Protective Effects Of Astragalus Essential Components Against BRL-3A Cells Oxidative Stress

[Objective]The objective of this study is to compare the protective effects of Astragalus essential component againstrat liver cells(BRL-3A cells)oxidative stress,and investigate the mechanismusing the best part within all the component studied above.[Methods]The crude polysaccharide(APS),total flavonoids(TFA)and total saponins(TSA)are extracted fromthe same batch of Astragalus,and converted to equivalent dose to detectthescavenging ability of DPPH radical,superoxide anion radical and hydroxyl radical,as well as the effects of cell viability and reactive oxygen species generation of the oxidative stressed BRL-3A cells.The effects on endogenous antioxidant enzyme systems and microsomal enzyme system are also detected using the better part(s)within all the three sorts of Astragalus component.And using the key active ingredient in best part within all the component studied above,the effects on endogenous antioxidant enzyme systems,ROS generation,CYP2E1 mRNA transcription and protein expression,and apoptosis are detectedin proper order.Meanwhile the expression of PI3K and Akt,and activity of caspase-3 are also detected to investigate its impact on the PI3K/Akt signaling pathway.The Changliver cells injury model induced by alcohol was established to verify the antioxidant effect of the test drug.[Results]Thecontents of APS,TFA and TSA,which are all extracted fromsame batch of Astragalus,are determined respectively with D-anhydrous glucose,rutin and Astragalus saponinⅣ.The radical scavenging and ROS generation tests showthatTFAandTSAhave better effect than APS,and the activities of endogenous antioxidant enzyme systems and CYP2E1,which has mediated oxidative stress,tests indicatethatTFA has better effect thanTSA.Moreover,the content of calycosin-7-O-β-D-glucopyranoside(CG)in TFA prepared previously complies with theChinese Pharmacopoeia.The effects of CG on oxidative stressed BRL-3A cells for improvement of endogenous antioxidant enzymes and inhibit of ROS generation show a dose-dependentrelationship,and thehigh concentrationCG group(40-mg-L-1)is better than thepositive controlgroup(DDB)with same concentration.Meanwhile,oxidative stress can lead to increase for mRNA transcription and protein expression of CYP2E1,while CG can make them significant decline;compared to the positive control group(DDB),the medium and high concentration CG groups have same effects on downregulation of CYP2E1 mRNA,and the high concentration CG group has same effect on downregulation of CYP2E1 protein.At the same time,according to the flow cytometry and DNA Ladder results,oxidative stress can also promote cells apoptosis but CG can significantly inhibit that trend.In addition,oxidative stress also leads to Akt downregulation and caspase-3 activity increase,but CG can make a significant reversal.CG has the same antioxidant effects in damaged Changliver cells,which induced by alcohol,as in the oxidative stressed BRL-3A cells.[Conclusion]Under the premise ofsamesource and concentration,compared with APS and TSA,TFA has better effects on free radicalsscavenging,antioxidant capacity enhancement of BRL-3A cells,and inhibiting of microsomal enzyme activityand ROS production.As the key active ingredient in TFA,CG also has significant intervention effects on BRL-3A cells against oxidative stress.Its mechanism may be that CG has suppressed the upregulation of CYP2E1induced byoxidative stress,then reducedROSproduction.So that theAkt downregulation caused by ROS has been recovered,thereby the caspase-3 activity has been reduced,and ultimately the immoderate cell apoptosisinduced byoxidative stress has been suppressed.