The Protective Effects Of N-acetylcysteine Against Methamphetamine-Induced Nerve Injury

BACKGROUNDMethamphetamine(METH,MA) is one of the Amphetamine Type Stimulants(ATS). It is usually called "ice" for its crystalline clear resemble ice.METH has multiple pharmalogical and toxicological effects.Firstly,it was used to be an anti-obesity drug and an anti-fatigue agent.Unfortunately,METH was found to be psychological dependence later which therefore made it became a forbidden drug.In previous study, Behavioral changes were observed following METH administration.Some reports revealed that METH could induce the injuries of heart,liver,lung and other organ, however,the most severe damage is nerve injury which includes neuron apoptosis, DA loss,DAT decreased and loss of TH activity.METH-induced neurotoxicity is a complex process,which included the oxidation of overload dopamine,excitoxicity caused by glutamate,mitochondrial dysfunction and apoptosis pathways activation in brain.However,the mechanism of METH-induced neurotoxicity is not very clear at present.OBJECTIVESeveral lines of evidence suggested that oxidative stress was the important mechanism of METH-induced neurotoxicity.Our previous studies indicated that METH could increase the activity of NOS and the level of NO,result in production of reactive oxygen species(ROS) and reactive nitrogen species(RNS) which reduced the SOD content and damaged the central nervous system.But the inhibitor of nNOS, 7NI,could attenuate the neurotoxicity.Some reports revealed that asymmetric dimethylarginine(ADMA) was an endogenous inhibitor of NOS and the metabolism of ADMA mainly occurs via hydrolytic degradation by Dimethyarginine dimethylaminohydrolase(DDAH).Our previous studies suggested that DDAH 1 was highly expressed in rat striatum in METH toxical animal model.Furthermore,some researches showed that ADMA was correlated with oxidative stress.But there were yet no reports whether METH induced neurotoxicity by interfering with DDAH/ADMA system.N-acetyl-L-cysteine was a potent antioxidant and radical scavenger,it converted into metabolites capable of stimulating glutathione synthesis in the body.Based on these contexts,we hypothesized that METH-induced neurotoxicity through oxidative stress on the effects NO production by interfering with DDAH/ADMA system.In present study,we made animal models to survey the neurotoxicity of METH by histopathological,molecular biology and neurochemical methods.We observed METH could induce oxidative stress by detecting not only the system influenced synthesis of NO in upstream,including the DDAH 1 expression, ADMA level and the activity of NOS,but also the production of NO in downstream, which included the level of peroxynitrite(ONOO~-) and neuron apoptosis in brain.The possible mechanism involved in the neuroprotective action of NAC and role of DDAH/ADMA system were also examined.Therefore,a new approach to study the mechanism of METH-induced neurotoxicity would be dicussed in our study.METHODS1 Animal protocol and METH toxicity observation. Wistar rats,weighting from 180g to 220g(the experimental animal centre of Southern Medical University,China) were randomly divided into 4 groups.Animals in NS group were injected with saline(1mL,at 12 h intervals),the same treatments were continued for 6 consecutive days.NAC group was the same administration as NS group,but with NAC(150mg/kg body weight) instead of saline.METH group was injected with saline(1mL,at 12 h intervals) during the first two days,but injected with METH(15mg/kg body weight,at 12h intervals) from the third day to the sixth day.NAC+METH group was similar to NAC group for the first two days,while NAC was pretreated before METH from the third day to the sixth day.The body weight, temperature and behavioral changes of rats were recorded.The histological changes of brain,liver,lung and kidney were observed by HE stain.High performance liquid chromatogram(HPLC) was used to measure the contents of DA and DOPAC.ROS fluorescence detection kit was used to detect the ROS changes in rat striatum.2 The protective effects of NAC against METH-induced nerve injury:role of the DDAH/ADMA System.We measured the changes of level ONOO~- in rat striatum by fluorescence detection. TUNEL method was used to observe the neuron apoptosis.The expression of protein DDAH 1 was detected by Western Blotting.HPLC was used to detect the ADMA level and the NOS detection kit was made to measure the NOS activity.RESULT1.Compared with the NS group,The body weight of rats significantly decreased (P=0.000) after METH administration.However,the body temperature increased significantly(P=0.000).The marked behavioral changes were observed such as the rats were hyperlocomotioned and easy to be irritated(P=0.000).The HE stain showed that swell of hepatic cell and neuron,puhnonary congestion and infiltration of inflammatory cell.It is also observed that ROS levels were increased significantly (P=0.000) and the levels of DA and DOPAC were significantly decreased(P=0.000; P=0.003).Pretreatment with NAC could attenuate the elevated score of stereotype behavior and ROS level(P=0.000;P=0.000) and prevent the loss of DA and DOPAC level(P=0.000;P=0.040).2.Compared with the NS group,repeated administration of METH could elevate the expression of protein DDAH 1 and decrease significantly the content of ADMA (P=0.000),the activity of NOS,ONOO~- level and neuron apoptosis increase significantly(P=0.000;P=0.000;P=0.000).Pretreatment with NAC(30 min before each METH injection) attenuated the elevated protein DDAH 1 expression,increased the content of ADMA(P=0.000),depressed the activity of NOS,ONOO~- level and neuron apoptosis(P=0.000,P=0.003,P=0.000).Furthermore,negative correlation between the level of ADMA and ONOO~- and positive correlation between the level of ONOO~- and neuron apoptosis were observed.CONCLUSION1.METH could cause the imbalance between ROS and antioxidants in striatum of rat brain.METH causes marked neurotoxicity,which included behavioral changes of rats, injuries of the neurons,increased level of ROS and loss of DA and DOPAC. However,the antioxidant NAC could attenuate METH-induced neurotoxicity.2.METH could influence the DDAH/ADMA system and the production content of NO in downstream.METH could increase the expression of protein DDAH 1 and the NOS activity,as well as ONOO~- level and neuron apoptosis.However,the ADMA content was decreased.But the changes above were attenuated by pretreatment with NAC,indicating the protective effects of NAC to METH-induced neurotoxicity. Furthermore,it's very interesting to find there is negative correlation between the level of ADMA and neuron apoptosis.3.Therefore,we could conclude that the DDAH/ADMA system would play an important role in oxidative stress caused by METH which could be the major mechanism of METH-induced nerve injury.