The Effect And Mechanisms Of Astragalus And Its Components On Energy Metabolism Of Rat Hepatocytes

Objective: This study is to observe the effect and mechanisms of Astragalus and its components on energy metabolism of rat hepatocytes in vitro.By the metabonomics techniques,this study explores the role of the various componts of Astragalus in playing warming effect,so as to provide evidence and ideas for the the study of medicinal properties of traditional Chinese medicine.Methods: We detected the activity of ATPase with spectrophotometer and the content of ATP,ADP,AMP with high performance liquid chromatography(HPLC)to observe the effects of Astragalus and its componets on energy comsumption and producation in hepatocyts.We observed mitochondrial NAO staining by fluorescence microscopy,the copy number of mtD NA and the expression of ATP synthase β subunit mRNA by RT-PCR to evaluate the efficacy of each component on the number and function of mitochondria in cells.The expressions of m RNA and protein of AMPK,p-AMPK,PGC-1α,NRF1,TFAM were detected via RT-PCR and Western Blot to explore the effect of each component on the promotion of mitochondrial biogenesis.The UPLC-Q Exactive Orbitrap-MS technique was used to study the effects of Astragalus and its components on the metabolism of hepatocytes.PCA and OPLS-DA were used to find out the different metabolites,then we used MetaboAnalyst 3.0 to analyze the metabolic pathways of different group to explore the common and different effects of each component of Astragalus on the metabolism of hepatocytes and to clarify the mechanism of “warm in nature” of each component.Results: 1.The activity of ATPase in normal rat liver cell line BRL-3A can be increased by all of the Astragalus component,especially the increase of Na+-K+-ATPase activity was most obvious,thereinto,The effect of Astragalus polysaccharids was the best Meanwhile,all of the Astragalus component can increase ATP content and energy charge of hepatocytes,the best of them is the Astragalus decoction.2.Astragalus decoction and its components can increase the number of mitochondria in hepatocytes,up-regulate the mRNA expression of ATP synthase β subunit,of which the effect of Astragalus polysaccharide most obvious.All of the Astragalus components can promote the expression of AMPK,p-AMPK,PGC-1α,NRF1 and TFAM.Among them,Astragalus polysaccharide up-regulated the expression of PGC-1α and NRF1 mRNA significantly,while the upregulation of AMPK and p-AMPK expression by calycosin was superior to other components.3.The results of metabonomics showed that,glutamic acid,glutamine,leucine,serine,choline,acetylcholine,phosphoethanolamine,and arachidonic acid may be the differerntial metabolites of Astragalus and its components.Glutamine and glutamate metabolism,glycerophospholipid metabolism,biyosynthesis of unsaturated fatty acids,arachidonic acid metabolism were the common metabolic pathways in Astragalus and its components groups.Glutamine and glutamate is the main metabolic pathways that regulate energy metabolism.Astragalus polysaccharides played a major role in the regulation of this metabolic pathway.Astragaloside and calycosin are mainly used to regulate metabolism of glycerol phospholipids and unsaturated fatty acids,and play a supporting role in affecting cell energy metabolism.Conclusion: 1.Both Astragalus Decoction and its components can promote the energy metabolism of hepatocyte,which can reflect the “warm in nature ” of Astragalus.This results is consistent with the new hypothesis about propertise of TCM.2.The mechanism of increasing the energy storage by Astragalusand and its components is related to promoting mitochondrial biogenesis and improving mitochondrial function.The Astragalus and its compounds can increase energy supply and reserves of hepatocytes by regulation the metabolism of amino acid,glycerophos-pholipids,unsaturated fatty acids and arachidonic acid.And in these metabolism,glutamine and glutamate metabolism is the main metabolic pathway that exert warming effects.3.The effects of Astragalus polysaccharides on the promotion of energy metabolism and increase mRNA expression of key proteins in the PGC-1α/NRF1 pathway are significantly superior to other components.Astragalus polysaccharides plays a major role in the regulation of important metabolic pathways for energy metabolism.So,we believe that Astragalus polysaccharides is the main active substance for Astragalus to play the warming effect.By up-regulating the expression of key proteins in the PGC-1α/NRF1pathway and regulating the metabolism of glycerophospholipids and unsaturated fatty acids Calycidose can promotes energy metabolism,which may be the material basis for Astragalus to play the warming effect.The effects of astragaloside IV is consistent with the Astragalus Polysaccharides and Calycidose,but the effect is not significant.Therefore,astragaloside IV plays an auxiliary role in promoting energy metabolism of Astragalus.