraneous autophagosomes,then the autophagosomes fused with lysosome for the wrapped cytoplasmic components to be degraded and recycled.As is known,autophagy participate in many physiological and pathological processes.Several studies have shown that autophagy can improve the energy conditions and function of nervous system,serving as a neuroprotective role.Nrf2 is not only a central molecule in the antioxidative system,but also a transcription factor of a variety of autophagy associated proteins.It has an interaction with autophagy.Recently,numerous reports have found that autophagy is involved in the regulation of pain and the role of opioids in the body.In our study,using the rats model of incision pain-remifentanil hyperalgesia as the research object,we determined the expression of some autophagy associated proteins and transcription factor Nrf2 in spinal cord of the experimental animals.And under the intervention of autophagy regulator 3-MA and the Nrf2 regulator tBHQ,corresponding molecules in the spinal cord of experimental animals and the behavioral changes responsing to pain stimuli were measured,then statistical analysis was conducted in order to understand the possible relationship among autophagy,Nrf2 signaling pathways and the remifentanil induced incisional hyperalgesia.Methods:There were two parts in the expreiment.The first part:The change of spinal autophagy in rats suffering incisional pain-remifentanil induced hyperalgesia and its effect on the hyperalgesia.40 rats were randomly allocated into the groups with 8 rats in each group:the control group?group C?,the remifentanil infusion group?group R?,the incision group?group I?,the incision+remifentanil infusion group?group RI?and the incision+remifentanil infusion+autophagy inhibitor 3-methyladenine?group MA?,.First,the models of rats with incision-remifentanil induced hyperalgesia were set up.At T_0-4?1d before molding,2h,6h,24h,48h after molding?,the behavioral indicators MWT?mechanical paw withdrawal latency?and TWL?thermal paw withdrawal threshold?were determined to assess the modeling results.At T₄,when the hyperalgesia reached its peak,the AGT expressions of LC3II,beclin-1 and p62 in the intumescentia lumbalis of spinal cord were measured by Western Blot immediately after behavial indicators measurement had been finished.Then we regulated the autophagy by the autophagy inhibitor 3-MA which was injected intraperitoneally to reverse the autophagy changes.We measured MPW and PWL at T_0-4,and autophagy associated protein p62,LC3II,Beclin-1 at T₄,then statistical analysis was conducted for the possible contaction between autophagy and the hyperalgesia.The second part:The change of the Nrf2-autophagy signaling pathway in rats suffering incisional pain-remifentanil induced hyperalgesia and its effect on the hyperalgesia.The rats were randomly divided into 3 groups:the incision group?group I?,the incision+remifentanil infusion group?group RI?,the incision+remifentanil infusion+Nrf2 agonist group?group tBHQ?.We measured MPW and PWL of the rats in 3 groups at T_0-4.At T₄ the protein and mRNA expression of Nrf2 and its downstream molecule HO-1,the expressions of Beclin-1 and LC3II,the content of MDA and SOD which are redox indicators were measured.Then Nrf2 agonist tBHQ was injected intraperitoneally to reverse the Nrf2 change,the expression of the related molecules described above were determined and the possible correlation between Nrf2-autophagy signaling pathway and the hyperalgesia was analyzed.Results:1.The first part:the behavior test results:compared with group C,the values of MPW and PWL in group I,group R and group RI were significantly reduced?p<0.01?.In group RI compared with group R and group I,as well as in group MA compared with group RI,MPW and PWL values were significantly reduced?p<0.01?.Western blotting results:Compared with group C,in group R and group RI the protein expression of LC3II and Beclin-1 were increased?all p<0.05?,of p62 was reduced?p<0.05?,while in group I only LC3?was increased?all p<0.05?;Compared with RI,in group MA the protein expression of LC3II and Beclin-1 were decreased while of p62 was increased?p<0.05?.2.The second part:Compared with group I,in group RI,the values of MPW and PWL were significanly reduced?p<0.05?,and the expression of LC3II and Beclin-1were increased,the protein expression of Nrf2 was decreased,the protein and mRNA expression of HO-1 both were decreased,SOD was decreased,and MDA was increased?all p<0.05?.Compared with group RI,in group tBHQ,the values of MPW and PWL were increased?p<0.05?,The protein expression of Nrf2,the protein and mRNA expression of HO-1 as well as protein expression of LC3II and Beclin-1 were all increased.SOD was increased while MDA was decreased.?all p<0.05?.Conclusion:1.Intraoperative infusion of remifentanil aggravates the postoperative incisional hyperalgesia in rats.2.Autophagy associated proteins LC3II,Beclin-1,p62 were significantly changed in the lumbar intumescent of incision-remifentanil infusion rats,show that remifentanil infusion may affect spinal autophagy activity in RI rats.3.Intraperitoneal injection of autophagy inhibitor 3-MA can worsen the hyperalgesia caused by incision-remifentanil infusion.Autophagy may play a protective role in the hyperalgesia and may be a new target in its prevention and treatment.4.The transcription factor Nrf2's transcription activity was restrained significantly in lumbar intumescent of incision-remifentanil infusion rats.5.The Nrf2 agonist tBHQ improved the hyperalgesia cansed by incision-remifentanil infusion,further activated autophagy in lumbar intumescent of RI rats.Nrf2-autophagy signaling pathway may be involved in the regulation of the hyperalgesia in RI rats.