The Oxidative Stress Mechanism Of Methcathinone-induced Neurotoxicity

BACKGROUNDMethcathinone(CAT)is a synthetic cathinone,which is called “bath salts”;It is also a kind of stimulant of beta-keto-amphetamines that has a similar structure with METH,and can produce euphoria,exercise increasing,and hallucinations effects,it also cause toxic symptoms such as high fever,memory loss,paranoid,anxiety and epilepsy,even disability and death.In the early,it was used as an antidepressant drug and was banned clinically for side effects in later.In recent years,the rigorous regulation and attack of traditional drugs,led to CAT gradually being abused as a substitute by people for its similar amphetamine stimulated function.So the social security and deaths caused by abuse of CAT have emerged in large numbers.Therefore,the study on the mechanism of CAT toxicity is particularly important.However,the studies about CAT toxicity mechanism are few,mostly stay in the structural analysis,pharmacological research and test analysis.So this study carried on the exploration of CAT neurotoxic mechanism.In chemical structure and pharmacology,CAT was similar to amphetamine.Both of them increase the concentrations of extracellular monoamine neurotransmitters dopamine and 5-hydroxytryptamine to produce toxic effects.Some study also found that amphetamine –type inhibitors can bring protective effects on CAT abuse patients.In addition,the experiment also found that CAT precursors will lead to activities change of oxidative stress related enzymes,such as Superoxide Dismutase,Catalase,Malondialdehyde,and generate neurons apoptosis.Early animal experiments have also shown that behavioral changes caused by CAT are associated with ROS products.A large number of experiments have confirmed that oxidative stress is the main neurotoxic mechanism of Methamphetamine(METH).Considering the similarities of chemical structure and toxicology between CAT and METH,Most scholars believe that CAT's neurotoxic mechanism may involve oxidative stress.In normal circumstances,the status of oxidation and antioxidant are keeping dynamic balance in body.Under the stimulation of chemical,physical and biological factors,oxidative stress is produced,that is,oxidizing active substance produces too much,and the antioxidants cannot completely eliminated it.Reactive oxygen species combine with cellular structures,such as DNA,will finally cause body damage.N-Acetyl-L-cysteine(NAC)is a powerful and effective small-molecule antioxidant,which can easy to pass through the blood-brain barrier,and it is often used to oxidative stress research.In this experiment,we use the antioxidant enzyme NAC pretreatment in the CAT-exposed rats,then detect the activity of enzymes related to oxidative stress,test the expression of autophagic protein and the apoptosis of neurons in each group to study the oxidative stress toxicity mechanisms of CAT.OBJECTIVE1.Basic toxicity symptoms observation of CAT on adult rats.2.The role of oxidative stress mechanism in CAT neurotoxicity 3.The changes expression of autophagy in rat neurotoxicity caused by CAT exposure.4.The apoptosis of neurons in the neurotoxicity of rats in CAT exposure.5.Protective value of antioxidant on CAT infected rats.METHODS1.Animal protocol and CAT basic toxicity observationMale Sprague-Dawley rats(N=24),weighting from 160 g to 200g(the experimental animal center of Huazhong university of science and technology,tongji medical school),were randomly divided into 4 groups,12-h light-dark cycle.Water and food were provided autonomously.Drug administration in all rats were given by intraperitoneal injection.The control group was treated daily with 0.9% NaCl(1ml,at a.m.8:00),for 15 consecutive days.Saline was injected on CAT group(1ml,at a.m.8:00)during first three days,but injected with CAT(0.5mg/kg body weight,at a.m.8:00)from fourth day to fifteenth day.NAC group were injected with NAC(150mg/kg body weight).In the inhibition + experimental group,only inhibitor NAC(150 mg/kg)was given in first three days,but NAC was injected 30 minutes before CAT administration on days 4-15.When 24 h after the last medication,decapitation treatment.The body weight,temperature and behavioral changes of rats were recorded daily.HE staining was performed to observe the pathological changes of brain tissue.2.The study on the mechanism of oxidative stress in neurotoxicity induced by CAT exposure.The activities of SOD,CAT,GSH-PX,GSH and MDA were detected by enzyme kits in brain tissue.Western blotting detects the expression of Atg-7,beclin-1 and LC3 in each group.TUNEL staining observes the apoptosis of neurons.3.Statistical analysisThe experimental data are expressed as mean ± SD.Date were analyzed using one –way ANOVA.Group analysis was performed with t-test.P<0.05 was considered statistically significant.RESULT1.There was no significant difference in body weight between groups after intraperitoneal injection of CAT.However,the body temperature significant increased after administration CAT.The marked behavioral changes were observed such as the rats were hyperlocomotioned,repetitively look up to exploration,and rotative.Then pretreatment with NAC could attenuate the evevated score of stereotype behavior and body temperature.The HE stain showed that swell of neuron,neuronal degeneration,ischemic changes,and neuronophagia.2.Compared with other three groups,CAT administration could significantly decrease the activities of SOD,GSH-PX,CAT and GSH.However,the levels of MDA was significantly increased(P<0.05).Pretreatment with NAC(30 minutes before each CAT injection),there was no significant digference in SOD,GSH-PX,CAT,GSH when compared with NS group.And there was also no significant change in the content of MDA.3.Compared with other three groups,CAT administration could significantly elevate the expression of protein Beclin-1 and Atg-7,and significantly elevate the ratio of LC?/LC?(P<0.05).Pretreatment with NAC(30 minutes before each CAT injection),,there was no significant digference in Beclin-1 and Atg-7 expression,and the ratio of LC?/LC?when compared with NS group.4.The TUNEL immunohistochemistry showed neuron apoptosis,and the number of positive cells was increased,and the positive index was significantly elevated(P<0.05).After NAC pretreatment,there was no significant digference in the number of apoptotic cells when compared with NS group.CONCLUSION1.CAT causes marked neurotoxicity,which included hyperthermia,stereotyped behaviorand corresponding pathological changes in brain tissue.2.Oxidative stress is one of the important mechanisms of neurotoxicity of methcathinone.3.CAT can cause neuronal apoptosis and autophagy increasing.4.Antioxidants can effectively antagonize oxidative stress,autophagy and apoptosisinduced by methcathinone.