Metabolomics Study On Cynomolgus Monkey Stress Using Nuclear Magnetic Resonance Strategy

Laboratory animals play an irreplaceable role in the field of life science,medicine and drug research and development.To a great extent,the results of animal experiments,such as drug safety evaluation and research results,directly or indirectly decide the fate of the certain drugs for development,therefore,all the factors except the drug self which may interfere with the results of the experiment must be reduced and controlled as far as possible.These factors mainly include environmental factors and internal factors,and one of factors was animal stress which is inevitable.Cynomolgus monkeys(Macaca fascicularis)are widely used in drug toxicology experiment and safety evaluation and research.In the process of using cynomolgus monkeys,a lot of inevitable exogenous factors may influence the results of the experiment.The most common and inevitable factors includes transportation between suppliers and the final facility,fasting and restraint during the experiment,etc.Researchs on the exogenous stimulus have a few number of published papers,and which was based on traditional evaluation strategy,such as evaluation of hematology,serum biochemical,to evaluate the impact of these stress on the body.Although the traditional evluation method has many advantages,it is relatively insensitive to slight metabolic disturbance.Metabolomics is another emerging genomics technologies after genomics,transcriptomics and proteomics,which is the quantitative measurement of the dynamic multiparametric metabolic responses of living systems to pathophysiological stimuli or genetic modification.In this thesis,we applied NMR-based metabonomics technology and combining with the traditional means to evluate the effects on transportation,restraint and fasting in cynomolugs monkeys.In study of transportation,we found that transportation lasting 8 hous could result in imbalance of oxidant and antioxidant and excess oxidative stress in cynomolgus monkeys,and the levels of MDA,PC and GSH increased,the activities of T-AOC,SOD,GSH-Px and CAT decreased;the concentrations of WBC,NUET,EOS,Retic,BUN and CK decreased,the contents of RBC,HGB,HCT,LYM,BASO,ALT,AST,ALP,CREA,LDH and TBIL increased;and plasma levels of lactate,alanine,glutamate,glutamine,pyruvate,myo-inositol,creatinine and urea increased,plasma levels of isoleucine and proline decreased;the levels of lactate,glycoproteins,pyruvate,citrate,choline,creatinine,?-ketoglutarate,taurine and allantoin increased,the levels of isoleucine,leucine,creatine,myo-inositol,3-hydroxybutyrate and hippurate decreased.These novel findings indicated that cynomolgus monkeys subjected to transport stress had a distinctive metabolic status,including altered energy metabolism,amino acid metabolism,lipid metabolism and perturbations in gut microbiota.These altered parameters would recovery to normal level after about 21 to 35 days.In study of restraint,a metabolomics approach based on NMR spectroscopy and pertinent multivariate data analyses were used to evaluate the impact of monkey chair restraint on metabolic alterations in cynomolgus monkeys.PCA and OPLS-DA showed that monkey chair restraint for two hours could induce metabolic perturbations.Compared with the control group,the levels of lactate,N-acetyl-glycoproteins,glutamate,glutamine,pyruvate,glycine,creatine,myo-inositol,creatinine,threonine and fumarate in monkey chair restraint group were significantly increased(P<0.05).Meanwhile the levels of isoleucine,alanine,choline,trimethylamine and dimethylamine were decreased significantly(P<0.05).Our results demonstrated that a single monkey chair restraint could influence the major energy metabolism and amino acid metabolism in cynomolgus monkeys.In study of fasting,impact of fasting on plasma and urine metabolic alterations,hematology,plasma biochemistry in cynomolgus monkeys were evaluated.Twelve male cynomolgus monkeys were divided into two groups,the fasting group and ad libitum groups.The urine were collected for 24 hours for both two groups.Plasma were collected at pre-fasting and post-fasting.PCA and OPLS-DA showed that plasma leucine,lipoproteins,glycoproteins,acetone,pyruvate,salt and unsaturated lipids muscle was significantly higher(P<0.05)compared with that in non-fasting,while lysine,arginine,glutamate,glutamine,myo-inositol and ?-glucose decreased significantly(P<0.05).Urine pyruvic acid,acetoacetic acid,choline,allantoin and creatinine were significantly higher than that in non-fasting group(P<0.05),while isoleucine,glycoprotein,citrate,dimethylamine,myo-inositol,formate and taurine significantly decreased(P<0.05).Hematology results showed that reticulocyte decreased significantly(P<0.01)in fasting group compared with that in non-fasting group,while WBC,RBC,HGB,HCT,MCH,MCHC,RDW,PLT,MPV,MONO,EOS,BASO and LUC increased or decreased in different degrees,but increased or decreased by less than 10%.Plasma biochemical results showed that ALT,AST,BUN,ALB,LDH,CK,GGT and Ca concentration increased or decreased in different degrees in fasting group,and the BUN,ALB and Ca concentrations(P<0.05)was significantly reduced.Elevated items in fasting group included ALP and P,while P is increased significant(P<0.05).Therefore,fasting could result the changes in hematological,plasma biochemical and disturbances of energy metabolism,amino acid metabolism,lipid metabolism,and imbalance of the intestinal microbial colonies.Our study might suggest that these influences should be taken into consideration in the design of experiments and the interpretation related blood and/or urine data when using cynomolgus monkey suffered transportation,restraint and fasting.