| Objective To screen the induce HL60 cells apoptosis active site of Bletilla striata and study the induce HL60 cells apoptosis of Active Fraction in Rhizoma Bletillae Striatae and Its Related Mechanisms.Methods CDUsing 95%alcohol as well as the distilled water to extract Bletilla striata by Backflow method,measure the growth inhibition effect of both extract by CCK-8;②Using the system resolver extraction method to divide the 95%alcohol extraction into the ligroin level,the chloroform level,the acetidin level,the n-butyl alcohol level and the water level,measure the growth inhibition effect of extract by CCK-8;③To observe the morphological changes of cell apoptosis by AO/PI staining and light microscopy after cells were treated with various parts of alcohol extract for 24h,then screen the induce HL60 cells apoptosis active site of Bletilla striata Combined with CCK-8 test results;④To observe the morphological changes of cell apoptosis by Hoechst33342 stain and ransmission electron microscope after cells were treated with active site of Bletilla striata for 24h;⑤The cells apoptosis was analyzed by flow cytometer after cells were treated with active site of Bletilla striata for 24h;⑥The expression of bcl-2 and Bax mRNA in cells was measured by RT-PCR,after cells were treated with active site of Bletilla striata for 24h;Results ①The 95%alcohol extraction of Bletilla striata can Effectively inhibit the proliferation of HL60 cells while water extraction can not have this pharmacologic actions,the IC50 of 95%alcohol extraction is 150.0 μg/mL;②The ligroin level,the chloroform level,the acetidin level can inhibit the proliferation of HL60 cells,their IC50 is 103.91 μg/ml,57.9μg/mL,101.8μg/ml respectively,the chloroform level have the most effect while the n-butyl alcohol level and the water level have very weak actions;③AO/PI staining cells showed typical morphological changes of apoptotic under a fluorescence microscope after cells were treated with The ligroin level,the chloroform level,the acetidin level of Bletilla striata,the chloroform level have the most obvious while the n-butyl alcohol level and the water level not have this phenomenon,results consistent with CCK-8 detection,the chloroform level is the induce HL60 cells apoptosis active site of Bletilla striata;④Hoechst33342 staining cells showed typical morphological changes of apoptotic under the fluorescence microscope and cells apoptosis in the transmission electron microscope.after cells were treated with the chloroform level of Bletilla striata;⑤Flow cytometry analysis shows that the chloroform level of Bletilla striata can effectively induce apoptosis of HL60 cells,the apoptosis rate of the different drug concentration(50、100、150pg/mL)was 24.9%,36.0%,51.3%represently;⑥The RT-RCR shows that the chloroform level of Bletilla striata can down regulation the bcl-2 mRNA expression and up-regulation the Bax expression.Conclusion Bletillae striata can induce HL60 cells apoptosis and refrain its multiplication,the chloroform level have the most obvious,the probably mechanisms is regulation the bcl-2 mRNA expression and up-regulation the Bax expression. |
