Intervention Study Of Purine Nucleotide Compensation On The Function Of Opioid Drug

The abuse of morphine and its chemical derivate heroin has caused serious medical and social problems.Heroin is an synthetic acetyl derivate of morphine,it's easier to pass the blood-cerebral barrier to play its pharmacological action,so the addiction of heroin is stronger.It has been the most widely abuse drug of the world. Drug addiction is a chronic and recurring brain disease.Its key feature is the compulsive drug abuse. Learing and memory has been witnessed to play an important role in the formation and maintain of the addiction .Hippocampus is the learing and memory centre,it may act an important part in addiction.We have got some achievements in the realationship between morphine and heroin and purine nucleotide metabolism.We tried to compensate AMP and GMP on the basis of our reseach to search their effect on heroin and explore the new pathway to lessen the tolerant and withdrawl symptom.The first part of this paper is the study on the tissue level.We established the heroin and compensate purine nucleotide models of rats. Adult male Wistar rats were randomly divided into 8 groups(10 rats each group).They were treated with①saline (NS) ,②AMP and GMP (A) ,③heroin (H) ,④heroin plus AMP and GMP (AH) ,⑤heroin (W3 ),⑥heroin (W9) ,⑦heroin (A3),and⑧heroin (A9) for 10 days, the①to④were killed on the 11th day , the⑤and⑥were killed on the 14th and 20th day , the⑦and⑧were treated with AMP and GMP for 3 and 9 days ,then were killed on the 14th and 20th day.We choosed the hippocampus as the target tissue .Firstly we studied the effect of heroin and AMP and GMP on the key enzyme of purine nucleotide metabolism, adenosine kinase (AK),hypoxanthine-guanine phosphate ribose transferase (HGPRT),Adenosine deaminase (ADA) and xanthine oxidase (XOD) on the protein and gene level. Secondly we studied the gene expression of proliferating cell nuclear antigen (PCNA) brain-derived neurotrophic facto(rBDNF)and an anti-apoptosis gene (BCL-2) by reverse transcribed polymerase chain reaction (RT-PCR). The last we studied the morphologic changes in hippocampus in NS,H and AH group,2 rats for each group.The second part of this paper is the study on the cell level. We determined the effect of heroin and AMP and GMP on C6 glioma cell of the above 7 gene expression by RT-PCR and PCNA,BDNF and BCL-2 changes by immuocytochemistry and immunofluorescence techniques.The third part of this paper is the study of proteome. Protein is the performer of biological functions. The changes of the cell proteome may be the real reason of drug addiction. So we applied two-dimensional electrophoresis to study the effect of heroin on C6 glioma cell .1. Uric acid and enzymes detection in plasma and hippocampus of heroin dependent ratsAs uric acid is the end product of purine nucleotide catabolism, determination of plasma uric acid concentration may reflect the catabolism state of purine nucleotides. In this study, we found that there was a significant increase in plasma uric acid of heroin treated rats as compared with controls . This result suggested that heroin addiction might cause the enhancement of purine nucleotide catabolism. What is the mechanism of heroin's effects on purine nucleotide catabolism? Does it affect the key enzyme ADA and XOD ? The results show us that During heroin administration , the levels of plasma and hippocampus ADA and XO increased significantly ,but coadministration of heroin with AMP and GMP decreased the activities of ADA and XOD in plasma and hippocampus, the activity of ADA in plasma and hippocampus is decreased in A3 and A9 group. This result implied that AMP and GMP can affect the function of heroin on purine nucleotide catabolism.2. Effects of heroin and AMP and GMP on gene expression of key enzymes of purine nucleotide metabolism in hippocampusRT-PCR was used to examine the gene transcripts of ADA,XOD,AK and HGPRT in hippocampus. Compared with that in the control group, the transcripts of ADA and XOD mRNA were significantly higher in rats treated with heroin. AMP and GMP had no significant effects on the ADA and XOD gene expression , but coadministration of heroin with AMP and GMP decreased the transcripts of ADA and XOD mRNA in hippocampus,this result is correspondence with its enzymes. It implied the influence might occur on transcript level . HGPRT and AK are the key enzymes of salvage pathway of purine nucleotide synthesis. There are only salvage pathways in brain. Compared with that in the control group, the transcripts of AK showed decreased tendency, while HGPRT mRNA was markedly decreased in rats treated with heroin.Coadministration of heroin with AMP and GMP decreased the transcripts of AK mRNA and increased the transcripts of HGPRT mRNA.In conclusion, heroin might promote the catabolism of purine nucleotide by increasing the gene expression of the key enzymes, ADA and XOD in this process, and inhibit the anabolism of purine nucleotide by decreasing the gene expression of the key enzymes, AK and HGPRT in this process in hippocampus . AMP and GMP might reverse this function of heroin.3. Effects of heroin and AMP and GMP on gene expression of PCNA,BDNF and BCL-2 in hippocampusRT-PCR was employed to assess the three gene expression in hippocampus . PCNA has the close relationship with anabolic metabolism,damage and repair of DNA. BDNF is a NF widely expressing in central nervous system (CNS )with wide physiological effects including the extension of neuron life, modulation of neuron phenotype and connection, and promotion of neuron regeneration. BCL-2 , as an important apoptotic suppressor gene , has been testified to have obviously relationship with the deterioration of the studing and memory function in the centrum . The three gene are very important in drug addiction . The results indicated that heroin could inhibit the three genes expression ,AMP and GMP could reverse this effect of heroin on PCNA and BDNF . The detail mechanism was not clear.4. Effects of heroin and AMP and GMP on the morphologic changes of hippocampusWe observed the ultra-microstructure of hippocampus in control,heroin and heroin plus AMP and GMP groups using the optical microscope and transmission electron microscope (TEM). Denaturation and necrosis were seen in hippocampus in heroin and AH group with optical microscope. The results of TEM showed that the neuron and neurofibra appeared the ischemic,anoxemia and retrogression changes in heroin group. At the same time,we found there are a large number of synapses and synaptic vesicles in heroin group. The structure of neuron and the plasticity of the synapses are the basic structure of studing and memory.The mechanism of drug addiction might be the changes of neuron and the synapses. The changes of ultra-microstructure in AH group was much better than it in heroin group.It implied purine nucleotide might improve the damage of heroin in hippocampus. 5. Effects of heroin and AMP and GMP on gene expression of key enzymes catalyzing purine salvage pathway and purine catabolism in C6 glioma cellsC6 glioma cells has already been used as a model system to study the mechanism of opioid action. ADA and XOD is the key enzymes of purine nucleotides catabolism and the major function of XOD is to produce uric acid. In this experiment, exposure of heroin to C6 glioma cells caused an enhancement of ADA and XOD gene expression. HGPRT and AK is the key enzymes of salvage pathway of purine nucleotide synthesis. Their transcript changes in C6 glioma cells after exposure of heroin for 24h showed decreased tendency. Coadministration of heroin with AMP and GMP decreased the transcripts of ADA and XOD mRNA . The results indicated that heroin could enhance the catabolism of purine nucleotide in cell level by regulating the gene expression of four key enzymes of ADA,XOD,HGPRT and AK. AMP and GMP can reverse this effect of heroin on ADA and XOD .These data were in accord with the results of animal experiments.6. Effects of heroin and AMP and GMP on gene and protein expression of PCNA,BDNF and BCL-2 in C6 glioma cellsRT-PCR was used to examine the gene transcripts of PCNA,BDNF and BCL-2 in C6 glioma cells . Immuocytochemistry and immunofluorescence techniques were used to test the changes of PCNA,BDNF and BCL-2 in C6 glioma cells. Compared with that in the control group, the transcripts of PCNA mRNA were significantly lower , while the transcripts of BDNF and BCL-2 mRNA showed decreased tendency after exposure of heroin for 24h. AMP and GMP has no significant effects on the BDNF and BCL-2 gene expression , but they decreased the PCNA gene expression .Coadministration of heroin with AMP and GMP increased the transcripts of PCNA and BCL-2 mRNA in C6 glioma cell ,while there was no changes on BDNF gene expression. The immuocytochemistry and immunofluorescence results demonstrated that heroin inhibited the expression of PCNA and BCL-2 ,while increased the expression of BDNF at protein level after exposure of heroin for 24h. Coadministration of heroin with AMP and GMP can reverse the function of heroin . This result was not correspondence with its gene expression. It implied the influence might occur in transcript and post-transcript level .7. 2-DE analysis of heroin on C6 glioma cell We applied two-dimensional electrophoresis to study the effect of heroin on C6 glioma cell .After analysis of the 2-DE profile protein spots 1.5 or more folds increasing or decreasing were found out . In the 2-DE profile imaged by coomassie blue staining 17 protein spots with significant difference were found , comparied with the control group 12 protein spots were decreased, while 5 protein spots were increased. 29 protein spots with significant difference were found by silver staining, 20 protein spots were decreased, while 9 protein spots were increased.Does those protein take part in the drug addiction? It needs more researches.In summary, hippocampus is an important tissue for drug addiction and tolerance . Heroin administration could increase the catabolism and decrease the anabolism of purine nucleotide by regulating four key enzymes─ADA,XO,HGPRT and AK in vivo and in vitro . The mechanism of drug addiction has some relationship with this process . Compensate AMP and GMP had some reverse function to heroin , the mechanism need to been explored .The use of heroin inhibited the PCNA,BDNF and BCL-2 gene expression , it might be the reasons of inhibiting cell proliferation , leading the changes of neuron plasticity and causing the changes of studing and memory . Those changes might take part in the formation of addiction memory. The study of protome is the cores of the postgenome era . The different proteins between normal and experimental tissue or cell might have some relations with heroin , they might be the labeled molecules or the target of drug design to solve the drug addiction,tolerant and withdrawal.