| Objective:The rat models of methamphetamine(METH)dependence on the basis of conditioned place preference(CPP)were established by different doses of METH.The untargeted metabolomic analysis was applied to analyze the changes of metabolites in serum and urine of METH dependent rat models using 1H NMR,and screen potential biomarkers and related metabolic pathways related to METH dependence.It may provide new ideas for forensic toxicant analysis and toxicological mechanisms of METH dependence.Methods:The design of the experiments adopted biased programming,and the non-preferred room as the dispensing room.Sixteen rats with poor mobility and unstable preference were excluded from 100 healthy male SD rats in the pre-test period,and residual rats(n=86)were randomly divided into 6 groups with 14 rats in each group according to the dosage per kilogram of body weight,including 20mg/kg METH group,10mg/kg METH,8mg/kg METH group,5mg/kg METH group,2.5mg/kg METH group and control group.The rats of different doses of METH group were intraperitoneally injected with METH interval of 1d at similar time period,with 10 injections in the conditioning period.The controls were intraperitoneally injected with the same saline at the corresponding period by repeating the above method.All rats were trained for 40min after each injection.The test was conducted for 15min on the second day after the last injection of normal saline.The METH dependent rats models were established successfully according to the behavior change,irritability,stereotyped behavior(SB)scores,and CPP scores.And,the optimal dose group of successful model establishment was selected as the treatment group.Urine was collected on ice in a metabolic cage and serum was obtained from the abdominal aorta after the successful model rats was fasted and drinking for 24h.Arterial blood was separated into serum,and the serum and urine were stored at-80℃ refrigerator until used.The urine and serum samples were analyzed by 1H NMR with CPMG spin echo sequence cpmgprld and the presaturated water peak suppression pulse sequence noesygpprlD,respectively.The MestReNova 12.0 and SIMCA 14.1 software was used to perform serum and urine sample data preprocess by univariate or multivariate statistical analysis statistical analysis,respectively.The characteristic variables were screened according to OPLS-DA(VIP>1)combined with t test(P<0.05),and were assigned and quantified by Chenomx NMR Suite8.5 software and related literature.MetaboAnalyst4.0 web analytic platform was performed differential metabolite-related metabolic pathway.Results:The control rats were normal activity and diet in the trial period.There was no statistical difference in body weight between the control group and the different METH group(P>0.05).The typical behavioral changes have been observed in rats of 20mg/kg METH group after the rats were first injected METH,including shaking and nodding head,lying down and stretching forward,continuous vertical tail,convulsions of body,rapid retreat or rotation,seizures,irritating states including jumping and hitting the wall with slight sound stimulation.Majority rats appeared salivation,paralysis,increased excrement,self-mutilation behavior.78.57%(11/14)rats died in 24h.There were similar typical behavioral changes among 10mg/kg METH group,8mg/kg METH group and 5mg/kg METH group,such as activity increasing,shaking and nodding head,fast or slow continuous exploration,enlarged genitalia,and irritable symptoms.The typical behavioral symptoms delayed occurrence when the number of METH administration increased.Some or individual rats appeared salivation,paralysis,and self-mutilation behavior between the 10mg/kg METH group and 8mg/kg METH group.The SB scores were all passed.50%(7/14)rats died after the rats injected fourth METH in the 10mg/kg METH group,and 64.29%(9/14)rats died after the rats injected seventh METH in the 8mg/kg METH group in the conditioning period.Therefore,the 20mg/kg METH group,10mg/kg METH group and 8mg/kg METH group may be not as the treatment group since more than half of the rats died in conditioning period.The 2.5mg/kg METH group was not also suitable for as treatment group because these rats lacked of typical drug dependence characteristics and half qualified of SB scores.The typical drug dependence characteristics,qualified of SB score,lack of death were observed in the 5mg/kg METH group during the trial period.Besides,there was no statistical significance between the CPP scores of the treatment group(0.2969±0.1073)and those of the control group(0.3160±0.1281)in the pre-test period(P=0.677);the CPP scores of the experimental group(0.7384±0.2007)was significantly increased than that of the control group(0.4091±0.2197)in the test period(P<0.0001).It was indicate that a stable METH dependent rats model could be established using intraperitoneal injection with METH(5mg/kg.time×10 times)interval of 1d in rats by the CPP experiment,and the next experiment could be carried out.So,the 5mg/kg METH group was used as the treatment group.The OPLS-DA was carried out a supervised pattern recognition model.R2Y and Q2 of OPLS-DA model in serum were 0.858 and 0.538,respectively.R2Y and Q2 of OPLS-DA model in urine were 0.916 and 0.691,respectively.The OPLS-DA model could distinguish serum and urine between treatment group and control group,and the clustering trend of the two groups was obvious.The PLS-DA response permutation test was verified.The model was not over-fitted and the model was robust and effective.Five small molecular substances(phosphocreatine,trimethylamine N-oxide,3-hydroxybutyrate,guanidoacetate,and glycolate)were screened as potential biomarkers in the serum of methamphetamine dependent rats using the double standards of univariate and multivariate statistics.And,four endogenous metabolites of creatinine,trimethylamine N-oxide,taurine,and N6-acetyllysine were screened from urine as potential biomarkers.The taurine and hypotaurine metabolism,arginine and proline metabolism,and glycine,serine and threonine metabolism pathways were revealed by MetaboAnalyst4.0 metabolic pathway analysis,which were involved in the pathophysiology of METH dependence.Conclusion:1.It was indicate that a stable METH dependent rats model could be established using intraperitoneal injection with METH(5mg/kg.time×l0 times)interval of Id in rats by the CPP experiment.2.Phosphocreatine,trimethylamine N-oxide,3-hydroxybutyrate,guanidoacetate,glycolate,creatinine,taurine,N6-acetyllysine in serum and urine may be potential biomarkers of METH dependent rats.Metabolic pathways such as taurine and hypotaurine metabolism,arginine and proline metabolism,and glycine,serine and threonine metabolism may be involved in the pathophysiological process of METH dependence. |
