Effect Of Epimedium Brevicornu On Material Energy Metabolism In Cold Syndrome Rat Model And Its Properties Research Based On Systems Biology

Objective:Metabolomics,proteomics and transcriptomics were utilized to investigate the effects of Epimedium brevicomum(EBM)and its split components on the material energy metabolism of cold syndrome model rats and to explore the potential targets and metabolic pathways.The significant foundation of EBM’s rational administration and clinical application was obtained via comprehensively analyzing and exploring the mechanism of action as well as assigning drug property categorization of each component.Methods:Based on the systematic biology,pharmacodynamics profile of EBM and its split components were studied involved with drug property attribution and material energy metabolism.EBM was extracted and divided to five uncrossed groups,and each group’s compound were identified by Unify software and uncrossed verified by HPLC,UPLC-TOF-MS and MarkerlynxTM software.The effects of EBM and its split components on the related indexes of basal metabolism and material energy metabolism of cold syndrome model rats were observed to preliminary assess its pharmacological properties.The UPLC-TOF-MS technique was used to study the urine of the cold syndrome rats with metabonomics.Moreover,the ITRAQ technique was used to study the proteomics of the liver of the cold model rats treated with EBM,and next-generation sequencing techniques were used to analyze the transcriptomics of the the liver’s mRNA in the cold syndrome model rats treated with EBM.The above three results had been assEBMled to analyze the intervention of EBM for the material energy metabolism of cold syndrome model rats.Taking"mRNA expression→protein coding gene→small molecule metabolism network" as the analytical ideal,the drug property of each component was attributed ultimately and the mechanism of improving cold syndrome pathological state of each component was also investigated.Results:1.EBM can be separated into five uncross segments which are the total EBM,EBM polysaccharide group,EBM non-flavonoids group,EBM large polar flavonoids group,EBM small polar flavonoids group.Unification was used to identify 19 compounds in the EBM non-flavonoids group.The large polar flavonoid group was mainly composed of 3,7-glycosidyl-substituted flavonoid glycosides represented by icariin,epmedin A,epmedin B,epmedin C and pterosinide B and pteroside I.The small polar flavonoid group is composed of the rouhuoside,large flower Icariin F,Astragaloside,Epimedium glycosides,Wushan Icariin,etc.,and its structural feature is that it is only substituted in the 7-position of the parent nucleus,and derived from the component without 3-glycon of large polar flavonoid group.The main substance in the EBM non-flavonoids group was alkaloids represented by Magnoflorine.2.In the basic metabolic experiments,the energy conversion rate of EBM large polar flavonoids group was significantly inhibited,and the tendency of basal metabolism was decreased.The energy conversion rate and the autonomic activity coefficient of EBM polysaccharide group was significantly increased,and there was a significant increase in heat production in the EBM non-flavonoids group,suggesting that the two component have the potential of promoting basal metabolism.In addition,the total EBM and EBM small polar flavonoid groups didn’t show significant change.3.In the related indexes of material energy metabolism experiments,the total EBM can reduce the levels of Acetyl-CoA and 5-HT,and inhibit the pyruvate oxidation stage and the autonomic nervous system to slow the metabolism of the body.EBM polysaccharide can ncrease the content of GCK and COX and promote the release of T3 in endocrine system,and promote glucose metabolism,oxidative respiratory chain and hormone secretion to speed up the metabolism of the body.EBM non-flavonoid group showed trend of affect the related indicators of material energy metabolism but not significant.EBM large polar flavonoid group decreased the levels of CS,PYGL,ATGL,Na+-K+-ATPase,and inhibit the tricarboxylic acid cycle,the synthesis and decomposition of glycogen,lipid metabolism pathway and cell respiration oxidative phosphorylation stage.EBM small polar flavonoid group decreased the level of PFK-1,PYGL,SCR,and inhibit the glucose oxidation into pyruvate stage,glycogen synthesis and decomposition,cell respiration oxidative phosphorylation stage.4.The result of metabonomics showed that,the urine profile of cold syndrome model rats changed significantly following intervention with EBM.The results of the PCA analysis showed that the model and the blank groups showed clustering respectively away from each other in the three-dimensional space.15 biomarks associated with material energy metabolism were indentified which were phosphatidyl serine(18:0/20:4),phosphatidylcholine(18:0/20:3),N-formylkynurenine,kynuric acid,α-oxoglutarate,etc..The total composition of EBM can not only reduce the level of α-ketoglutaric acid(α-KG)and glucosamine-6-phosphate,thereby slowing the process of tricarboxylic acid cycle,but also rise the level of hydrogenated Leucodopachrome,thereby promoting the tyrosine metabolism process.EBM polysaccharide could increase the levels of phosphatidylserine(PS),phosphatidylcholine(PC),UDP-N-acetyl-D-mannosamine and α-ketoglutarate(α-KG)which were related to the level of energy metabolism,and promote glycerol phospholipid metabolism,tricarboxylic acid cycle,amino sugar and nucleotide glucose metabolism,EBM non-flavonoid group promoted glycerol phospholipids and tryptophan,tyrosine metabolism by increasing the expression levels of phosphatidylserine(PC),N’-Formylkynurenine and Leucodopachrome.It can also inhibit the metabolism of starch and sucrose by reducing the expression level of β-D-Fructose 6-phosphate,4-Hydroxyphenylacetylglutamin.EBM large polar flavonoid significantly reduced the content of glucosamine 6-phosphate,β-D-Fructose 6-phosphate and 2-phenylglucuronide,which indicated that the inhibitory effect on the material metabolism.EBM small polar flavonoid group comparing with model ratios could significantly reduced the level of β-D-fructose 6-phosphate and 2-phenylethanol glucuronide,indicating that it may be similar to that of large polar flavonoid group.5.Proteomics results showed that,The total EBM inhibit glucose metabolism,arachidonic acid metabolism and steroid biosynthesis by decreasing triglyceride 3-phosphate dehydrogenase(GAPDH),alkane 1-monooxygenase(AlkB),cytochrome CYP4502C.However,the cytochrome c oxidase subunit 2(COX Ⅱ)and phenylalanine-4-hydroxylase(Pah)were up-regulated and the oxidative phosphorylation and abnormal phenylalanine metabolism of the cold syndrome model were improved.Overall,the total EBM had the tendency of inhibiting the material energy metabolism of organism.Epimedium polysaccharide can increase the levels of seven proteins that is glucokinase(Idnk),glucuronosyltransferase(UGT),hexokinase(HK3),soluble epoxide hydrolase(Amdhdl),and promote glucose metabolism,pentose phosphate metabolism,oxidative phosphorylation,arachidonic acid metabolism,steroid biosynthesis.The EBM non-flavonoid group could up-regulated Dihydroxyacetone kinase(Tkfc),sulfonated quinone oxidoreductase(Sqrdl),and down-regulated acetyl-CoA acyltransferase 2(ACAA2),long chain acyl-CoA synthase(Acsl1),because of the opposite trends with sugar and lipid metabolisms,suggesting that the material energy metabolism kept a flat state.EBM large polar flavonoid group were capable of reducing the levels of 8 proteins that is glyceraldehyde 3-phosphate dehydrogenase(GAPDH),glucokinase(Idnk),alpha-amylase(Amy la),acetyl-Co A acyltransferase 2(ACAA2),glycogen phosphorylase(Pygb).It had the tendency of inhibiting material energy metabolism.The EBM small polar flavonoid could decrease the expression of long chain acyl-CoA synthase(Acsl1),glucuronosyltransferase(UGT),phenylalanine4-hydroxylase(Pah),cytochrome CYP4502C,and expressed significant inhibitory trend of the material energy metabolism.6.The result of transcriptomics showed that there were the most different genes between the total EBM and the EBM polysaccharide groups,which indicated that the mechanism of their potency might based on the same target gene.There was the least different genes between the EBM small polar flavonoid group and the EBM non-flavonoids group,suggesting that the pathway of interfereing target gene has no intersection.The high expression of Tf gene and low expression of Cyp1a1,Aacs,COMT and Acaca in the total EBM group,indicating that it inhibit the transformation of free fatty acid into acetyl-CoA,then prevent the tricarboxylic acid cycle(TCA)pathway from oxidation for energy,and inhibit the decomposition of ATP.The high expression of Cpt1a,Ephx2,Mcu and Ehhadh genes and low expression of Akr1b7 in the EBM polysaccharide group indicate that it promotes the degradation of fatty acids and the β-oxidation in the mitochondrial matrix to achieve the final energy generation.The high expression of Akr1b8,Aacs,Cps1 genes and low expression of Sqle gene in the EBM non-flavonoids group indicated that it inhibited oxidative stress and lipid peroxidation,and promoted the lipid metabolism and the clearance of toxic substances,and promoted the transformation of arginine into glutamine and the synthesis of steroid.The low expression of Bdhl,Hsd17b2,Hsd17b7,Elovl5,Srd5a1,Gda genes in the EBM large polar flavonoid group indicated that it inhibited the metabolism of estrogen and the production of progesterone and also inhibited the fat metabolism.The high expression of Cptla and the low expression of Cyp17a1,Fads2 and Car7 genes in the EBM small polar flavonoid group indicated that it inhibited the biosynthesis of steroid hormone and the synthesis of unsaturated fatty acid.7.Integrative Omics data analysis show that,through the integration analysis,it was found that the pathogenesis of cold syndrome is mainly related to the glucose metabolism and the lipid metabolism,and has the effect of regulation and inhibition of the tricarboxylic acid cycle,starch and sucrose metabolism and other multiple pathways.The imbalance of material energy metabolism in the level of three omics induces the decline of the organism function and potential lesions of the major systems and organs,such as skeletal muscle system,nervous system,cardiovascular and cerebrovascular system.The effect mechanism of the total EBM group is lowing the expression of Acaca and Aacs genes,thus the level of Acetyl-CoA is significantly reduced,which reduces the amount participating in cycle of tricarboxylic acid and slows down the metabolic stage,which shows the characteristics of inhibiting material energy metabolism.The EBM polysaccharides group increases the expression level of the glucuronosyltransferase,then increases the expression of glucose kinase and mannosine diphosphate,thereby accelerates glycolysis and metabolism,and it also up-regulating glyceraldehyde 3-phosphate dehydrogenase,and promote the sugar metabolism and tricarboxylic acid cycle,while the release of α-ketoglutarate increased,and promote the formation of succinyl CoA,to promote the release of high-energy phosphate bond.The EBM non-flavonoids group raises expression of Cps1 gene to increase the affinity to ATP,and increases oxidative energy supply,and heighen expression of Aacs gene,promotes the conversion from free fatty acid to Acetyl-CoA,thus into the tricarboxylic acid cycle to oxidative energy supply,and increased expression of COX Ⅱ,and catalytics fatty acid oxidation condensation into the tricarboxylic acid cycle to release energy.The EBM large polar flavonoid group inhibit the α-amylase activity to delay the degradation of starch and other substances and absorption of glucose in the body,and decline the level of glucose kinase and 2-phenyl glucuronide,slowing the sugar metabolism.Glycogen phosphorylase is the rate-limiting enzyme in the process of glucose metabolism,and its down-regulation can slow down the glycolysis process,while the levels of glucose Amine-6-phosphate andβ-D-fructose-6-phosphate reduced.The EBM small polar flavonoid group down-regulates the glycogen phosphorylase to slow down the glycolysis and gluconeogenesis,and decreases the levels of β-D-fructose-6-phosphate and 6-phosphofructokinase in fructose and mannose,and down-regulates the level of glucuronosyltransferase to decrease the level of 2-phenyl glucuronide then inhibit the starch and sucrose metabolism.Conclusions:EBM has different effects and interfere targets on the material energy metabolism of the cold syndrome model rats.It is proved that the total EBM is cold,EBM polysaccharide is warm,EBM non-flavonoids is warm,EBM large polar flavonoid is cold,EBM small polar flavonoid group is cold.The biological mechanism of each group was explored on the level of the three omics.The main mechanism was to change the activity of the protein by changing some mRNAs in the upstream and finally to make the metabolism of the downstream small molecule metabolites different then significantly impact the glucose metabolism and the lipid metabolism.