Molecular Mechanisms Of Naloxone And Opioid Drugs Involve In Regulation Of Gastrointestinal Motility In CNS And Peripheral In A Rat Model Of Postoperative Pain

Objective Incisionial pain, postoperative opioid analgesia, and naloxone administration are quite common in patients who undergo surgeries. Accurate evaluation of endogenous motilin expression both in peripheral sites and CNS during pathophysiological conditions would help in understanding, preventing, and treating GI disorders caused by these inevitable factors. The present study attempted to simulate the clinical procedure involving all mentioned factors to analyze their interactive effects on motilin expression in peripheral hippocampal and spinal cord regions. The study aimed to investigate effects of intrathecal morphine and fentanyl combined with low-dose naloxone on expression of motilin and its receptor in a rat model of postoperative pain. The study results provided understanding on the variation trend and potential relationship between motilin and CNS, and they provided basis for potential mechanism of multilevel GI motility regulation.Methods(1)156 healthy,male,adult SD rats,weighing 180-220 g,were randomized into two groups: Control group(group C,n=78) and incisional pain group(group P,n=78),the rats in P group received incision on the right plantaris.Six rats,chosen randomly from each group were measured for Paw Withdrawal Mechanical Threshold(PWMT) and Paw Withdrawal Thermal Latency(PWTL) at different time points of 24 hours before operation(T0) and 1 hour(T1),6 hours(T2),24 hours(T3),48 hours(T4) and 72 hours(T5) after operation.Twelve rats were chosen from eachgroup at T0-5 and sacrificed and the plasma、the mucosal tissues of gastric body and duodenum was prepared for motilin concentration measuring by means of ELISA.(2)72 healthy male Sprague-Dawley rats(weight 180~220 g), successfully intrathecally catheterized, were randomly divided into 6 groups(n=12 in each group): normal saline group(NS group), incisional pain group(P group), morphine, fentanyl + incisional pain group(MF group), MFN1 group(morphine, fentanyl, naloxone + incisional pain), MFN2 group(morphine, fentanyl, naloxone + incisional pain), MFN3 group(morphine, fentanyl, naloxone + incisional pain). All groups except NS group were needed to be made the model of incisional pain on the right plantar surface. At 24 hours before intrathecal cathetherization(T0), 24 hours before modelling(T1), 1 hours(T2), 3 hours(T3), 6 hours(T4), 24 hours(T5), 48 hours(T6) and 72 hours(T7)after modelling respectively, Paw Mechanical Withdrawal Threshold(PMWT) and Paw Withdrawal Thermal latency(PWTL) were detected on the rats’ right hind paw in 6 rats of all groups. The other 6 rats in each group were sacrificed 6 hour after operation, with plasma, stomach, duodenum and hippocampus of rats were quickly extracted for determination of the expression of motillin using ELISA.(3)72 healthy male Sprague-Dawley rats(weight 180~220 g), successfully intrathecally catheterized, were randomly divided into 6 groups(n=12 in each group): normal saline group(NS group), incisional pain group(P group), morphine+fentanyl(MF group), naloxone+incisional pain group(NP group), morphine+entanyl+incisional pain group(MFP group), morphine+fentanyl+naloxone+incisional pain(MFNP group). 3 groups involving incisional pain received an incision on the right plantar surface of each rat. Took 6 rats from each group for Paw Mechanical Withdrawal Threshold(PMWT) and Paw Withdrawal Thermal latency(PWTL) detection at 5 different time points: 24 hours before intrathecal cathetherization(T0), 24 hours before modelling(T1), 1 hours(T2), 3 hours(T3), 6 hours(T4) after modelling respectively. Deheaded the rest 6 rats of each group for plasma withdrawal as well as tissue extraction from stomach, duodenum and spinal cord. Prepared the samples for further motilin concentration detection with ELISA. Meanwhile,using western Blog to analyse spinal motilin receptor expression.Results(1) Compared with group NS, PWMT、 PWTL and motilin levels in plasma and gastric body were significantly decreased at T1-4(P<0.05). Motilin levels in duodenum were significant increased at T1-4(P<0.05),and no significant change was found at T0 and T5 in P group(P>0.05).The motilin levels were positively correlated with PWMT and PWTL(r=0.9407,r=0.9918,respectively,P<0.01)in plasma in P group; The motilin levels were positively correlated with PWMT and PWTL(r=0.9702,r=0.9307,respectively,P<0.01)in gastric body in P group;The motilin levels were negatively correlated with PWMT and PWTL(r=-0.9913,r=-0.9748,respectively,P<0.01)in duodenum in P group.(2) Compared with NS group, the PMWT of P and MFN3 groups were significantly shorter(P<0.05) at T2, T3, T4, T5 and T6, while the PMWT of MFN2 group were significantly shorter only at T4. Compared with NS group, the PWTL of P group were significantly shorter(P<0.05) at T2, T3, and T4, while MFN3 at T2, T3, T4, T5 and T6. Compared with P group, the PWTL of MFN3 group were significantly longer(P<0.05)at T2, T3, T4 and T6. At 6 hours after operation, the expression of motillin in hippocampus and duodenum in P group were significantly higher than NS group(P<0.05). The expression of motillin receptor in hippocampus in MF, MFN3 and MFN2 groups were significantly higher compared with NS group, with MFN2 group the most.(3) In P group PWMT at T0 and T1 were comparable.However,T2、T3 and T4 have significant differences when compared to T0 or T1 seperately(P<0.05). In NP group PWMT at T2 is not significantly different when compared to T0 or T2(P>0.05), however T3 and T4 showed significant differences toward T0 and T1(P<0.05). In MFPN group, PWMT at T4 showed no difference when compared to T0 or T1(P>0.05). There are significant differences on spinal cord motilin expression between groups of P, MFP and NS group(P<0.05), however no differences were found between MFPN and NS group(P>0.05). When it comes to spinal cord motillin receptor expression, it was significantly upregulated in all groups except for NS group( P<0.05).Conclusion(1) The levels of motilin in a rat model of acute incisional pain is decreased in the plasma and gastric body and increased in duodenum.(2) In a rat model of incisional pain, intrathecal injection of naloxone at 1 ng/kg could inhibit the up-regulation of motillin in hippocampus and peripheral tissues caused by morphine, fentanyl and incision pain, and up-regulate the PTWL, enhancing the analgesic effects of opioids.(3) Spinal cord motillin and its receptor may participate in gastrointestinal regulation. Opioids, naloxone and acute incisional pain are involved in the regulation of motillin and its receptor expression on the level of spinal cord; however their individual influence was not quite consistent.