The Role And Mechanism Of Tetrodotoxin Or Combined With Sufentanil In A Rat Model Of Acute Postoperative Pain

BackgroundWith the rapid development of modern surgery,the number of patients with postoperative pain is gradually increasing,but the current postoperative pain management is still not the most ideal.Nearly 10% of patients who undergo surgery worldwide each year develop chronic postoperative pain,which may be partly due to inadequate management of acute postoperative pain.At present,opioids are still the most important drugs in clinical acute postoperative pain management,but side effects such as nausea and vomiting greatly limit the application,and there is a risk of drug abuse.Therefore,there is an urgent need for new methods of analgesia in clinic.Tetrodotoxin is a natural neurotoxin that blocks voltage-gated sodium channels on the excitatory cell membrane with high selectivity and affinity.More and more studies have shown that TTX has significant analgesic effects in a variety of pain models,such as acute and inflammatory pain,neuropathic pain,cancer pain and so on,by affecting TTX-S sodium channels.However,there are no studies on the role of TTX in acute postoperative pain.We speculate that the antinociceptive effect of TTX may be related to its anti-inflammatory factors and blocking TTX-S sodium channels.In recent years,with the introduction of the concept of "multimodal analgesia",there are more choices of drugs and techniques for postoperative pain management.In theory,due to the different mechanisms and side effects of different drugs,the use of combinations of analgesic drugs with different pharmacological effects to manage perioperative pain can improve the safety and effectiveness of pain treatment.The endogenous opioid system,which has the strongest analgesic effect,is a widely concerned object in pain research.Some studies have shown that sodium channels may be involved in the regulation of endogenous opioid peptide system.Therefore,we then studied the antinociceptive effect of TTX combined with low-dose sufentanil in acute postoperative pain model,and we speculated that TTX may enhance the antinociceptive effect by enhancing endogenous opioid peptide system and opioid receptors.Therefore,in this study,a rat model of acute postoperative pain was established,and subcutaneous injection of TTX alone were used to detect the effects of TTX alone on inflammatory factors and TTX-S sodium channels.Preenkephalin gene PENK,μ opioid receptor gene(opioid receptor μ 1,OPRM1),δ opioid receptor gene(opioid receptor δ 1,OPRD1)and κ opioid receptor gene(opioid receptor κ 1,OPRK1)were detected when combined with sufentanil.The purpose of this study is to explore the role and mechanism of TTX in acute postoperative pain,and the combination of sufentanil is expected to develop new postoperative analgesic drugs and interpret the combination of sodium channel blockers and strong opioid drugs from a new point of view.Methods and ResultsPart I: Study on the Effect and Mechanism of Subcutaneous TTX Injection on Acute Postoperative Pain in RatsMethods: 1.Forty adult male SD rats were randomly divided into control+NS group,control+TTX group,PI+NS group and PI+TTX group,with 10 rats in each group.The method of administration was as follows: control+NS group: subcutaneous injection of NS;control+TTX group: subcutaneous injection of TTX(6μg/kg);PI+NS group: 5 minutes after plantar incision,subcutaneous administration of the same volume of NS;PI+TTX group:TTX(6μg/kg)was subcutaneously given after the same PI model.2.The mechanical paw withdrawal threshold was measured before operation(-3d~-1d)and at 1 h,2 h,4 h,6 h and24 h after operation.The levels of IL-1β,TNF-α in DRG tissue of L4-L6 segment and the TTX-S sodium channel were detected by q PCR and Western Blot methods.The levels of IL-1β and TNF-α in serum samples were detected by ELISA method.Results: 1.Behavioral results: compared with control+NS group,MPWT in control+TTX group had no statistical difference(p>0.05),MPWT in PI+NS group began to decrease significantly at 1 hour after operation(p<0.05);compared with PI+NS group,MPWT in PI+TTX group increased significantly at 1 hour and lasted until 24 hours after operation(p<0.05).2.Western blot and q PCR were used to detect the DRG tissue 24 hours after operation.Compared with control+NS group,IL-1β and TNF-α m RNA levels in DRG tissue of PI+NS group were significantly increased(p<0.05),while IL-1β and TNF-αm RNA and protein levels of PI+TTX group were significantly lower than those of PI+NS group(p<0.05).The q PCR results of TTX-S sodium channel showed that the m RNA of Nav1.1,Nav1.6,Nav1.7 in PI+NS group was significantly higher than that in control+NS group(p<0.05),and the m RNA of Nav1.1,Nav1.3,Nav1.6,Nav1.7 in PI+TTX group was lower than that in PI+NS group(p<0.05).ELISA detection of serum showed that there was no significant difference in the levels of IL-1β and TNF-α between control+TTX group and control+NS group(p>0.05),but the levels of IL-1β and TNF-α in PI+NS group were significantly higher than those in control+NS group(p<0.05),while the levels of IL-1β and TNF-α in PI+TTX group were significantly lower than those in PI+NS group(p<0.05).Part II: Effect and Mechanism of Tetrodotoxin Combined with Sufentanil on Acute Postoperative Pain in RatsMethods: 1.Forty adult male SD rats were randomly divided into PI+NS group,PI+TTX group,PI+ Suf group and PI+TTX+ Suf group,with 10 rats in each group.The methods of administration were as follows: PI+NS group: rat model of plantar incision pain was established under isoflurane maintenance,and the same volume of NS was given subcutaneously 5 minutes after surgery.PI+TTX group: PI model was also established and TTX(6μg/kg)was given subcutaneously;PI+ Suf group: PI model was also established and Suf(5μg/kg)was given subcutaneously.PI+TTX+ Suf group: PI model was also established and TTX(6μg/kg)+ Suf(5μg/kg)were subcutaneously given.2.The threshold value of mechanical paw withdraw threshold was measured before surgery(-3d~-1d)and 1h,2h,4h,6h and 24 h after surgery.The Preproenkephalin(Penk),μ opioid receptor 1(OPRM1)and δopioid receptor 1(OPRD1)and κ opioid receptor 1(OPRK1)in DRG tissues were detected by q PCR and Western Blot 24 h after surgery.Results: 1.Behavioral results: When TTX was combined with sufentanil,the postoperative MPWT in PI+TTX+ Suf group increased more significantly and lasted longer(p<0.05).2.Molecular level detection results: when combined with sufentanil,Western blot and q PCR results showed that compared with PI+NS group,there was no statistically significant difference in the levels of PENK,OPRM1,OPRD1 and OPRK1 in PI+TTX group(p>0.05),while there was down-regulation of PENK and OPRM1 in PI+ Suf group(p<0.05).PENK,OPRM1,OPRD1 m RNA and protein levels were up-regulated in PI+TTX+Suf group compared with PI+Suf group(p<0.05),but there was no statistical difference in KOR1 m RNA and protein levels among all groups(p>0.05).ConclusionsThe behavioral results showed that the PI model was established successfully,and subcutaneous injection of TTX could relieve the mechanical pain threshold,block the Nav1.1,Nav1.3,Nav1.6,and Nav1.7 of the TTX-S type sodium channel,and reduce the level of IL-1β and TNF-α.TTX combined with sufentanil can enhance and prolong the postoperative analgesic effect of low dose sufentanil,and this effect is related to the up-regulation of the expressions of proenkephalin,μ opioid receptor and δ opioid receptor.