Studies Of The Inhibitory Effect Of Melatonin On Morphine Withdrawal Syndromes And Signal Transduction In Morphine Dependent Rats

Objective: Morphine is an opioid, which is commonly used to relieve pain. But using it for long time will lead to tolerance, psychological and physiological dependence, withdrawal syndromes and hunting for drugs, which is not only a problem related to medical science, but a serious social problem. Drug dependence is a long term adaptation of central nervous system, which depends on the production of neonate peptides or proteins resulted from signal transduction, gene transcription and translation. Previous studies were conducted to investigate opioid receptor in brain and endogenous opioid peptides (EOP), but recent investigations had shown the changes of opioid receptor in brain and EOP couldn't explain the mechanisms of drug dependence. So the post-receptor mechanisms have become the focus in this field. Some studies indicated that cAMP, cGMP, Ca2＋and CaM took important effects during morphine dependence period. Melatonin (MT) acting as an important active material secreted by pineal, has extensive physiological and pharmacological effects. MT can inhibit opioid's withdrawal syndromes, but the mechanism remains unclear. The present study was undertaken to investigate the changes of cAMP, cGMP in cerebellum and striatum, the level of [Ca2＋]i, CaM in hippocampus and brain stem of morphine dependent and withdrawal rats, analyzed the signal transduction mechanisms of MT attenuating morphine withdrawal syndromes.Method: 60 healthy Wistar rats, half male and half female, weighting from 250g to 280g, were divided into six groups at random: NS control group (NS group), morphine dependence group (MOR group), nalxone precipitation group (NAL group) and MT treatment group (MT25 group, MT50 group, MT100 group). Five incremental dose (10mg·kg－1, 20mg·kg－1, 30mg·kg－1, 40mg·kg－1, 50mg·kg－1) of morphine was injected subcutaneously to establish the physical dependent model in rats. Withdrawal syndromes were induced by intraperitoneal injection of nalxone (5mg·kg－1), the intensity of withdrawal syndromes was evaluated according to the number of rats whose body weight loss is more than 2% and the diarrhea incidence. Rats were anesthetized and killed to get cerebellum and striatum after experiment. cyclic AMP (cAMP) and cyclic GMP (cGMP) contents were determined by radioimmunoassay (RIA). 20 healthy Wistar rats, half male and half female, weighting from 200g to 230g, were divided into four groups at random: NS control group (NS group), morphine dependence group (MOR group), nalxone precipitation group (NAL group) and MT treatment group (MT group). Cytoplasmic free calcium ([Ca2+]i) was measured using flow cytometry, the cells were stained with Furo-2 prior to be detected. The calmodulin activity in brain tissues was investigated with the help of flow cytometry after binding with trifluoperazine (TFP). The results of experiment were analyzed with SPSS statistics software, and a level of P<0.05.was considered statistically significant.Results1. The establishment of physical dependent model in rats and the inhibitory effect of MT on morphine withdrawal syndromesThe body weight loss and diarrhea of morphine dependent rats with nalxone precipitation were increased. Treatment with MT (25mg·kg－1,50mg·kg－1 and 100mg·kg－1) could decrease diarrhea, and MT100 was more effective than MT25; the body weight loss (vs NAL group, P<0.05 or P<0.01) was inhibited by MT50 and MT100, the effect of latter was stronger.2. The effect of melatonin on cAMP level in cerebellum and striatum of morphine withdrawal ratsThere was no change in cAMP content in cerebellum of morphine dependent rats (vs NS group, P>0.05). No difference was investigated between NAL group and MOR group (P>0.05). Increase of the cAMP contents in striatum was detected (vs NS group, P<0.05) in MOR group (738.2±189.3 pmol·g-1). The increase of the cAMP was more significant in NAL group (905.6±164.3 pmol·g-1). After treatment with MT 100 mg·kg－1(730.5±133.4 pmol·g-1), the up-regulation was decreased (vs NAL group, P<0.05)