The Roles Of Thioredoxin-1 Inducers In Regulating Addiction Induced By Environmental Toxicant Morphine

Morphine is an opioid analgesic drug.The long-term use of morphine can lead to addiction and the higher rate of relapse.Morphine is an alkaloid which is the earliest purified from opium poppy and is one of the main components of heroin.Cultivation of a large number of poppies,extraction of papaverine can cause environmental pollution,destroy the living environment of animals and human beings,and harm human health.The pollutants and its metabolism in the environment play the destructive role in human health and ecosystem.There are many factors that affect morphine addiction,including the drug itself,the environment in which it is administered,and other drug-related cues.After a period of withdrawal,the user will develop replase when re-exposed to drug-related environmental cues.Therefore,understanding the mechanism of the relapse is crucial for the treatment of morphine addiction.Morphine addiction is related to dopaminergic and glutaminergic systems in the ventral tegmental area（VTA）,nucleus accumbens（NAc）,prefrontal cortex（PFC）and Hippocampus（Hip）.The dopaminergic system and glutaminergic system are associated with reward effect and relapse after withdrawal.Thioredoxi-1（Trx-1）is a redox regulating protein,not only maintains the homeostasis balance of oxidation and reduction,but also is involved in the regulation of transcription factors,inhibition of apoptosis and the protection of neurons.Our previous studies have shown that Trx-1 overexpression transgenic mice resist the expression of conditioned place preference（CPP）induced by morphine and methamphetamine.Geranylgeranylacetone（GGA）induces the expressions of Trx-1and Heat shock protein 70（Hsp70）and resists the reward effect induced by morphine.However,it is not clear whether GGA blocks morphine-CPP expression is associated with Trx-1,and whether GGA has an effect on the reinstatement of morphine-CPP.The aim of this study is to explore that Trx-1 inducer blocks morphine-CPP expression involved in Trx-1 by using PX-12,a specific inhibitor of Trx-1,as well as the effect and molecular mechanism of Trx-1 inducer resisting to morphine addiction.The main research results of this paper are as follows:（1）Trx-1 inducer,Panax Notoginseng Saponins（PNS）or Naltrexone（NTX）administration alone blocked the expression of morphine-induced CPP in mice,however,the combinated administration of the two drugs did not block the expression of morphine-induced CPP.Western blot showed that PNS induced the expression of Trx-1 in the VTA,NAc and PFC.Morphine induced increases of Trx-1,tyrosine hydroxylase（TH）,Dopamine receptor 1（D1R）,ΔFosB,and Cyclin-dependent kinase5（CDK5）expression in the VTA,NAc and PFC,which were inhibited by NTX or PNS pretreatment alonely.Interestingly,combined administration NTX with PNS pretreatment reversed the inhibition of NTX or PNS on the increasing expressions of Trx-1,TH,D1R,ΔFosB,CDK5 induced by morphine.These results suggest that NTX and PNS blocks the expression of morphine-CPP by regulating Trx-1,TH,D1R,ΔFosB,CDK5 expression.（2）GGA,an inducer of Trx-1,blocked the expression of morphine-CPP.GGA pretreatment significantly inhibited the expression of morphine-CPP in mice,while PX-12 administration reversed the inhibition of GGA on morphine-CPP expression in mice.Western blot showed that GGA and morphine induced the expression of Trx-1 in the VTA and NAc in mice,whereas Trx-1 expression was not further increased by morphine together with GGA,PX-12 administration counteracted the regulating effect of GGA on the expression of Trx-1 induced by morphine.At the same time GGA administration inhibited the increases of TH,D1R,ΔFosB,CDK5,and activities of dopamine-and cAMP-regulated phosphoprotein Mr 32 kDa（DARPP-32）,cAMP-responsive element-binding protein（CREB）induced by morphine,while PX-12administration reversed the inhibition of GGA on increases of TH,D1R,p-DARPP-32,p-CREB,ΔFosB,CDK5 induced by morphine in the VTA and NAc.These results suggest that GGA occluds the expression of morphine-CPP by inducing the expression of Trx-1 and regulating the D1R/DARPP-32/CREB pathway.（3）GGA,an inducer of Trx-1,blocked the reinstatement of morphine-CPP.GGA treatment after CPP extinction blocked the reinstatement of CPP induced by low priming dose of morphine,while PX-12 treatment reversed the inhibition of GGA on reinstatement morphine-CPP.Western blot showed that GGA could induce the expression of Trx-1 in the NAc and Hip in mice after the reinstatement of morphine-CPP.Morphine induced the expression of Trx-1 and N-methyl-D-asparate receptor 2B subunit（GluN2b）,and activities of Ca²⁺/calmodulin-dependent protein kinase II（CaMKII）,extracellular signaling regulated kinases（ERK）and CREB in the NAc and Hip,whereas these increases were suppressed by GGA pretreatment.PX-12administration inhibited the expression of Trx-1 by morphine together with GGA.Theseresults suggest that GGA blocks the reinstatement of morphine-CPP by inducing the expression of Trx-1 and regulating GluN2b/CaMKII/ERK/CREB pathway.GGA or PNS play roles in regulating the expression and reinstatement of morphine-induced CPP by inducing the expression of Trx-1.Combined administration of PNS and NTX counteracted the role of PNS to the expression of morphine-CPP.Trx-1 inducer resisted reward effect induced by morphine through inducing Trx-1expression and regulating dopaminergic system.Trx-1 inducer inhibited morphine-CPP reinstatement through inducing Trx-1 and regulating glutamatic system.Therefore,Trx-1 inducer plays an important role in resisting morphine addiction.Trx-1 inducer is an important candidate drug for the treatment of morphine addiction.This study has the theoretical and practical significance for the treatment of morphine addiction.