Metabolomic Study On Model Combining Disease With Syndrome Of Both Qi-yin Deficiency And Blood Stasis Of Type 2 Diabetes Mellituss

Objective To investigate the metabonomics differences of urine and plasm between syndrome of both Qi-yin deficiency and Blood stasis of type 2 diabetes mellitus's rats by metabolic method,analyse the relationship between the TCM syndrome and metabonomics differences in order to determine the metabolic characters and small molecular marker of both Qi-Yin deficiency syndrome and Blood stasis syndrome and explore the changes of endogenous metabolites in the same disease with different methods,thus to provide experimental evidence for essence of type 2 diabetes mellitus with Qi-Yin deficiency syndrome and Blood stasis syndrome and establish a technology Platforms of metabonomics for TCM syndrome based on Gas Chromatography-Mass Spectrometry(GC-MS)and Nuclear Mgnetic Resonance(NMR).Methods 80 male SD rats(200±20g))were randomly divided into two groups including the normal group and type 2 diabetic model group.The type 2 diabetic model rats were induced by injected streptozotocin(STZ,30mg/g)plus high-fat and sugar diet.Fasting blood glucose(FBG)which is higher than 11.1mmol/L was identified as type 2 diabetes.According to FBG,the type 2 diabetes model rats were randomly divided into three groups including type 2 diabetic model group,Qi-Yin deficiency syndrome group and Blood stasis syndrome group.Qi-Yin deficiency syndrome model was induced by hypodermic injection of adrenaline plus ice water bath,and Blood stasis syndrome model was induced by hypodermic injection of L-thyoxine plus sleep deprivation.Shengmai decotion and Xuefuzhuyu decoction were choosed as a treatment compound for Qi-Yin deficiency syndrome and Blood stasis syndrome respectively.Urinary and blood samples were collected at four week.The reliability of model of syndrome of Qi-yin deficiency and Blood stasis of type 2 diabetes mellitus was evaluated by the laboratory,histopathology,general behavior and the curative effects.GC-MS and NMR were used to analyze metabonomics character of the urine and plasm samples.Principal components analysis(PCA),partial least-squares discriminant analysis(PLS-DA)and Orthogonal partial least squares-discriminant analysis(OPLS-DA)were applied to identify potential biomarkers.The relationship between metabonomics differences in Qi-Yin deficiency syndrome and Blood stasis syndrome was further analysed in order to explore the metabolic characteristics respectively.The reliability of model of syndrome of Qi-yin deficiency and Blood stasis of type 2 diabetes mellitus was confirmed by effects of Chinese herbal compound.Results 1.Compared with the normal group,the whole blood viscosity was increased in type 2 diabetes mellitus's rats with blood stasis syndrome(P<0.01).The physical signs such as growth retardation,hair withered and dull action in type 2 diabetes mellitus's rats is consistent with the clinical features of Qi and Yin deficiency.The effects of Chinese herbal compound showed that laboratory indexes and the general condition were significantly improved,the reliability of model of syndrome of Qi-yin deficiency and Blood stasis of type 2 diabetes mellitus' rats was confirmed once again.2.Results of urine metabolic analysis based on GC-MS:PCA and PLS-DA analysis methods have good discrimination and prediction.The urine metabolic profiles were completely separated in Qi-Yin deficiency syndrome group,Blood stasis syndrome group and the normal group.It is showed that the urine metabonomics can be used to identify the TCM syndrome types.And two TCM syndrome types had different metabolic patterns.Proline,tyrosine,phenylalanine,arginine,aspartic acid,glutamic acid,glycine,sorbitol,hippuric acid,adenine and cytosin were potential biomarkers of Qi-Yin deficiency syndrome.Tryptophan,sphingosine,myoinositol,succinic acid,glutarc Acid,lactate and urea were potential biomarkers of Blood stasis syndrome.The effects of Chinese herbal compound showed that metabolic profile changed significantly in two TCM syndrome types.Compared with type 2 diabetic patients with TCM syndrome,metabolic profile of type 2 diabetes mellitus's rats with Qi-Yin deficiency syndrome and Blood stasis syndrome showed clustered closely,but the patients's were dispersed.It showed that individual differences existed in clinical patients.3.Results of plasm metabolic analysis based on NMR:PCA and PLS-DA analysis methods have good discrimination and prediction.The plasm metabolic profiles were completely separated in Qi-Yin deficiency syndrome group,Blood stasis syndrome group and the normal group.It is showed that the plasm metabonomics can be used to identify the TCM syndrome types.There are great difference in the plasm metabolits between Qi-Yin deficiency syndrome and Blood stasis syndrome.The metabonomics results showed that amino acid metabolism,energy metabolism and glucose metabolism was disorder.Leucine,isoleucine,valnine,glutamine,acetate,aspartate,alanine,pyruvate and lactate were potential biomarkers on syndrome differentiation of Qi-Yin deficiency syndrome and Blood stasis syndrome.Leucine,isoleucine,valnine,aspartate and lactate were the main compounds of the two types of syndrome differentiation.The effects of Chinese herbal compound showed that metabolic profile changed significantly in two TCM syndrome types.Conclusion The model of syndrome of Qi-yin deficiency and Blood stasis of type 2 diabetes mellitus was established.The metabonomics differences of urine and plasm between syndrome of both Qi-yin deficiency and Blood stasis of type 2 diabetes mellitus's rats was analysed by metabolic method based on GC-MS and NMR.The urine and plasm samples metabolic profiles were completely separated in Qi-Yin deficiency syndrome group,Blood stasis syndrome group and the normal group.The differential metabolites were potential biomarkers on syndrome differentiation of Qi-Yin deficiency syndrome and Blood stasis syndrome.The analysis of biomarkers and metabolic pathway will provide new explanation on etiology and pathogenesis on Qi-Yin deficiency syndrome and Blood stasis syndrome and reveal the essence of TCM syndrome.The study validated the feasibility of TCM syndrome by metabolic method.It will provide a powerful support for clinical diagnosis and syndrome differentiation of TCM syndromes of type 2 diabetes.