Study On The Relationship Between Phosphatidylethanolamine Binding Protein And Opioid Dependence

Drug addiction is a chronic, relapsing disorder in brain in which compulsive drug-seeking and drug-taking behavior persists despite serious negative consequences. Prolonged or repeated use of addictive substances, such as opioids, induces adaptive changes in the central nervous system that lead to tolerance, physical dependence, sensitization, craving and relapsing. Although the exact mechanisms are still unknown, researches in recent years have shown that these adaptations not only happened in the opioid receptor system itsself, but also in other non-opioid systems such as excitatory amino acids receptor , dopamine receptor, acetylcholine receptor and imidazoline receptor systems.Proteomic analysis of our previous study revealed that the expression of phosphatidylethanolamine binding protein (PEBP) changed in some brain regions of morphine dependent rats. Many researches have shown that PEBP played a variety of biological roles. It is the precursor protein of hippocampal cholinergic neurostimulating peptide (HCNP), which may play an important role in the septal cholinergic development of the hippocampus. Also PEBP is associated with an increasing number of diseases through its involvement in several cell signaling cascades (Raf-1/MEK/ERK, GPCRs). Thus the aim of this study is to find out whether PEBP is involved in the pathogeny of opioid dependence and to investigate the possible mechanisms.The first step in this study was to identify the result of proteomics. Results of Western blot showed that among six brain regions studied, PEBP was significantly up-regulated by chronic morphine in hippocampus and nucleus accumbens (60%, 22% increase vs. saline group respectively), while down-regulated in medial prefrontal cortex (35% decrease vs. saline group). On the other hand, there were no significant changes of PEBP expression in striatum, brain stem and cerebellum. Although these results were not completely consistent with proteomic analysis, we confirmed that PEBP was regulated by chronic morphine in special brain regions.According to the results of proteomics and Western blot, the expression of PEBP was up-regulated significantly by chronic morphine in hippocampus. To learn more about the regulation of PEBP in opioid dependence, the changes of PEBP expression in hippocampus during different time courses were observed by Western blot in morphine dependent and natural withdrawal rat models. Results showed that PEBP was up-regulated 4 d and 8 d after morphine injection compared with untreated rats. While 3 d after the final morphine injection (3 d of natural withdrawal), the expression of PEBP decreased to the control level and then up-regulated again 7 d after natural withdrawal. This up-regulation of PEBP remained until 28 d after natural withdrawal. However, the expression of PEBP showed no change during the same time courses of saline-treated rats. Meanwhile, we observed withdrawal symptoms with the same animal models to investigate the possible behavioral significance of this regulation. Compared with saline-treated control group, morphine dependent rats displayed significant withdrawal symptoms in the early 9 days of natural withdrawal period. The highest score was obtained on the second day. Also the weight of the natural withdrawal rats decreased from the first day and reached the lowest level on the second day. These results suggested PEBP might have negative correlation with the expression of morphine withdrawal syndroma.Since above results indicated the expression of hippocampal PEBP altered during morphine dependence and withdrawal, antisense oligonucleotide strategy was used to study whether down-regulation of PEBP in hippocampus influence the development and/or expression of physical dependence. Softwares such as Sfold, Mfold and RNA structure were used to design ASODN targeting the coding sequence (CDS) of PEBP. Then the effective ASODN was screened by RT-PCR and Western Blot in PC12 cells. Finally precipitated withdrawal behavior of morphine dependent rats was observed after down-regulation of PEBP by continued microinjecting of ASODN in hippocampus. It was showed several (wet dog shakes, irritability and sialorrhea) but not all withdrawal symptoms were markedly enhanced in PEBP-down-regulation rats. The overall withdrawal score was significantly increased in ASODN-treated morphine dependent rats, while this did not happen in SODN-treated group.Then results of Western blot identified down-regulation of expression of PEBP in hippocampus of ASODN-treated morphine dependent rats compared with NS-treated group and morphine-treated group.HCNP is an 11 amino acid peptide which is released from N-terminal of PEBP. It plays an important role in the septal cholinergic development of the hippocampus by enhancing the activity of ChAT. Previous studies have found that the hippocampal cholinergic system was involved in the development and expression of opioid physical dependence. Thus we continued to observe activity changes of the hippocampal cholinergic system during morphine dependence and natural withdrawal to see if PEBP was involved in opioid dependence through the hippocampal cholinergic system. Using different antibodies of PEBP, we observed the level of HCNP by Western blot. Results showed that the changes of HCNP level in hippocampus of morphine dependent and withdrawal rats had a similar trend to the changes of PEBP. Then ChAT activity was measured to observe if this enzyme activity changed with the level of HCNP. We found ChAT activity in hippocampus varied with the level of HCNP: it was increased significantly 4 d and 8 d after morphine administration, then decreased to the control level after 3 d and increased again after 14 d which continued until 21 d of natural withdrawal. This time-dependent changes in HCNP level and ChAT activity were similar to those in PEBP expression, suggesting PEBP regulated morphine dependence and withdrawal through the hippocampal cholinergic system.In conclusion, in the present study we found hippocampal PEBP participated in morphine dependence at first time. The expression of PEBP in hippocampus had different changes during morphine dependence and natural withdrawal. Down-regulation of PEBP in hippocampus enhanced the expression of precipitated withdrawal symptoms, and the modulation to hippocampal cholinergic system functions mediated the involvement of PEBP in morphine dependence.