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Background and Purpose: the Blue Rabdosia (Rabdosia japonica (Burm. f.) Hara var. Glaucocalyx (Maxim.) Hara) Lamiaceae (Labiatae) Isodon genus, stomach, detoxification, blood, antibacterial anti-inflammatory activity, for the treatment of gastritis, abdominal pain, bloating, hepatitis early, cold and fever, mastitis, joint pain and other diseases. In recent years, a large number of literature reports the Plectranthus genus generally have anti-tumor activity, kaurane shaped diterpenoid compounds is the active ingredient. Blue Rabdosia Antitumor Activity of rarely reported. From the in vitro level study the main Blue Rabdosia diterpenoids glaucocalyxin-Su (glaucocalyxin; of GLA) glaucocalyxin C prime (glaucocalyxin C; GLC) anti-tumor effect. Using a variety of methods to detect the GLA and GLC-induced apoptosis, and apoptosis mechanisms as well as reactive oxygen species (reactive oxygen species; ROS) in GLA-induced apoptosis of HL-60 cells in depth study. Trends in pharmaceutical research using molecular biology methods treasure trove of traditional medicine in the motherland of Chinese herbal medicine research to give a new interpretation of its pharmacological mechanism, the development of new anticancer drugs, carry forward the Chinese traditional medicine. In this context, depth to explore the molecular mechanisms of the anti-tumor effect of the prime glaucocalyxin a promising research value of the subject. Method: First, MTT assay and compared the cytotoxicity of GLA and GLC. In addition, we use molecular docking technology (autodock), Bcl-2 target proteins investigated GLA, or GLC their binding capacity; respectively by AO / EB double staining, DAPI staining, TUNEL assay, DNA ladder and Annexin V- FITC / PI double staining method, from nuclear morphology, biochemical changes detected cell apoptosis, and the use of flow cytometry apoptosis rate. Spectrophotometric method to detect the activity of caspase 3,8 and 9. Flow cytometry and fluorescence microscopy to detect the outbreak of the mitochondrial membrane potential and ROS. Oxygen electrode method to detect the oxygen consumption, simultaneous detection of MDA content and antioxidant enzymes (CAT and SOD) activity. Immunofluorescence assay the prime of cytochrome C glaucocalyxin release of impact. Detected by Western blotting the the prime of Bax and Bcl-2 protein expression glaucocalyxin. RT-PCR was used to detect the the prime of Fas gene expression glaucocalyxin. While using the MTT assay, flow cytometry and fluorescence microscopy to detect antioxidants the NAC glaucocalyxin A-induced apoptosis of HL-60 cells. RESULTS: GLA on the choice of 15 tumor cell lines having a significant anti-tumor activity, and a concentration-dependent effect of HL-60 to its most sensitive cell lines, the IC50 of only 0.33μg/ml, and in selected the concentration of, GLC 15 tumor cells had no effect. In addition, molecular docking (autodock) GLA with a combination of the Bcl-2 protein docking free energy and Ki values ??were lower than the GLC, the GLA easier binding to inhibit the function of Bcl-2, Bcl-2 and promote apoptosis. GLA can induce apoptosis in HL-60 cells the apoptotic percentage rose from 7.80% (0μg/ml) to 40.60% (10μg/ml), a concentration-dependent effect, but the GLC has no effect on HL-60 cells. GLA can lead to a decline in HL-60 cell MMP, ROS outbreak, the release of mitochondrial cytochrome C, Bax/Bcl-2 rise Fas gene upregulation and caspase 3,8 and 9 activity increased. Antioxidant NAC inhibits GLA induced ROS production and survival of HL-60 cells, the mitochondrial membrane potential increased apoptosis rate. Conclusion: This experiment showed that GLA can inhibit the growth of HL-60 cells and induce apoptosis, but GLA structure is very similar to GLC no anti-tumor activity. GLA intrinsic pathway (mitochondrial pathway) and the extrinsic pathway (death receptor pathway) induced apoptosis in HL-60 cells. ROS involved GLA induced HL-60 cells through the mitochondrial pathway of apoptosis, and plays an important role.
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