The Mechanism Of Morphine Inhibition On PC12 Cell Proliferation And The Experimental Research Of Purine Nucleotides Compensation Treating Morphine Physical Dependence

Morphine is classical opiate receptor agonist. It can produce conspicuous analgesia and mitigation on central nervous system, and inhibit hyperalgia and uncomfortable symptom by psychological agent such as tension, restlessness and anxiety in patient. But it will induce physical dependence and psychological dependence, and the consumers feel uncomfortable and crave the drug severely when they are withdrawaled. The scientific researchers are looking for antinociceptive drug which can replace opioid and can't induce drug addiction. Purine nucleotides are compotent of nucleotides. Many research make clear that purine nucleotides have effect on relieving ache, so they are can be antinociceptive drug clinically. They have biological function by purinergic neuroreceptor mainly, and there is close relation between purine nucleotides and opioid system in relieving pain, and the two type receptors have cross reaction. So this research investigateed the mechanism of morphine on inhibiting cell proliferation and reducing intracellular purine nucleotides firstly. Then nucleotide metabolism, antinociceptive response and withdrawal symptom were observed in morphine dependent cells and rats by exogenous compensation with purine nucleotides.1.Morphine inhibited cell proliferation in rat pheochromocytoma (PC12)Cell viability was determined with MTT colorimetry, and cell survival rate was detected by trypanblue method, and cell cycle change was measured by flow cytometry in PC12 cells which were effected on in different concentration of morphine. RT-PCR and immunocytochemistry were used to examine the gene transcripts of proliferating cell nuclear antigen (PCNA).This experiment results were following:μmol·L^-1 morphine has obvious inhibiting effect on PC12 cell proliferation. The higher morphine concentration was, the lower cell viability was. Cell survival was decreased and dead cell was increased in high content morphine (100μmol·L^-1). The number of cells in G1 phase increased and the number of cells in S phase decreased in 10μmol·L^-1 morphine concentration or higher. The expression of PCNA mRNA became lower from 24 hours(P<0.05), and significantly lower in 48 and 72 hours (P<0.01) than control group. Furthermore, PCNA mRNA expression has no seeming changes (P>0.05) comparing to control when PC12 cells were treated with naloxone blockade then morphine or morphine then naloxone blockade.The above results indicate that 10μmol·L^-1 morphine can inhibit PC12 cell proliferation, and block cells in G1 stage and decrease cell in S stage, so morphine mainly repressed the cell proceeding of G1 stage to S stage. PCNA is one agent of cell proliferation. There is some relationship between cell proliferation inhibition and PCNA expression decreasing in chronic morphine administration through the lower expression of PCNA mRNA. There is some relationship between G1 stage cell increasing and PCNA production decreasing in flow cytometry since PCNA is close related to cell process. The changes of PCNA mRNA expression by morphine was attenuated by naloxone, a opioid receptor antagonist, which certify reversion effect of naloxone on decreased PCNA mRNA by morphine. The above phenomena suggest that morphine may regulate PCNA gene transcript byμreceptor, which is one reason of PC12 cell proliferation deceasing induced by long-term morphine.2.The effect of gene expression of purine nucleotide metabolic key enzyme in PC12 cell by morphineThe effect of morphine on purine nucleotide anabolism and catabolism was analyzed by examining mRNA expression of adenylate kinase ( AK ) and hypoxanthine-guanine phospho ribosyl transferase ( HGPRT ), which were purine nucleotide anabolic key enzyme, and adenosine deaminase ( ADA ) and xanthine oxidase ( XO ), which were purine nucleotide catabolic key enzyme after PC12 cells were treated by morphine in 12h, 24h, 48h and 72h.AK mRNA expression was downregulated since PC12 cells were treated 48 hours, and HGPRT mRNA expression was downregulated since 24 hours. ADA mRNA expression was upregulated since PC12 cells were treated 24 hours, and XO mRNA expression was upregulated since 48 hours. So in conclusion, chronic morphine administration decreased intracellular nucleotide anabolism by lessening mRNA expression of AK and HGPRT, and increased intracellular nucleotide catabolism by adding mRNA expression of ADA and XO. Purine nucleotide is one constituent of DNA and RNA, and the reduction of DNA and RNA was certain to cell proliferation inhibition. So we drew conclusion that there were possible relation between cell proliferation inhibition by morphine and nucleotide anabolism decreasing and catabolism increasing.3.The effect of compensative purine nucleotides on cell proliferation and purine nucleotide metabolism key enzyme gene expression PC12 cellsPurine nucleotide is one basic constituent of cell and essential material of cell proliferation. Morphine caused cell purine nucleotide scant synthesis and over disassociation, so purine nucleotide deficit is induced in long-term use. Purine nucleotide different modality ( AMP and GMP, ATP and GTP ) were compensated in this test. Cell viability was determined by using MTT in PC12 cells treated with morphine and purine nucleotides. RT-PCR was used to examine the gene transcripts of AK and HGPRT, ADA and XO.PC12 cell proliferation ratio was decreased in 10μmol·L^-1 morphine. The PC12 cell proliferation rate was no significant difference ( P>0.05 ) in the group of AMP+GMP and ATP+GTP at the sometime morphine treatment comparing to that in control group and single morphine group.AK mRNA and HGPRT mRNA expression were decreased in morphine group comparing to that in control group ( P<0.05, P<0.01 ). AK mRNA expression was no difference in morphine+AMP+GMP group comparing to that in morphine group and contol group. HGPRT mRNA expression in morphine+AMP+GMP group was higher than that in morphine ( P<0.05 ) and return to the level of control group. AK mRNA expression in morphine+ATP+GTP group was still lower than that in control group ( P<0.01 ). HGPRT mRNA expression in morphine+ATP+GTP group was no difference comparing to that in morphine group and control group ( P>0.05 ).ADA mRNA and XO mRNA expression were increased in morphine group comparing to that in control group ( P<0.05 ). ADA mRNA expression was no difference in morphine+AMP+GMP group comparing to that in morphine group and contol group. XO mRNA expression in morphine+AMP+GMP group was lower than that in morphine ( P<0.01 ) and return to the level of control group. ADA mRNA expression in morphine+ATP+GTP group was still higher than that in control group ( P<0.05 ). XO mRNA expression in morphine+ATP+GTP group was lower than that in morphine group and reture to control level..According to the above results, the inhibition of cell proliferation by morphine was relieved when PC12 cells were treated with morphine and purine nucleotide. Exogenous purine nucleotide can ameliorate the downregulation of purine nucleotide anabolic key enzyme gene transcripts and the upregulation of purine nucleotide catabolic key enzyme gene transcripts by morphine in PC12 cells. Purinergic nerve receptor maybe make great function. They improve the side effect of morphine in cell proliferation and nucleotide metabolism partly.4.Establishment of animal administration model and antinociceptive test48 clean male Wistar rats whose body weight were 180-200g were randomly divided into 6 groups: control group ( normal saline injection ), morphine group, s.c. morphine hydrochloride on the back bid in progressive dosage, purine nucleotide + morphine group, i.p. AMP + GMP equimolar at the same time morphine administration, control withdrawal group, morphine withdrawal group and purine nucleotide + morphine withdrawal group were treated drugs in the same style with corresponding above group, and were acut had withdrawal after the last administration. Body weight was measured in each group during this experiment. Rats tail pressure-pain threshold was detected in the 1st , 3rd and 6th hour after the first administration, and in the 3rd hour on the 1st,3rd,5th and 7th day after every morning administration by YLS-3E electric pressure pain instrument. Rat anti-thermo-pain ability was examined in the 3rd hour on the 2nd,4th,6th,and 8th day by tail flick test after the morning administration. Acute withdrawal symptom induced by i.p. 5mg·kg-1 naloxone was observed at the 4th hour after the last drug administration in the 4, 5 and 6 groups. The withdrawal symptom was recorded in 30 minutes including in rearing, tooth chattering, stretch, jumping, sneezing, body shake, ptosis, sialosis and diarrhea, and general score was evaluated.Rats weight has no significant variance in each group comparing to control group during in the early administration. The weight of morphine group rats were lessened from 6th day comparing to control group, and the weight of morphine + purine nucleotide group rats was similar to that of morphine group. Nociceptive response disappeared in the 1st hour after morphine treatment in the first day in rats. The pressure-pain threshold in morphine group was higher in the 3rd hour than that in control group, and returned to normal in the 6th hour. There was no statistical difference ( P>0.05 ) in morphine + purine nucleotide group and morphine group. The pressure-pain threshold in morphine group was higher than that in control group during drug administration ( P<0.01 ), and that in morphine + purine nucleotide group was higher than that in morphine group in the same day from the 3rd day ( P<0.01 ). The latency of tail flick was increased in morphine group from the 2nd day ( P<0.05 ), and the latency was longer in morphine + purine nucleotides group than in morphine group from the 6th day (P<0.05) than that in morphine group. The score of rearing, stretch, body shake, ptosis, sialosis and general score were lower in morphine + purine nucleotide withdrawal group than that in morphine withdrawal group ( P<0.05 or P<0.01 ).The above results make clear that purine nucleotide compensation have no significant adding function on the weight of morphine dependent rats. It can raise pressure-pain threshold and anti-thermo-pain ability in morphine dependent rats, and lessen some withdrawal symptom. So there is close relation in opioid system and purine nucleotide system, which displays the cross response in their receptors.5.The effect of purine nucleotide compensation on nucleotide metabolism in ratsExogenous purine nucleotide compensation can ameliorate the mRNA upregulation of purine nucleotide catabolic key enzyme ADA and XO by morphine in PC12 cells. This research examined the effect of purine nucleotide on the activity of ADA in plasma, brain cortex and small intestinal in morphine dependent and withdrawal rats and the concentration of uric acid ( UA ) which was purine nucleotide catabolic production. The concentration of Blood Urea Nitrogen ( BUN ) and Creatinine ( Cre ) in plasma which was related to renal function closely was detected.The ADA activity in plasma, brain cortex and small intestine of morphine group and its withdrawal group was higher obviously than that in corresponding control group ( P<0.05 or P<0.01 ). The ADA activity in plasma, brain cortex of morphine + purine nucleotide group and its withdrawal group was lower than that in corresponding morphine group ( P<0.05 or P<0.01 ). There was no difference in the ADA activity in small intestine between morphine + purine nucleotide group and other group ( P>0.05 ). The concentration of UA in plasma in morphine group and its withdrawal group was higher obviously than that in corresponding control group and that in corresponding morphine plus purine nucleotides group (P<0.05). The concentration of BUN and Cre in each group had no difference (P>0.05).The above results made clear that exogenous purine nucleotides compensation can degrade the enhancement of purine nucleotide catabolism by morphine in rats'plasma and some organs. Purine nucleotide catabolic production was not increased. On the contrary it tend to normal level, which mechanism has relation to signal conduction of receptors probably. There was no variance in the content of BUN and Cre in plasma in each group, which means that the change of UA was not caused by impaired renal function, but by the change of nucleotide catabolism by exogenous morphine and purine nucleotides.6.The effect on purine nucleotide compensation on neurotransmitter in rats'mid brainMesocorticolimbic dopaminergic system ( MLDS ) is the core location of psychological dependence induced by opiate, and it is neurophysiological base of all drug addiction. The synthesis and releasing of many neurotransmitter are changed during the course of opioid addiction and withdrawal, and the changes of dopamine ( DA ), norepinephrine ( NE ) and serotonin ( 5-HT ) are the base of addiction, dependence, tolerance and relapse. This experiment detected the concentration of DA, NE and 5-HT in rats'midbrain in purine nucleotides compensation during morphine dependence and withdrawal by fluorospectrophotometry.The content of DA in morphine group and morphine + purine nucleotide group was lower than that in control group ( P<0.05 ). Morphine withdrawal group and morphine + purine nucleotide withdrawal group was lower than that in corresponding control group ( P<0.05 ) too. The content of DA in morphine + purine nucleotide group and its withdrawal group was higher than that in corresponding control group ( P<0.05 ) too, and has no difference comparing to that in corresponding morphine group. The content of NE in morphine group was lower than that in control group ( P<0.01 ), and there was no statistic difference in the content of NE between morphine + purine nucleotide group and other group ( P>0.05 ). The content of NE in morphine withdrawal group was higher than that in control withdrawal group ( P<0.01 ). The content of NE in morphine + purine nucleotides withdrawal group was higher than that in control withdrawal group, and was lower than that in morphine withdrawal group ( P<0.01 ). There was no statistical difference in the content of 5-HT in control group, morphine group and morphine + purine nucleotides group ( P>0.05 ). The content of 5-HT in morphine withdrawal group and morphine + purine nucleotides withdrawal group was higher than that in control withdrawal group ( P<0.01 ).The above results confirm that purine nucleotides compensation has no significant effect on DA and 5-HT in morphine dependent and withdrawal rats'midbrain, and lightened the variance of NE content, so body metabolism tended to balance. DA was base of psychological dependence by morphine, and NE was close related to physical dependence. Purine nucleotides compensation made the content of NE close to control group, which indicated purine nucleotides can cure physical dependent symptom in morphine dependent rats, and lessen withdrawal response.According to above opinion, morphine can inhibit PC12 cells proliferation, which one mechanism was preventing cells in G0/G1 stage. Morphine inhibited proliferating cell nuclear antigen (PCNA) mRNA and protein expression which was closely relative to cell proliferation. It decreased cell purine nucleotide anabolism and increased its catabolism, so nucleotides was deficiency in cells. Exogenous purine nucleotides compensation attenuated the effect of morphine on nucleotide metabolism in PC12 cells. Exogenous purine nucleotides compensation enhanced antinocicetive effect of morphine in rats, and lightened morphine dependent rats'withdrawal symptom. It weakened purine nucleotide catabolism augment in plasma, brain cortex and small intestine by morphine in rats. It also lessened the variance of NE in midbrain in morphine dependent rats. So purine nucleotides not only reinforced the antinociceptive effect of morphine, but also relieve physical dependence symptom by chronic morphine treatment. They can be a project of associative analgesic drug, and a perspective method and strategy in clinic.The new point of this paper: (1) A new therapy of compensating purine nucleotides AMP and GMP was proposed from the change of pathology and physiology of purine nucleotide biochemical metabolism in morphine dependent rats. (2) It confirms firstly that purine nucleotides compensation can strengthen analgesia effect of morphine. (3) It lightened physical dependent symptom when addiction. (4) It obviously relieve the variance of norepinephrine in mid brain.The shortage of this paper: (1) The activity of purine nucleotide anabolic key enzyme AK and HGPRT can't be detected because of short of its kit. (2) The activity of purine nucleotide catabolic key enzyme in all animal'tissue and organs can't be examined since time was limited. The mRNA expression and activity of ADA and XO and mRNA expression of de novo synthesis and salvage synthesis key enzyme will be detected in every tissue and organs in morphine dependent and purine compensation rats in next test in order to observe the therapeutic efficacy of purine nucleotides..