| Objectives and Significance of ResearchFatigue belongs to“lao zheng”in traditional Chinese medicine,mostly caused by“qi and blood deficiency”or“zang-fu disorder”,and it is often treated with prescriptions for invigorating“qi”and“blood”.Bone marrow type acute radiation sickness belongs to“blood deficiency”in TCM,mainly due to the absence of new blood.Traditional Chinese medicine formula with the functions of promoting hematopoiesis,anti-oxidation and enhancing immunity has certain application value in the protection and treatment of ionizing radiation injury.Danggui Buxue Decoction?DBT?was first described by Li Dongyuan in<Neiwaishangbianhuolun Shushangweiqilun>in 1247.It consists of astragalus and angelica sinensis,and used for nourishing“qi”,enriching“blood”and treatment of“lao juan nei shang”.According to the 2015 edition of Chinese pharmacopoeia,the oral liquid of DBT is used for the treatment of“qi and blood deficiency”.It has the functions of improving immunity,anti-oxidation,promoting hematopoiesis of bone marrow and improving chronic fatigue syndrome,which involved multi-component,multi-target and multi-path collaboration.However,the active components of DBT are complex,and the explanation of the mechanism is only limited to a single component or biochemical appearance,failing to reflect the holistic and systematic view of TCM compound therapy.The mechanism of anti-fatigue and anti-radiation of DBT still needs further analysis.In order to explore the regulatory mechanism of DBT on biochemical pathway changes caused by fatigue and radiation injury,metabolomics analysis base on GC-MS and LC-MS combined with chemometrics methods was first applied to find endogenous metabolites with significant changes in abundance.Then,metabolic pathways were constructed to analyze the metabolic network related to the effects of DBT on the metabolism profile of fatigue and radiation injury.And further to explore the metabolic regulation of DBT on the disease from the level of metabolites.The purpose of this paper was to investigate the effect mechanism of Danggui Buxue Decoction on chronic fatigue syndrome,exercise fatigue and radiation injury.Methods and Results Research1.Metabolomics analysis of serum reveals the effects of DBT and single drug on rats with chronic fatigue syndrome1).A HPLC method for the determination of Astragaloside IV and Ferulic acid in DBT was established.The method was sensitive and accurate showed by systematic adaptability experiment and methodological investigation,providing a guarantee for DBT quality control.2).The rat model of chronic fatigue was established by basic diet combined with weight-loading swimming.The serum metabolic profile of rats with normal and chronic fatigue syndrome were determined by LC-MS and GC-MS,and the metabolites were screened out by multivariate statistical analysis and variance analysis.The result showed that there were 24 metabolites associated with chronic fatigue syndrome.3).Then,the effects of DBT with low,medium and high doses,single drug of Huangqi and single drug of Dangggui on the levels of metabolites in rats with chronic fatigue syndrome were compared by using the metabolomics analysis based on LC-MS and GC-MS.The results showed that the high-dose DBT could significantly regulate the metabolic disorders caused by chronic fatigue syndrome,and the effects were better than low,medium dose of DBT and single drug.4).Metabolites significantly regulated by DBT were analyzed and metabolic network was constructed.The results showed that the 5 metabolic pathways with high correlation with the effects mechanism of DBT were phenylalanine,tyrosine and tryptophan biosynthesis,proline,leucine and isoleucine biosynthesis,glycine,serine and threonine metabolism,taurine and hypotaurine metabolism and phenylalanine metabolism.5).Biochemical results showed that DBT significantly improved leukopenia,the degeneration of thymus and spleen caused by chronic fatigue syndrome,and significantly down-regulated the levels of TNF-?and IL-6 in rats with chronic fatigue syndrome.Combined with the changes in serum metabolites and metabolic pathway analysis,indicating that DBT could improve thymus function by regulating the metabolism of threonine and serine,which restored leukocyte differentiation and improved immune function.Secondly,the metabolism of choline was regulated to normal to improve the degeneration of the spleen.Then,the inflammatory reaction,caused by chronic fatigue syndrome,was inhibited by the enhanced function of the spleen.The levels of TNF-?and IL-6 were down-regulated after that.In addition,DBT could help to eliminate free radicals and reduce the oxidative stress response by up-regulating the levels of precursor of glutathione and taurine in this paper.Then,the level of MDA was significantly down-regulated and SOD and GSH-Px were significantly up-regulated.DBT could also improve energy metabolism and regulate the pathway of phenylalanine and tyrosine biosynthesis to normal.And the level of norepinephrine was up-regulated to improve the exercise endurance of rats chronic fatigue syndrome.2.Metabolomics analysis of urine reveals the effects of DBT and single drug on rats with chronic fatigue syndrome1).In order to supplement the results of serum metabolomics research,metabolomics analysis based on LC-MS was established to determine the urine metabolic profile of rats with normal and chronic fatigue syndrome,and the metabolites related to chronic fatigue syndrome were screened by multivariate statistical analysis.The result showed that there were 13 metabolites significantly changed in the urine of CFS rats.The level of citrulline,sperminr and guanidine were significantly increased,and the level of glutamic acid,carnitine,2-oxadipate,2-oxoglutaramate,citrate,fumarate,leucine,proline,kynurenine and tryptophan were significantly decreased.And the changes of tryptophan,citric acid and 2-oxadipic acid were consistent with the results of serum analysis.2).The effects of DBT with low,medium and high doses,single drug of Huangqi and single drug of Dangggui on the levels of metabolites in rats with chronic fatigue syndrome were compared by using the metabolomics analysis method based on LC-MS.The results showed that the high-dose DBT could significantly regulate the 13metabolites in rats with chronic fatigue syndrome.3).Metabolites significantly regulated by DBT were analyzed and metabolic network was constructed.The results showed that the top 5 metabolic pathways associated with the effects mechanism of DBT were glutamine and glutamic acid metabolism,alanine,aspartic acid and glutamic acid metabolism,tryptophan metabolism,arginine and proline metabolism and TCA cycle.DBT could restore the tryptophan metabolism pathway by up-regulating the levels of tryptophan,kynuric acid and 2-oxoadipate to promote the synthesis of5-hydroxytryptamine to improve the disorder of Corticosterone metabolism caused by the abnormal hypothalamic-pituitary-adrenal axis.Then DBT exerted the effect on rats with chronic fatigue syndrome.DBT could also act on the liver by regulating metabolites which are on the metabolic pathways of arginine and proline,that made the urea cycle to normal.Then,DBT could reduce the production of RNS by reducing the production of NO to alleviate the level of oxidative stress and inflammatory reaction in rats with chronic fatigue syndrome.3.The metabolomics study of the effects of DBT on exercise-induced fatigue mice1).The model of exercise-induced fatigue was established by weight-loading swimming.The metabolic profile of normal and exhausted mice were determined by GC-MS and 14 metabolites significantly changes in serum were screened out by multivariate statistical analysis.2).The method base on GC-MS and multivariate statistical analysis was used to compare the changes of the level of metabolites in mice with different doses of DBT.The results showed that DBT could significantly increase the levels of proline,citric acid,glycine,creatinine,urea,threonine,alanine,tyrosine and malic acid in fatigue mice,and significantly decrease the levels of pyruvate,lactic acid,succinic acid,oxalic acid and acetic acid.3).Metabolites significantly regulated by DBT were analyzed and the constructed metabolic network showed that the metabolism of phenylalanine,tyrosine and tryptophan metabolism,glycine,serine and threonine metabolism,glyoxylic acid and dicarboxylic acid metabolism,pyruvate metabolism and TCA cycle were involved.Among them,DBT was firstly to improve the swimming time of exhaustion by improving the metabolic level of TCA cycle by regulating pyruvate,a metabolite of key nodes.And the production of ROS was reduced by decreasing blood oxygen concentration after DBT treatment.In addition,it could decrease the concentration of H+in muscles by accelerating the clearance of lactic acid,to improve the release of Ca2+and regulate it coupled with troponin.Then,the purpose of eliminating fatigue was achieved.4.Metabolomics study of serum on mice with radiation injury1).The bone marrow type acute injury was established by exposure to 60Co?with a dose of 6.5 Gy.And the metabolic profiles of normal and model mice were analyzed by metabolomics analysis of serum based on GC-MS.17 metabolites with significant changes in levels were screened,which involved in amino acids,organic acids and fatty acids.Among them,the significantly decreased ornithine could be a potential biomarker for the excessive generation of free radicals caused by radiation damage.2).The metabolic network constructed by metabolites with significant changes showed that amino acid metabolism,fatty acid metabolism and energy metabolism disorders were significantly correlated with radiation damage.Under exposure to radiation,the levels of serine,glycine and aspartic acid were significantly down-regulated in C57 mice,resulting in disorder of purine metabolism and pyrimidine metabolism.Then,the fuction of DNA replication was damaged and the differentiation and proliferation of bone marrow cells were inhibited,causing the reduction of red blood cells,white blood cells and platelets.Secondly,the significantly decreased aspartate and lysine would lead to a decrease in carnitine content,which blocked the?-oxidation of fatty acids.Under the high concentrations of ROS,the metabolic levels of fatty acids involved in lipid peroxidation,which leaded to produce a large number of cytotoxins.Cells biological membrane damage caused by cytotoxins could affect the cell and organ function,immunity and weight.At the same time,lipid peroxidation leaded to mitochondrial damage,and the synthesis of ALA was inhibited,which is an important precursor of heme.And the content of hem was also decreased.Then that leaded to impaired the function of red blood cell.5.The metabolomics study of the anti-radiation effects of DBTThe anti-radiation effects of DBT were evaluated by a metabolomics method base on GC-MS.The results showed that DBT could significantly regulate the levels of 9metabolites related to the radiation injury:?-alanine,proline,citric acid,cholesterol,lysine,stearic acid,palmitic acid and oleic acid.Conclusion1.In this paper,a metabolomics analysis method was established for the first time to investigate the effects of DBT on rats with chronic fatigue syndrome.LC-MS and GC-MS were adopted to analyze the serum and urine in rats.The results showed that 34differentially expressed metabolites were closely related to the effects of DBT on rats with chronic fatigue syndrome.The analysis of metabolic pathways and construction of metabolic networks revealed that the target organs of DBT for chronic fatigue syndrome were thymus,spleen and liver.2.In this paper,for the first time,the metabolic pathways of glycine,serine and threonine was selected as the metabolic regulatory center of DBT to improve immunity,oxidative stress and inflammatory response caused by chronic fatigue syndrome.3.In this paper,the metabolomics analysis based on GC-MS was established to investigate the effects of DBT on exercise fatigue for the first time,and the results indicated that pyruvic acid was the key node of metabolic regulation of DBT to improve exercise endurance and accelerate fatigue elimination.4.The metabolomics analysis based on GC-MS was used to explore the molecular mechanism of decreased hematopoietic function caused by radiation injury.The result showed that 17 metabolites involved in amino acid metabolism,fatty acid metabolism and energy metabolism were closely related to the phenotype of radiation-induced diseases. |
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