Effect And Mechanism Of Heroin And Morphine On Gene Expression Of ADA In C6 Glioma Cell

The abuse and addiction of opiates is a serious social and medical problem, which threatens the health and happiness of people. The chronic exposure to opiates such as morphine and heroin can cause psychological and physical dependence to addicts, it's an important subject to study the basis of addiction and dependence.At present, there are little research studying the relationship of the dependence of opiates and receptor antagonist as well as add adenosine. We inject sensitive antagonist into receptor, cultivate it in vitro cell with heroin, to find whether heroin has inhibitory effects on the cell proliferation using the MTT colorimetry method. In addition, we test the antagonist NTI on the c6cell which effected by heroine for 24h, and examine the influence of antagonist on the cell. Opioid drugs caused the excessive decomposition of adenosine, and there's an obvious change on the enzyme's activity and gene expression which is related to adenosine metabolism. Some literatures show the opiate addiction may be related with adenosine metabolism, so we add adenosine and guanosine to heroin, and examine the influence. We choose the ADA pathway as study object which has a strong metabolic process, observe the effect on the inhibition of cell proliferation as well as the gene expression of ADAmRNA.The experiment on receptor antagonist is divided into four parts: the cyprodime of mu-receptor-antagonist 1h then heroin 24h; the NTB of detal-receptor-antagonist 1h then heroin 24h; the NTB of detal-receptor-antagonist 1h then morphine 24h; the heroin 24h then the NTI of detal-receptor-antagonist 24h. there are control group and heroin(morphine) group in every part, the antagonist with four dose: 1nmol/L ,10n mol/L, 100nmol/L, 1000nmol/L were given for 1h then heroin was added. While the NTI was given 20ug/ml for 24 then antagonist was added. We choose the heroin with the concentration of 20ug/ml, concluded from the experiment at which it inhibits the proliferation of c6 cell significantly without leading to the cell death.The experiment of MTT confirms the heroin inhibition of cell proliferation is not merely through a receptor, the antagonist weakens the effect of heroin on c6 glioma cells in inhibiting cell proliferation, and there's an increasing trend with the dose increase, there're marked difference among the cyprodime of mu-receptor-antagonist group, the NTB of detal-receptor-antagonist group, the NTI of detal receptor antagonist group and the grads group of NTI with the concentration of 1000nmol/L, 1nmol/L. there's marked difference only in the NTB group with the concentration of 100nmol/L and 10nmol/L in the neighbor concentration group of antagonist. The inhibition effect of heroin and antagonist on cell proliferation and DNA synthesis may be caused by the adverse reaction, which means that the drug leads to the cell death. To exclude the possibility, we determine the cell vigor by trypan blue method. The results indicate that there is no significant deviation between control and heroin group and cell vigor are all over 90 percent, which confirms that the decrease of cell growth is not caused by cell death.Therefore, we believe that heroin results in the inhibition effect not through a single opiates receptor. At present, we study only with the mu-receptor and detal-receptor, we will make further research on other receptors, and observe the gene expression related to cell proliferation, finding heroin's inhibition effect on C6 glioma cell proliferation sensitively.The achievement of adenosine catabolism is through the pathway of adenosine deaminase ADA and adenosine kinase AK. ADA's effect on adenosine catabolism is superior to AK. ADA is the key enzyme of purine nucleotide catabolism, it catalyzes the adenine nucleotide into hypoxanthine nucleoside, and then decompose into hypoxanthine. In this experiment, we compare the group of added AMP, GMP with the group of heroin in c6 cell, the expression level of ADAmRNA lowered, and an increasing dose of AMP,GMP will weaken the effect of heroin on the expression level of ADAmRNA. We observe the inhibition effect on cell proliferation with the adenosine and guanosine added using the MTT colorimetry method. but not found pattern changes, which may be the result of that gene expression behaves more obviously than cell proliferation in short time.There is no obvious difference on the expression level of ADAmRNA between the group with high dose adenosine and guanosine added and the control group. The ADA/β-actin1 in control group is 0.4877, while 0.5009 in the group with adenosine, 10.7270 in the group with heroin, 0.5227 in the group with heroin and high dose adenosine and guanosine(100ug/ml), 0.5830 in the group with heroin and low dose adenosine and guanosine(25ug/ml).In conclusion, the mu-receptor and detal-receptor that we choose effect by antagonist and then heroin(morphine), the cell proliferation is weakened, and there's a similar circumstance as the heroin is firstly given. Though it weakens the damage to the cell by the opiates, there is still a marked difference compared with the control group, the inhibition of cell proliferation decreases more in the high dose antagonist compared with the low dose group. With the decrease in the concentration of antagonist, the inhibition of cell proliferation is more obvious, there is usually no marked difference between the neighbor group. The c6 cell treated with heroin and then AMP,GMP can weaken the catabolism of adenosine, behaves in that the mRNA expression of the metabolism key enzyme ADA decreases compared with the one simply giving heroin. The more the AMP and GMP is added, the smaller the mRNA expression of ADA is. There is no obvious change between the group treated only with AMP and GMP and the control group.