| Objective:Animal model of heroin addiction and heroin metabolites of test method were established to study the toxic effects of heroin addiction for central nervous system, and explore physiological and biochemical mechanism of heroin addiction.Methods:1. On the first day, each rat was done muscle injection heroin 10.00 mg/kg,2 times a day by sequentially dosing method. On the second day later, each increased 5.00mg/kg, daily dose of 135.00 mg/kg, maintaining the dose for 7d, heroin addictive rats model was established.2. The Gas-MS (Gas Chromatography-Mass Spectrometry Analysis, GC-MS) was used in determination of heroin metabolites.3. Establishment of heroin metabolites of simple test was chromogenic method, thin layer chromato-graphy, and high performance capillary electrophoresis method.4. By immunohisto-chemical method, heroin addiction was detected Bax and c-fos expression in brain tissue.5. After being given with reserpine, heroin was given to observe the; addiction of animals, levels of adrenaline (A), noradrenaline (NA), dopamine (DA), cyclic adenosine phosphate (cAMP), and cyclic guanosine phosphate (cGMP) in blood and brain tissues with RIA method.6. By the Dossy-2H more pathways physiological parameters acquisition storage system, it was test for heroin addiction electroence-phalogram (eeg), electrocardiogram (ecg), and muscle spindle in rats after the change of discharge diagram.Results:1. The day after the last to heroin,2 hours after intraperitoneal injection of naloxone hydrochloride 4.00 mg/kg, rat showed body torsion, wet dog kind shake, grind teeth, jump and stand withdrawal symptoms, and addiction animal model is successful.2. GC-MS detection, morphine and 6-single acetyl morphine are heroin mainly metabolites in the body.3. The chromogenic method, thin layer chromatography and high performance capillary electrophoresis method to detect heroin metabolites (morphine) effect is sensitive, simple and easy operation.4. The immunohistochemical method to detect heroin showed that Bax and c-fos expressive positive cells in the brain were more in heroin group than in control group significantly (p<0.01), IOD absorbance (integral), MA (the average absorbance) significantly enhanced(P< 0.01).5. Detection of radioimmunoassay showed that,1) compared with the control group, the expression levels of cAMP in the blood of heroin and reserpine group were significantly higher (P<0.01), and the rise extent of reserpine group was larger than that of heroin group. The expression levels of cGMP in the blood of heroin (P>0.05) and reserpine group (P<0.05) were less, and the decrease extent of reserpine group was larger than that of heroin group. The expression levels of A and NA in the blood of heroin and reserpine group were changed. But the changes of the heroin group were significantly increased (P<0.01). However, the changes of the reserpine group were significantly decreased (P<0.01).2) compared with the control group, the expression levels of cAMP in the midbrain ventral tegmental area (VTA), the prefrontal cortex (PFC) and hippocampus (Hipp) of heroin and reserpine group were significantly higher (P<0.01), and the rise extents of the above tissues of the reserpine group were larger than those of heroin group. The expression levels of cGMP in the above tissues of the reserpine group were significantly decreased (P<0.01), and the expression levels in the heroin group were also decreased. The decrease extents in the midbrain ventral tegmental area (P<0.01) were larger than those of the prefrontal cortex and hippocampus (P<0.05). On the whole, the decrease extents of the above tissues of the reserpine group were larger than those of heroin group.3) compared with the control group, the expression levels of DA in the prefrontal cortex, hippocampus, striatum and nucleus accumbens (NAc) of the reserpine group were significantly decreased (P<0.01), and the decrease extents of the above tissues of the reserpine group were larger than those of heroin group. The expression levels of DA in the prefrontal cortex, hippocampus and nucleus accumbens of the heroin group were increased, but the increase extents of the prefrontal cortex and nucleus accumbens (P<0.01) were larger than that of the hippocampus(P<0.05). However, the expression levels in the striatum was decreased (P>0.05). Generally, the expression levels of DA of the above tissues of the reserpine group were less than those of heroin group.6. Neurophysiological studies show that EEG, ECG and muscle spindle discharge figure of the heroin addiction rat were obviously changed.7. The mechanism of heroin addiction may be that heroinâ†'6-acetyl morphineâ†'receptorâ†'G proteinâ†'ACâ†'ATPâ†' cAMPâ†'PKAâ†'DA, A and NA synthesis and release of target organs, withdrawal symptoms (addiction).Conclusion:1. We have established that the rapid modeling approach on an animal model of heroin addiction, in which the modeling time is short, the withdrawal symptoms is obvious, and the operation is simple and easy.2. The heroin metabolites (single-acetyl morphine or morphine) is detected by the chromogenic method, thin layer chromatography and high performance capillary electrophoresis, in which the effect is sensitive, and the operation is simple and easy.3. Heroin addiction has significantly upregulated the expression of Bax and c-Fox in the brain tissue, which led to brain damage.4. Prefrontal cortex, ventral tegmental area, hippocampus, striatum and nucleus accumbens are involved in the process of heroin addiction.5. Heroin addicts will change the expression levels of the DA system regulation-associated with hormones, neurotransmitters and other factors in the blood and brain tissue of the addiction animals. After using the drug rifampicin intervention, the expression levels of the DA system regulation-associated with hormones, neurotransmitters and other factors in the blood and brain tissue also occured some changes, but the intervention would improve withdrawal symptoms of the addiction animals.6. The normal physiological function of brain, heart and muscle spindles of the addiction animal would be affected by heroin addicts.7. DA system plays a very important role in the process of heroin addiction. The mechanism of heroin addiction may be that heroinâ†'6-acetyl morphineâ†' receptorâ†'G proteinâ†' ACâ†' ATPâ†'cAMPâ†'PKAâ†'DA, A and NA synthesis and release of target organs, withdrawal symptoms (addiction). |
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