| Endogenous or exogenous reactive oxygen species（ROS） in cells can not onlywidely involve in regulating cell signal pathways,but also participate in the regulationof apoptosis signal transduction through the modification of proteins or the change ofintracellular redox state.In this study,proliferation rate,Cell cycle and apoptosis,the ROS andGlutathione（GSH） have been tested in HepG-2cells treated by Berberine or combinedwith the N-acetylcysteine （NAC）.The results will helpful to explain the inhibitionmechanism to HepG-2of Berberine.The proliferation rate of cell was detected by SRB assay. Cell morphology andnuclear morphology were observed by the inverted microscope and fluorescent dyeHoechst33258. Cell cycle, cell apoptotic ratio were detected by Flow cytometry （PIsingle staining, Annexin-V-PI double staining）, intracellular ROS content anddistribution were tested by flow cytometry and observed by the fluorescencemicroscope staining with DCFH-DA, respectively.The concentration of intracellularGSH in HepG-2cells was measured by colorimetric method.The results are shown as follows:（1） The cytotoxicity results with SRB assay showed that Berberine on HepG-2cells has high cytotoxicity and in time-dose dependent way. The IC50is44.10μmol·L-1treated and8.53μmol·L-1in24h and48h, respectively. The lethal effects emergedwhen the concentration of drug was more than160μmol·L-1.（2） The morphology of cells and nuclei observed by inverted microscope dyedby Hoechst33258staining exhibited that treated cells by Berberine became round andshrunken, poor adherence, chromatin condensation, showed marginalization and DNAfragmentation. These were hallmarks of apoptosis. But NAC have relief apoptosischaracteristics induced by Berberine.（3） After AO/EB fluorescence double staining HepG-2cells treated by Berberineshowed apoptosis also and when protected by the NAC,apoptotic cells numberdecreased. The proliferation rate of HepG-2treated by Berberine only and NACcombined results show that the rate proliferation of cell was improved in theprotection of NAC. （4） Cell cycle assay by flow cytometry with Propidium iodide （PI） stainingdisplayed that the “Sub-G1peak”（cells in SubG1phase） appeared when cells weretreated by Berberine(45μmol·L-1and160μmol·L-1).The percentage of cells in SubG1phase were in positive correlation with the dose and exposure time, and cell cyclearrest occurred at the S phase cycle. The combination of Berberine and NAC reducethe Sub-G1phase cell percentage of HepG-2decreased from15.53%to10.93%, and20.13%to14.73%,compared with45and160μmol·L-1Berberineonly,respectively.Annexin-V-PI double staining results showed that HepG-2cellswere induced to apoptosis by the45μmol·L-1of the concentration berberine.Buttreated by160μmol·L-1cells mainly in the process of early to the late apoptosis. Andwhen protected by the NAC, cell apoptosis rate is decreased.（5） Flow cytometry assay and fluorescence microscopy observations by DCFH-DA staining suggest that the intracellular ROS content and the effect of Berberinewere significant dependent by time-dose when HepG-2cells were treated by45μmol·L-1and160μmol·L-1Berberine for6h,12h and24h. And NAC reduced ROScontent raised by Berberine. The colorimetric assay showed that there is a time-dosedepented negative effects of the intracellular GSH content with berberine. And NACcan improve GSH content reduced by Berberine.All the results above demonstrated,Berberine induced cells to apoptosis throughthe impacting on the intracellular redox system,promoting the generation of ROS andreduceing the content of GSH in HepG-2cell. |
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