Influence Of Oxymatrine On Proliferation And Energy Metabolise Of Human Hepatoma Cell Line HepG-2

Objective: Investigate the influence of oxymatrine(OM) on theproliferation of and energy metabolise of human hepatoma cell line HepG-2.Methods:1.Using ODS-SP C18chromatography column(4.6×250mm,Particle size5μm) the mobile-phase was100mM phosphate buffer solution:methonal=99:1.Culumn temperature was30℃, Flow rate was set at0.6ml/s, simple volumewas20μl,Establish a reversed high performance liquid chromatography(RP-HPLC) methods for determination of adenosine triphosphate (ATP) andadenosine diphosphate(ADP) in standard;Create a peak area-concentrationstandard curve, Detect ATP and ADP content in HepG2cells.2.Human hepatoma cell line HepG2were cultured in vitro. Theexperimental group were added different dose of OM,but no any medicine innegative control group, to examine the influence of OM on HepG2by MTT andCCK-8methods respectively, determined lactic acid concentration of cellsupernatant After treating with1.0mg/ml oxymatrine snd Antimycin A indifferent culture medium at different time by enzymatic colorimetric method. And ATP in HepG2cell were detected after different dose of OM (0.5mg/ml,1.0mg/ml,2.0mg/ml,4.0mg/ml)were added for48hours by RP-HPLC.Result：1.With the chronmatographic condition in this experiment, thechronmatographic peak of ATP and ADP was obvious and separated. In addition,other components of cells did not disturb the chronmatographic peak of ATP andADP. The method had good linearity with the concentration ranges of7.5-100μg/ml for ATP and ADP.2. The CCK-8assay showed that: The inhibition rate of OM on humanhepatoma cell line HepG2proliferation with the concentration of0.5mg/ml、1.0mg/ml、2.0mg/ml、4.0mg/ml and8.0mg/ml for48h and72h was9.14%、17.9%、39.61%、59.55%、71.86%, and15.4%、32.37%、48.37%、72.44%、91.55%, respectively. Compared with negative control group, there is significantdifference in inhibited effect of OM on the proliferation of human humanhepatoma cell line HepG2, respectively (P <0.05), and with the increase ofOM concentration and prolonging the time of action, the inhibition effect of OMon the human human hepatoma cell line HepG2was more obvious, with time-and dose-dependent.48hIC50=3.08±0.09mg/ml. The MTT assay showed that:The inhibition rate of OM on human hepatOMa cell line HepG2proliferationwith the concentration of0.5mg/ml、1.0mg/ml、2.0mg/ml、4.0mg/ml and8.0mg/ml for48h and72h6.93%、16.95%、33.74%、53.78%、65.95%and9.40%、20.58%、38.82%、57.80%、86.56%, espectively.48h IC50=3.08±0.09mg/ml.Similar results with CCK-8assay, But Compared with48hIC50valuesdetermined by CCK-8assay, there is significant difference between48hIC50values determined by CCK-8and MTT assay, the48hIC50values determinedby CCK-8was lower than that of MTT assay(P=0.01<0.05); Simultaneously, when human hepatoma cell line HepG2treated with low concentrations ofOM(0.5mg/ml）during48h，72h, Compared with the negative control group,there is significant difference in inhibited effect of OM on the proliferation ofhuman human hepatoma cell line HepG2by CCK-8assay (P=0.014<0.05),whereas no obvious difference by MTT assay（P>0.05）.3. Lactic acid concentration in the culture supernatant of HepG2detectedby enzymatic colorimetric method:on lactate production,HepG2cells wereincudated with GM or GFM medium,GM cells presented a10-fold increased inthe rate of lactate formation Compared with GFM cells(P<0.05),treatment ofGM cells with OM(1.0mg/ml) led to3-fold decrease in lactateformation(p<0.05),while GFM cells resulted in1.5-fold decrease in lactateformation(p<0.05); treatment of GM cells with Antimycin A(2ug/ml) caused1.7-fold increased(p<0.05),And GFM cells has increased5-fold(p<0.05).4.Treated with HepG2cells different the concentration of OM (0.5mg/ml,1.0mg/ml,2.0mg/ml,4.0mg/ml) and maintained for48hours, The level ofintracellular ATP lower than the negative control group, there is statisticallysignificant, respectively (P <0.05), and with the increase of OM concentration,the Intracellular ATP content decreased more obvious.Conclusion:1.RP-HPLC can effectively detect the ATP、ADP content in vitro cell,peaktime of ATP appear at12.12s, and peak time of ADP appear at13.45s.2.Both CCK-8assay and MTT assay confirm that OM inhibit theproliferation of HepG2cells and with time-and dose-dependent. Expermententresult showed that CCK-8assay is more sensitive than the MTT assay.3.The mechanism of the OM anti-hepatoma may be that throughtargeting of the glycolytic pathway and results in decreased ATP.