Opioid Addiction Formation Related Syndromes And The Neurobiology Mechanism

Along with the exogenous opioid abuse becomes the public disruption in the world,in China the abusers is increasing sharply.Drug addiction is a chronic and recurrent encephalopathy,which is difficult to solve in modern medicine.Over the last 20 years,due to the general research in the pathogenesis and the therapeutics to drug abuse,detoxification can be cured completely,and the acute withdrawal syndrome,protracted physical dependence can be treated directly too.But the obsessive drug-craving,the main reason for relapse,is the hang-up to cure the drug addiction radically.The treatment based on syndrome differentiation is the characteristic of the Chinese medicine.Drug abuse is treated for hundreds of years by Chinese medicine,but the cognitions about the etiopathogenisis and pathogenesis are only confined to personal experiences,which is the shackle to treat craving objectively and systematically.To cure drug-craving in modern medicine and Chinese medicine,the study on syndrome of drug craving systematically and objectively is necessary.The research explores the syndrome differentiation of drug craving in both theory and experiment,which will afford objective evidence and systematical theory to guide the treatment of drug craving in clinic.The thesis contains theoretical and the experimental study:in the theoretical part, deduction,analogism and the citation were used to elicit the hypothesis on the pathogenesis and syndrome of drug-craving;in the experimental part,the method to detect the syndrome by formula was used to certify the hypothesis.First,according to the research of syndromes for years clinically,the effective formula to prevent relapse,named "hu men he ji" was divided into four sub-formulas aiming at different syndromes,called "invigorating the kidney formula" "dispersing the depressed liver-energy formula" "dissipating phlegm formula" and "supporting heart formula".And then we assessed the syndromes by detecting the effect of the above sub-formulas to conditioned place preference induced by morphine and analyzed pathogenesis of related syndromes,for the nucleus accumbens(NAc) and Cortex of frontal lobe(PFC) are the crucial encephalic regions in drug addiction,and the synaptic plasticity is the fundamental pathogenesis of drug-craving,the analysis focused on the relation between the drug-craving and the synaptic plasticity in NAc and PFC.PartⅠThe theoretical study on the syndromes of drug-craving Theoretical study one:the relation between entrails and drug addictionMethod:By literature study,the relation between all kinds of withdrawal symptoms and functional disorder of entrails was analyzed by induction,and the related organs to drug-craving was unkown,together with the core mechanism of drug-craving,analogism,deduction,and citation were used to analyze the relation between the "liver controlling dispersion" and the formation of drug-craving.And the relation between "liver managing deliberation" "kidney controlling aspiration" and the formation of drug-craving is analyzed by deduction,and citation too.Result:The formation of drug-craving is the result of functional disorder of "liver controlling dispersion" "liver managing deliberation" and "kidney controlling aspiration".Conclusion:Drug-craving is an emotional disease,the core syndrome of which is "liver failing to control dispersion",and other related syndromes are "Yin and Yang deficiency of kidney".Theoretical study two:Phlegm should be responsible for persistence of drug-cravingMethod:By literature study,the production and morbigenous characteristic were analyzed,analogism and deduction were used to analyze the relation between the pathogen of phlegm and the persistence of drug-craving.Result:Phlegm is a patho-product of diseases, which influences the disease as a pathogen too.The persistence of drug-craving coincides with the morbigenous characteristics of phlegm.Conclusion:The phlegm,which resulted from drug addiction,is the main reason for the persistence of drug-craving.The conclusion of theoretical part:Different withdrawal symptoms are related to different entrails,and the core syndrome of fonnation of drug-craving is "liver failing to control dispersion",and other related syndrome is the "Yin and Yang deficiency of kidney". The phlegm,which resulted from drug addiction,is the main reason for the persistence of drug-craving.In experimental study,the theoretical conclusion will be verified further.PartⅡThe experimental study on the syndromes of drug-craving Experiment one:Optimization and evaluation of the conditioned place preferenceMethod:Based on the classical conditioned place preference(CPP),automatic test system of CPP was set up to make use of the behavioral equipment and software from NODULE corporation.According to unbiased design,white compartment was designed as the morphine-paired compartment.Morphine(5mg/kg,10mg/kg,or 15mg/kg,i.p.) was separately administrated in white compartment and saline on the other side.In control group,0.9% sodium chloride(10mg/kg,i.p.) was administrated on both sides.Conditioning consisted of 50-min sessions,and testing was for 15min.Result:Real-time recording and hipping analysis to CPP came true in the automatic test system.CPP could be induced successfully by morphine of the above three doses.CPP induced by morphine of 10mg/kg and 15mg/kg were more obvious,but rats injected by morphine of 15mg/kg showed irritability and low reactiveness,so the 10mg/kg was optimized dose for CPP in the study.Conclusion:The automatic test system of CPP are more accurate and objective,for it can make real-time recording and hipping analysis to behavior,and eliminate the disturbance of unconditioned clues.10mg/kg is the optimized dose to induce the CPP by i.p..The above optimized scheme will be used in further study.Experiment two:the relation between the malfunction of liver and kidney and the formation for drug cravingMethod:The optimized design by experiment one was used to set up the CPP.Method to detect the syndrome by formula were used to certify the theoretical study.According to clinical study on syndromes of opiod dependence for years,the effective formula to prevent relapse,named "hu men he ji" were divided into four sub-formulas aiming at different syndromes,called "invigorating the kidney formula" "dispersing the depressed liver-energy formula" "dissipating phlegm formula" and "supporting heart formula".And then modulation of the four formulas to rewarding effect in CPP induced by morphine was detected.Result: The time staying in white compartment of model group was significantly longer than that of control(P＜0.01).Compared to model group,the time staying in white compartment of both "invigorating the kidney group" and "dispersing the depressed liver-energy group" was significantly reduced(P＜0.01).And the mean time staying in white compartment of dissipating phlegm group reduced too,but there was no significant difference to the model group,and the supporting heart formula had no effect.Conclusion:"invigorating the kidney formula" and "dispersing the depressed liver-energy formula" can alter the CPP induced by morphine,together with the previous study,"liver failing to control dispersion" is considered as the core syndrome of the formation of drug-craving,and other related syndrome is the "Yin and Yang deficiency of kidney".Experiment three:The relation between synaptophysin and the syndromes in formation of drug-cravingMethod:After behavioral test of CPP in experiment two,in each group 8 rats were randomly chosen to be detected by the immunohistochemistry,and 5 rats were randomly chosen to be taken out the NAc and PFC,the left of which were detected by western blot and the right of which were detected by real time PCR.Result:In immunohistochemistry,in NAc and PFC the synaptophysin's positive products were buffer granules,which distributed in cytoplasm,and had no significant difference in each group.In western blot,compared to control group,the protein of synaptophysin of model group increased in NAc(P＜0.05),but not in PFC.Compared to model group,in NAc the protein of synaptophysin of the invigorating kidney group and the invigorating liver group decreased significantly(P<0.05), and had no chang in PFC.No significant differences were found in other groups.In real time PCR,there was no significant difference between control group and model group.But compared to the model group,the expression of synaptophysin's mRNA were down-regulated in invigorating liver group in NAc(P<0.05),and had no difference in other encephalic region. Conclusion:In the study,synaptophysin,related to synaptic plasticity,is up-regulated in NAc, so it probably is a main reason for drug-craving,but the further pathology still need be studied. Invigorating kidney formula can down-regulate the protein of synaptophysin,and the invigorating liver formal can down regulate both the protein and the gene expression,and other formulas have no effect.The above proves the theoretical study that the kidney and liver, but not heart,should be responsible for formation of drug-craving,and the dysfunction of kidney and liver is correlated to the synaptic plasticity due to the increase of the synaptophysin in the NAc.Experiment four:The relation between regulatory subunit of NMDA receptor and the syndromes in formation of drug-cravingMethod:After behavioral test of CPP in experiment two,in each group 8 rats were randomly chosen to be detected by the immunohistochemistry,and 5 rats were randomly chosen to be taken out the NAc and PFC,the left of which were detected by western blot and the right of which were detected by real time PCR.Result:NR2A and NR2B regulatory subunit were studied.In immunohistochemistry,in NAc and PFC,NR2A's positive products had no significant difference in each group.Compared to control group,NR2B's positive products increased in both NAc and PFC(P<0.05),and compared to model group, invigorating kidney formula down-regulated NR2B's positive products in NAc(P<0.05), invigorating liver group down-regulated NR2B's positive products in both NAc and PFC(P<0.05),other formulas didn't have effect.In Western blot,compared to control group, the protein of NR2B increased significantly in NAc(P<0.01),and increased in PFC too(P<0.05).Compared to model group,invigorating kidney and invigorating liver formulas could down-regulate the protein of NR2B in both NAc and PFC(P<0.05),but still higher than the control group in NAc in invigorating kidney group(P<0.05).In real time PCR,compared to the control group,the expression of mRNA of NR2B increased significantly in NAc(P<0.01),and increased in PFC too(P<0.05).Compared to model group,invigorating liver formula could down regulate expression of mRNA in both NAc and PFC(P<0.05).Conclusion:in NAc and PFC,the change of NR2B in protein and gene level is related to the formation of drug-craving,not NR2A.In NAc and PFC,invigorating kidney formula can down-regulate the protein of NR2B,and invigorating liver formula can down-regulate both protein and gene of NR2B,Other formulas had no effect.The experiment proves that the kidney and liver should be responsible for drug-craving,but not heart,which means the pathology of drug-craving is the dysfunction of kidney and liver.The essence of "liver failing to control dispersion" is the up-regulation of NR2B in both protein and gene level,and "Yin and Yang deficiency of kidney" is due to the up-regulation of NR2B in protein level.Experiment five:The relation between BDNF and the syndromes in formation of drug-cravingMethod:After behavioral test of CPP in experiment two,in each group 8 rats were randomly chosen to be detected by the immunohistochemistry,and 5 rats were randomly chosen to be taken out the NAc and PFC,the left of which were detected by western blot and the right of which were detected by real time PCR.Result:In immunohistochemistry, compared to the control group,in NAc the reactive products of BDNF increased significantly in model group(P＜0.01),and in PFC the products increased too(P＜0.05).Compared to model group,in both NAc and PFC,invigorating kidney and invigorating liver formulas could down-regulate the reactive products(P＜0.05),but the products of invigorating liver group were still higher than the control group(P＜0.05).Other formulas had no effect.In western blot, compared to the control group,in NAc and PFC,the protein of BDNF increased significantly(P＜0.01).And compared to model group,in NAc invigorating kidney and invigorating liver groups down-regulated the protein of BDNF(P＜0.05),and in PFC down-regulated the protein remarkably(P＜0.01).In real time PCR,compared to the control group,in model group the gene expression of BDNF increased significantly(P＜0.01). Compared to model group,in NAc and PFC,invigorating liver formula could down-regulate the gene expression of BDNF(P＜0.05),but in PFC the gene expression of BDNF was still higher than control group(P＜0.05).Conclusion:In NAc and PFC,the increase of protein and gene of BDFN is related to the formation of drug-craving,which represents dysfunction of BDNF is probably the main reason of drug-craving.And in NAc and PFC,invigorating kidney formula only can down-regulate the protein of BDNF,but invigorating liver formula can regulate in both protein and gene level.Along with the experiment about synaptophysin and NMDA receptors,the research verify that the core syndrome of the formation of drug-craving is "liver failing to control dispersion",and other related syndrome is the "Yin and Yang deficiency of kidney",whose neurobiology essence is the synaptic plasticity due to dysfunction of related proteins like synaptophysin,NR2B and BDNF in PFC and NAc.The research makes breakthrough in five sides:first,based on the classical CPP, automatic test system of CPP is set up to make use of the behavioral equipment and software from NODULE corporation,which are more accurate and objective,for it can make real-time recording and hipping analysis to behavior,and eliminate the disturbance of unconditioned clues.Second,provide theoretical conclusion to syndromes of drug-craving by deduction, citation and analogism,and then use formulas to verify that the core syndrome of the formation of drug-craving is "liver failing to Control dispersion",and other related syndrome is the "Yin and Yang deficiency of kidney".Third,the neurobiology essence of drug-craving is considered as the increase of NR2B and BDNF in both protein and gene level in PFC and NAc,and also related to increase of synaptophysin in protein level only in NAc.Four,the neurobiology essence of "liver failing to control dispersion" in formation of drug-craving is the synaptic plasticity due to dysfunction of related proteins such as synaptophysin,NR2B and BDNF in PFC and NAc in both protein and gene level,and "Yin and Yang deficiency of kidney" due to the dysfunction of the above in protein level.And above that,the research expand the essence of the kidney and liver that the two ZANG-organs are related to motive circuit in nerve center.