| BACKGROUND: Licorice,an edible and officinal plant,is the root or stem of Glycyrrhiza glabra(G.glabra)with a pleasing flavor.Because of its sweet favor,licorice is widely used in beer,beverage,candy,ice cream,baking cookies,soy protein imitation meat products,soy sauce and other foods,and also is used in tobacco and cosmetics.As a traditional Chinese medicine,licorice exhibits various pharmacological effect and biological function.The aging of the world's population is getting worse.How to effectively delay aging or healthy aging has becoming the central issue of the people.Aging is a kind of specifically cell protein degradation in organ.The research showed that the protein abundance was significantly different between the young rats and aging rats.It is the main reason that the organism cannot remove the harmful proteins.Furthermore,thefree radical damage and antioxidant are also closely related with aging.A large number of researches also indicate that D-gal induced aging rats have more similar natural aging characteristics,especially the oxidative damage and the decline in cognitive ability.While the D-gal was long term administrated to rats which leaded to the massive free radical production and induced the occurrence of oxidative stress,further resulted in the neural dysfunction and the debasement of learning and memorizing ability.Furthermore,the excessive free radical can modify the amino groups of proteins and peptides without enzyme to form complicate advanced glycation end products(AGEs).AGEs can enhance oxidative stress damage and led to the changes of the physiological activity and physiological function.At the same time,studies indicated that D-gal also can accelerate neuronal apoptosis and neurodegenerative,which are similar to natural aging and widely used to establish the aging rat model.Metabolomics is a powerful systemic approach that can identify the effects of medicines intervention and can help in understanding how global changes in the metabolites of animals are influenced by nutrition,genetics,environment,and others.The aging process is accompanied by the disorder of metabolism.Alterations of physiological status can disrupt homeostasis,leading to perturbations in the levels of endogenous biochemicals involved in different key metabolic processes.Therefore,the metabolomics were used to study the process of the biological and biochemical changes by endogenous small molecule metabolites.OBJECTIVE: Therefore,in this study,the molecular biology and metabonomics techniques were used to evaluate pharmacological effect and mechanism of licorice extracts in improving learning and memory functions on D-gal induced aging rats.METHOD: The chemical composition of licorice extracts was analyzed by LC-MS.Taking SD rats as the research object,aging rat model was established by using D-gal.After one week acclimatization,the rats were randomly divided into five groups(n=15): the control,the model and three licorice groups.The model group and three licorice groups were received the D-gal at the dose of 300 mg/kg for 5 consecutive weeks while the control group was received the same volume of physiological saline.At the same times,three licorice groups were administrated licorice extracts dissolved in the water at the three doses of 1,5 and 10 g herb/kg respectively by oral gavage after the D-gal injected intraperitoneally.The rats in the control group and the model group were administrated with the same volume of water.The abilities of spatial and learning memory were evaluated by Morris water maze test and Object-place recognition test,and then collect the urine of the rat for 24 h.After behavioral test,all rats were anesthetized and sacrificed,and serum and tissue samples were collected.Neuronal cells injury hippocampal region and liver injury were observed by Hematoxylin and eosin(HE)staining.The SOD,GSH-Px,CAT and AChE activity and the content of MDA in the hippocampus and liver were determined by assay kits.The expression levels of Bax,Bcl-2,Caspase-3 and Cyt-C in the hippocampus were detected by western blot.In order to explore the mechanism of its action,the liver tissue,serum and urine were analyzed by metabolomics.First,the principal component analysis(PCA)was used to analyze the variation regular pattern of metabolic profile on serum urine and liver tissue.Then,the orthogonal signal correction filtered partial least squares discrimination analysis(OPLS-DA)combined with "S-plot" were used to different metabolites in serum,urine and liver tissue.And then screened associated with aging potential diagnostic markers.The Pathway Enrichment analysis was used to find out the significantly correlated with the metabolic pathway and verified preliminarily.RESULTS:(1)The 21 compounds were analyzed and identified by LC-MS inlicorice extract.(2)The results of behavior test showed that the D-gal treated rats impaired their cognitive and memory as indicated by the Morris water maze and the Object-place recognition test,while the administration of licorice extracts could recover the age-related cognitive impairment caused by D-gal.(3)HE staining features of cell loss in the liver are shown that compared with control group,the necrotic cavity,structure obviously damaged denatured and necrotic were appeared in the liver tissue of the model group,while the administration of licorice extracts,the results of HE staining features of cell loss in the liver were improved significantly.HE staining features of cell loss in the hippocampus are shown that the rats in the control group had full hippocampus neurons,which were arranged tightly and morphologically intact.Pyramidal neurons presented round and large nuclei and clear nucleoli.Widespread damage was visible in the hippocampus of the model group treated with D-gal.Intercellular space largened,and cells were loosely arranged.Pyramidal neurons either presented a densely stained shrunken appearance with minimal cytoplasm or had disappeared.However,licorice extracts were administered after D-gal injected,the neuronal cells were rescued in the hippocampus compared with model groups.(4)SOD,CAT,GSH-Px activities and MDA content in the hippocampal and liver tissue were determined as the biomarkers of oxidative stress in D-gal induced aging rats.The results indicated that the D-gal was consecutively received and induced a significant decrease of SOD,CAT and GSH-Px activities and increase of the MDA level compared with the control group.A significant increase of SOD,CAT and GSH-Px activity was observed in licorice extracts administered group compared with the model group.(5)The western blot analysis revealed that D-gal led to significantly increased in Bax/Bcl-2 ratio,Caspase-3 and Cyt-C in the model group compared with the control group.Licorice extracts administration remarkably reduced Bax/Bcl-2 ratio,Caspase-3 and Cyt-C levels compared with D-gal treated rats.These results indicated that licorice extracts have the effects of anti-apoptosis and neuroprotection to through regulating the expressions of apoptosis-related protein.(6)Metabonomics of liver,serum and urine showed that there was an obvious separation in PCA of the control group and the model group,indicated D-gal induced the significantly changed metabolites in model groups compared with the controls.The OPLS-DA was applied to maximize the difference of metabolic profiles and show inter-group metabolic differences.The variable importance for the projection(VIP)values and S-plot were used to find the differential biomarker.?-glucose,phosphatidylethanolamine,glycogen,creatine,betaine,valine,3-hydroxybutyricate,choline,glutathione,succinate,glycine,taurine and phosphatidylcholine were detected and identified in liver tissue.Glycine,creatinine,lacate,taurine,guanidoacetate,N-methylnicotinamide,?-glucose,citrate,fumarate,creatine,dimethylglycine,?-ketoglutarate,acetate and alanine were detected and identified in urine.Isoleucine,Leucine,valine,alanine,acetate,glutamate,pyruvate,citrate,?-ketoglutarate,TMAO,cystine,taurine and glycine were detected and identified in serum.(7)To further explore the impacts of these selected metabolites and to identify possible biochemical pathways that were affected by aging,MetPA(Metabolomics Pathway Analysis)was constructed to reveal the most relevant pathways affected by aging induced by D-gal.In this study,the threshold of the potential target of metabolic pathways was set to 0.10.The value of metabolic pathway is above 0.1,which was considered as the potential target of metabolic pathways.The potential target metabolic pathway of urine includes:(1)Taurine and hypotaurine metabolism,(2)Valine,leucine and isoleucine biosynthesis,(3)Glycine,serine and threonine metabolism,(4)Alanine,aspartate and glutamate metabolism.The potential target metabolic pathway of serum includes:(1)Valine,leucine and isoleucine biosynthesis,(2)Taurine and hypotaurine metabolism,(3)Glycine,serine and threonine metabolism,(4)Glyoxylate and dicarboxylate metabolism,(5)Pyruvate metabolism,(6)TCA cycle,(7)glycolysis and gluconeogenesis.The potential target metabolic pathway of urine includes:(1)Taurine and hypotaurine metabolism,(2)Glycine,serine and threonine metabolism,(3)Glyoxylate and dicarboxylate metabolism.(8)The metabolic pathway enrichment analysis showed that taurine metabolism was most associated with the aging in D-gal induced rats in liver,serum and urine.The level of taurine in serum and urine was significantly decreased in D-gal induced aging rats compared with the controls,which indicate that the D-gal induced the metabolic disorder of taurine in aging rats.Some key enzymes involved in this taurine cycle,such as CDO1,CSAD and GAD,were determined by the enzyme-linked immunosorbent assay in serum.The activity of CDO1,CSAD,and GAD1 were significantly decreased compared with controls in serum,which indicates that the synthesis of taurine were disordered in aging rats induced by D-gal.The licorice were administrated,the activity of CDO1 and CSAD were significant increased in licorice extract administered group compared with the model group.However,the activity of GAD1 had no significant change in all licorice groups compared with the model group.CONCLUSION:(1)Licorice extracts could prevent the effect by enhancing the cognitive performances,regulating oxidative stress and cholinergic system,displaying anti-apoptotic effects.Taken together,these data suggest that licorice extracts maybe a beneficial candidate for the treatment of aged-related conditions.(2)The taurine metabolic pathway was significantly correlation with the D-gal induced aging rats by metabolomics and multivariate statistical analysis.(3)The taurine and the taurine metabolism may be responsible and significant for aging in rats induced by D-gal,which is also a major mechanism of licorice in anti-senility effect. |
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