Metabolites Of Diabetic Peripheral Neuropathy Treated By

Purpose:This study was to establish a rat model of diabetic peripheral neuropathy,simulate and characterize the corresponding Qi deficiency and Blood stasis syndrome model.Through metabolomics studies of the model of plasma specimens, to look for characteristic metabolites of diabetic peripheral neuropathy with Qi deficiency and Blood stasis syndrome lesion. Through metabolic changes of product analysis, to explore the mechanism of their different metabolic pathways related to physiological and pathological changes. Explore the molecular changes of diabetic peripheral nerve with Qi deficiency and Blood Stasis syndrome,and further reveals syndromes essence of diabetic peripheral neuropathy syndromes with Qi deficiency and Blood stasis syndrome. With the use of metabolomics techniques and methods,from the perspective of endogenous metabolites to explain the mechanisms and targets with Mudan granule to treat diabetic peripheral neuropathy.To provide a scientific basis for the systematic study of the mechanism of diabetic peripheral neuropathy with traditional Chinese medicine.Material and method:1 Preparation of animal disease modelsSelected 40 healthy male SD rats weighing(180 ± 20) g, were randomly divided into two groups by the random number table method after adaptive feeding 1 day: the control group(n= 10) and model group(n = 30). The blank control group, normal diet for 8 weeks,the model group were given high fat and high sugar diet for 8 weeks. Before modeling the fasted for 12 hours, the model group was given a single intraperitoneal injection of streptozotocin solution(Streptozotocin, STZ) to induce diabetes. The rat tail vein blood glucose were measured by the blood glucose monitor after 72 hours, blood glucose greater than 16.7mmol / L were included in the observed object. The control group 10 rats after 12 hours fasting were injected intraperitoneally with the same volume of 0.1mol / L of citric acid buffer solution.Then the model group fed cabbage single day 15 ~ 20 g per mouse, free access to water and swim to the endurance limit(half sinking). And doubleday continued high sugar high fat diet feeding, a total of 4 weeks and to establishdiabetic peripheral neuropathy with Qi deficiency and blood stasis syndrome model. To observe and record general status, the biological characterization of qi deficiency and blood stasis syndromethe,the interval time of licking the claws,the rats blood glucose, the changes of triglyceride,low density lipoprotein, total cholesterol and high density lipid protein, to determinate the nerve conduction velocity and blood rheology in rats.Animal anatomy confirmed degeneration or necrosis of nerve cells in rats pathological changes by electron microscopy and light microscopy.All the experimental data are used in IBM SPSS Statistics 19 software package for statistical analysis.Each experimental data was presented as mean ± standard deviation.ANOVA was used to process the data Mean difference between the groups.P <0.05 when determining the difference was significant, P <0.01 was considered very significant difference.2 Metabolomics studies in diabetic rat model of diabetic peripheral neuropathyThe rat plasma samples of the model group(n =7) selected from successful preparation of diabetic peripheral neuropathy with Qi deficiency and Blood Stasis syndrome and the control group(n = 7) were analyzed.The total number of ions generated by Total Ion Chromatogram( TIC) treated by Masslynx V4.1 workstation after peak matching, peak alignment, normalization and other treatment, were imported to SIMCA-P11.5(Umetrics,Umea, Sweden) software for principal component analysis(PCA).To produce score plot to obtain samples classified information, to produce the loading plot to identify potential biomarkers. The identification of metabolic marker in the rat plasma-based metabolism UPLC-MS spectrum,MS information through primary and secondary metabolic markers,combined database searches, literature and reference confirmation were completed to trace back to its source and metabolic pathway. First of all, according to retention time and mass to charge ratio, extraction ion chromatography peaks in the total ion chromatogram extracted metabolic markers in the map. The second step, through a mass spectra of extracted ion chromatogram peaksobtainedmetabolite, to find its ionic excimer and related ions, metabolites of molecular weight determination. The third step, two order mass scanning on metabolic markers,obtain the structure information of metabolites. The fourth step, the use of domestic and international relevantmetabolomicsmarkers onlinedatabase,retrievalbymetabolites of molecular weight, given the number of candidate compounds. The two order massinformation comparison of metabolite, remove unrelated compounds, complete the metabolite identification. The fifth step, for the control of goods available, under the same experimental conditions plasma samples and the control sample were analyzed to carry on the confirmation of the identification results through the comparison of the retention time, the information of the primary and secondary mass spectrometry.3 Study on application of Mudan granule intervention on diabetic peripheral neuropathy in rats with the metabolomic approach.The successful preparation of rats model of diabetic peripheral neuropathy with Qi deficiency and blood stasis syndromes were divided into two groups : the model group(n=7) and model+ traditional Chinese medicine group(n=7). According to the traditional Chinese medicine dosage of the clinical patient per day theequivalent dose were converted into in rats 0.59g/100 g body weight per day, Every rat of the model plus traditional Chinese medicine group was fed once, for 4 consecutive weeks. Through the UPLC-MS technique, to observe the difference variation trend of small moleculemetabolites in plasma group, model of diabetic peripheral neuropathy with Qi deficiency and blood stasis syndrome through Mudan granuletreatment after the intervention. The system stability analysis method with plasma metabolites in rat spectrum was validated. At the same time, the stability precision of the instrument, repetitive,plasma samples and system stability were investigated.Results:1. The model group rats appeared after the start, the endurance of swimming half settling time decreased day by day, food less, accidie, spirit cachexia, the light brightness of body hair subsided, soft or pond stool. After 4 weeks, the rats in the model group began to appear blood stasis symptom such as ear vascular dilatation and congestion, black lips, claw and tail with dark purple.2. After 6 weeks, fasting blood sugar increased in model group.Triglyceride, low density lipoprotein, total cholesterol and high density lipoprotein were abnormal changed, The model is completed the model group had elevated blood glucose.Animal anatomy by light microscope and electro microscope analysis showed that thedegeneration and necrosis of nerve demyelination, Nerve conduction velocity decreased and blood rheology changed.3. Plasma metabolite profiling study found that arginine, phenylalanine, tryptophan, cholic acid and LPC in the blank group and the model group are available through UPLC-MS.Compared with blank control group rats, L-arginine,tryptophan,cholic acid and lysophosphatidylcholine(LPC) 18:2 levels in model group rats, increased significantly;phenylalanine, LPC 18:0, LPC 18:1 and LPC 18:2 level decreased significantly.The results suggest that several substances exist abnormal changes in diabeticperipheral neuropathy of diabetes with Qi deficiency and blood stasis syndrome in rat plasma, possibly as a biomarker associated with diabetic peripheral neuropathy with Qi deficiency and blood stasis syndrome.4.Plasma metabolite profiling study found through UPLC-MS, compared with the rats in the model group, the level of tryptophan, LPC(20:4) and PC(36:4) with intervention group rats plasmaincreased significantly;the levels of phenylalanine, LPC(14:0), LPC(16:0/0:0), LPC(0:0/18:0),LPC(18:0/0:0), LPC(18:1), LPC(0:0/18:2), LPC(18:2/0:0) and cholic acid significantly reduced.The results suggested that content in the plasma of several substances occurring after the application of Mudan granule intervention in the treatment of diabetic peripheral neuropathy in rats with Qi deficiency and blood stasis syndrome changed, may provide a new basis for studying the mechanism of Mudan granule in the treatment of diabetic peripheral neuropathy.Conclusion:1. In this study, using a combination of methods Syndrome, the evaluation of the "biological characterization" was introduced.Based on the preparation of qi deficiency and blood stasis model by using the method of the research of traditional Chinese medicine is recognized as fatigue internal injuries "half settlement", combined with small dose injection of streptozotocin to establishcompliance with relevant pathological indicators of diabetic peripheral neuropathy model. The results of the successful establishment of animal model of diabetic peripheral neuropathy with Qi deficiency and blood stasis syndrome were both to meet the criteria of diabetic peripheral neuropathy, and also the biological characteristics with Qi deficiency and blood stasis model representation.2. Arginine, phenylalanine, tryptophan, cholic acid and a variety of LPC and other substances was found in the experiment may be metabolites of diabetic peripheral neuropathy in rats with Qi deficiency and blood stasis syndrome characteristic. Studies results provide a new thinking method for early diagnosis and treatment of diabetic peripheral neuropathy with application of the metabolomic approach.3. Through research and analysis of the plasma markers of metabolic pathways and metabolic mechanisms in rat experimental model of group showed the pathogenesis of diabetic peripheral neuropathy may be the result of the combined effect of the following factors:Brain monoamine neurotransmitter serotonin(5-HT) dysregulation; t he synthesis of dopamine(DA) and norepinephrine(NE) decreased, false neurotransmitter octopamine and benzene ethanol amine generated,and affected the normal function of the nervous system;phenylalanine metabolic abnormalities lead to a lack of vitamin B12;vasomotor dysfunction, vascular permeability, endothelial function injury;the synthesis and release of nitric oxide(NO) reduced; The activity of Na+-K+-ATP reduced, the oxygen free radical injury, oxidative stress, increased vascularinflammatory reaction; glycolipid metabolic abnormally changed.4. The treatment intervention of Mudan Granule on diabetic peripheral neuropathy showed characteristics of many ways, multi-target, two-way regulation etc. Not only can make the tryptophan, LPC(20:4) and PC(36:4) level increase significantly; at the same time also make phenylalanine, LPC(14:0), LPC(16:0/0:0), LPC(0:0/18:0), LPC(18:0/0:0), LPC(18:1), LPC(0:0/18:2), LPC(18:2/0:0) and bile acid levels significantly reduce. Through the characteristic trend of the downstream metabolic after the intervention appeared to infer the characteristics of Mudan granule mechanism.5. Using learning method of the spectrometry metabolomics technology of ultra high performance liquid chromatography mass is one of effective means of TCM animal model ofcombination of disease and syndrome, According to the variation of diabetic peripheral neuropathy metabolites guess its possible pathogenesis, disease and related research to reveal the mechanism of diabetic peripheral neuropathy and traditional Chinese Medicine has established a good platform.