| Opiate abuse such as heroin can make the addicts shape physiological dependence and psychic dependence to heroin, which result in damages and changes to multisystem inevitably. There is still no ideal drug for opiate addiction in clinical therapy so far. Previous research work of our lab showed that both heroin and morphine promote purine nucleotide catabolism and inhibit purine nucleotide anabolism in several tissues especially in brain tissues; this suggested that purine nucleotide may be in deficiency in heroin dependent rat brain tissues. This deduction was verified in our research work, heroin decreased the content of purine nucleotide in rat brain tissues, while purine nucleotide compensation can improve this deficiency of purine nucleotide in rat brain tissues. More and more evidence demonstrated that monoamine neurons, especially the serotoninergic neurons have close relationship with the mechanisms of opiate addiction and the therapy for opiate addiction. In this study, effects and mechanisms of purine nucleotide compensation on 5-HT system in heroin dependent rats were investigated to provide new theoretical insight to the mechanisms of opiate addiction and the therapy for addiction.1. Establishment of heroin dependence modelAdult male Wistar rats were randomly divided into control group (ip normal saline for 9 days), heroin administration group (ip heroin for 9 days), heroin and AMP+GMP administration group (ip heroin and AMP and GMP mixture for 9 days), heroin and AMP administration group (ip heroin and AMP for 9 days), heroin and GMP administration group (ip heroin and GMP for 9 days). Abstinence symptoms evoked by naloxone were observed to test the establishment of heroin dependence model.After 9 days heroin treatment, naloxone treatment evoked abstinent symptoms such as rearing, wet-dog shake, chewing, tooth chattering, ptosis and diarrhea. Purine nucleotide compensation decreased the number of chewing, tooth chattering and ptosis remarkably.These results showed that rats shaped physiological dependence after exposure to heroin for 9 days and the dependence model was established successfully; purine nucleotide compensation partially alleviated the abstinent symptoms.Body weight record showed that heroin treated rats had alower increasing rate of body weight than control ones; purine nucleotide compensation alleviated this effect of heroin on rat's body weight.2. Effects of heroin and purine nucleotide compensation on the content of 5-HT and 5-HIAA in rat brain tissuesIn heroin administration group, content of 5-HT was decreased dramatically compared with control group. Content of 5-HT was increased in heroin and AMP+GMP administration group, heroin and AMP administration group, heroin and GMP administration group compared with heroin administration group. This result demonstrated that heroin can decrease the content of 5-HT in rat brain tissues and purine nucleotide compensation inhibited this effect of heroin on 5-HT content.There are two reasons for the reduction of 5-HT content, the one is the synthesis of 5-HT is decreased and the other is 5-HT catabolism is increased. We measured the content of 5-HIAA, which is a metabolite of 5-HT. The result showed that content of 5-HIAA was decreased dramatically in heroin administration group compared with control group. In heroin and AMP+GMP administration group, heroin and AMP administration group, heroin and GMP administration group, content of 5-HIAA was increased compared with heroin administration group. Content of 5-HIAA changed in the same direction as 5-HT, this demonstrated that the reason for the reduction of 5-HT is the reduced synthesis but not the increased catabolism.3. Effects of heroin and purine nucleotide compensation on the expression of tryptophan hydroxylase in rat brain tissuesIn heroin administration group, content of TPH was decreased dramatically compared with control group. In heroin and AMP+GMP administration group, heroin and AMP administration group, heroin and GMP administration group. TPH content was increased compared with heroin administration group.Percentage of TPH-positive cells was decreased in heroin administration group compared with control group. However, in heroin and AMP+GMP administration group, heroin and AMP administration group, heroin and GMP administration group, the percentage of TPH-positive cells was increased compared with heroin administration group.TPH mRNA level was decreased in heroin administration group compared with control group. In heroin and AMP+GMP administration group, heroin and AMP admin-istration group, heroin and GMP administration group, TPH mRNA level was increased compared with heroin administration group. Aboved-mentioned results showed that heroin affected the synthesis of 5-HT by inhibition of TPH gene expression at both protein and transcript level. However, purine nucleotide compensation up-regulated the expression level of TPH gene and alleviated the effect of heroin on TPH gene expression.4. Effects of heroin and purine nucleotide compensation on the content of purine nucleotide and mRNA level of GTP cyclohydrolaseâ… in rats brain tissuesIn heroin administration group, content of AMP, GMP and GTP was decreased compared with control group. In heroin and AMP+GMP administration group, heroin and AMP administration group, heroin and GMP administration group, the content of AMP and GTP was increased compared with heroin administration group. This result demonstrated that purine nucleotide compensation alleviated the deficiency of purine nucleotide in rat brain tissues caused by heroin.In heroin administration group, GTP cyclohydrolaseâ… mRNA level was decreased compared with control group. In heroin and AMP+GMP administration group, heroin and AMP administration group, heroin and GMP administration group. GTP cyclohydrolaseâ… mRNA level was increased compared with heroin administration group. This result showed that purine nucleotide compensation inhibited this effect of heroin on the level of GTP cyclohydrolaseâ… mRNA.5. Effects of heroin and purine nucleotide compensation on morphology of rat brain tissuesMorphological observation showed that the morphology of rat brain tissues of control group were normal under electron microscope.Under electron microscope, rat brain tissues of heroin administration group showed that the heterochromatin were increased in the nucleus of neurons, the rough endoplasmic reticulum were decreased, there is ecphyma and cavitation in the neuropil, the neurofilament were decreased, the mitochondria were swelling.There was no conspicuous change in the rat brain tissues of heroin and AMP+GMP administration group, morphology of neurons and neuropil were normal, there were affluent rough endoplasmic reticulum and mitochondria.6. Effects of heroin and purine nucleotide compensation on the expression of glial fibrillary acidic protein in rat brain tissues In rat brain tissues of heroin administration group, the color of GFAP-positive particles was darker and the OD is higher than control group. In heroin and AMP+GMP administration group, heroin and AMP administration group, heroin and GMP administration group, the color of GFAP-positive particles was lighter and the OD is lower than heroin administration group.Aboved-mentioned results showed that heroin increased the expression of GFAP in rat brain tissues; purine nucleotide compensation alleviated this effect of heroin on GFAP expression. Level of GFAP expression is in positive correlation with the damaged degree of nervous tissues. This result demonstrated that purine nucleotide can alleviate the damage caused by heroin in rat brain tissues; purine nucleotide has neuroprotective effect.7. Effects of heroin and purine nucleotide compensation on the expression of caspase-3 in rat brain tissuesIn heroin administration group, percentage of caspase-3-positive cells was increased compared with control group. However, in heroin and AMP+GMP administration group, heroin and AMP administration group, heroin and GMP administration group, the percentage of caspase-3-positive cells was decreased compared with heroin administration group.These results demonstrated that heroin induced apoptosis in rat neurons:purine nucleotide compensation can alleviated this apoptosis-inducing effect of heroin. Not only were the changes of neurotransmitter but also apoptosis induced by heroin involved in its neurotoxicity effect on the nervous system.8. Effects of heroin and purine nucleotide compensation on the proliferation of C6 glioma cells in vitroHeroin inhibited the proliferation of C6 glioma cells in dose-dependent manner. However, purine nucleotide attenuated this inhibitory effect of heroin on the proliferation of C6 glioma cells in dose-dependent manner.9. Effects of heroin and purine nucleotide compensation on the expression of caspase-3 in C6 glioma cellsIn heroin administration group, percentage of caspase-3-positive cells was increased compared with control group. However, in heroin and AMP+GMP administration group, heroin and AMP administration group, heroin and GMP administration group, the percentage of caspase-3-positive cells was decreased compared with heroin administration group.All these results showed that heroin inhibited the proliferation of C6 glioma cells in vitro; its mechanism may involve the induction of apoptosis by heroin in C6 glioma cells.In conclusion, the neurotoxic effects of heroin educed in rat brain tissues were as follows:decreased the content of 5-HT, 5-HIAA and TPH; downregulated the expression of TPH and GTPCH gene at transcript level; upregulated the expression of GFAP and caspase-3. Heroin inhibited the proliferation of C6 glioma cells:its potential mechanism may involve the upregulation of caspase-3. Purine nucleotide compensation attenuated these effects of heroin to some extent; alleviated some abstinent symptoms and pathological changes in rat brain tissues caused by heroin. All these results suggested that changes of neurotransmitter 5-HT content and 5-HT synthesis-related gene expression level play important roles in the development of heroin dependence; and purine nucleotide compensation attenuated these effects caused by heroin and may be a potential therapy for heroin addiction.
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