| OBJECTIVE:To investigate the effect of shikonin on the proliferation and apoptosis in human cervical carcinoma HeLa cells and protective and therapeutic effect of chemotherapy injury in vascular smooth muscle cell (VSMC) and possible mechanisms.METHODS:1. Primary culture of rabbit vascular smooth muscle cells, vascular smooth muscle cell morphology was observed by optical microscopy, with vincristine sulfate (VCR) based chemotherapy injury model;2. MTT colorimetric method for the determination of shikonin in different concentrations and different action time on vascular smooth muscle cells in the prevention and treatment of chemotherapy induced injury;3. Cell lactate dehydrogenase (LDH) mechanism and determination of Lithospermum free intracellular Ca2+concentration in prevention and treatment of vascular smooth muscle cells to chemotherapy;4. Determination of shikonin in prevention and treatment of vascular smooth muscle cells damaged by chemotherapy in the cell cycle and apoptosis rate by flow cytometry.5. Human cervical carcinoma HeLa cells were cultured in vitro, the morphological changes of Hela cells were observed by optical microscope;6. MTT colorimetric method for the determination of shikonin in different concentrations and different action time inhibitory effect on the proliferation of Hela cells in vitro;7. Through the determination of Caspase-3, activity of-8,-9, to investigate the pathway of apoptosis;8. Analysis of the changes of determination of shikonin induced cell apoptosis and cell cycle of Hela by flow cytometry.RESULTS:1. To observe the shape of vascular smooth muscle cells under optical microscope, the non adherent cells were round or oval, cytoplasmic density, opaque, nucleus is oval, with multiple nucleoli; cytoplasmic adherent growth of transparent, long spindle shaped, irregular in shape, radioactive growth, bundles of cells arranged in parallel part of the regional, multi overlapping, part of the district was single, ups and downs, accord with those of vascular smooth muscle cells morphological characteristics,"peak valley" showed the typical shape growth. After HE staining, nuclei stained blue, cytoplasm stained red, confirmed the vascular smooth muscle cells. The alpha-SM-actin immunofluorescence identification, cytoplasm, showed positive reaction, observed at high magnification, the cytoplasm of a cell, and the brown long axis parallel fiber filament, namely smooth muscle alpha actin filament, which confirmed that the cultured cells to vascular smooth muscle cells.2. MTT on VSMC cell activity assay of different drug concentration and action time, the results showed the chemotherapy drug VCR (0.8μg·ml-1) on damage of cells over60%, the protective effect of shikonin effects and treatment showed potent, of which5μg·ml-1(action time0.5,1h) better than10μg·ml-1(action time0.5,1h).3. Determination results of lactate dehydrogenase activity of LDH display, prevention group of shikonin and shikonin+VCR LDH activity determination results were1349.2±107.7,1590.5±40.8, and VCR chemotherapy group3004.8±129.0, with statistical significance (P<0.05), and the blank control group1398.4±64.0, with no statistical significance (P>0.05);In the treatment group, shikonin and VCR+shikonin LDH activity determination results were1351.0±31.4,1966.7±93.0, and VCR chemotherapy group3598.0±87.0, with significant difference (P<0.01), and the blank control group1529.4±40.2, with no statistical significance (,P>0.05).Free intracellular Ca2+concentration was measured by norepinephrine (NE) and Vera Pammy (VER) as positive control, group NE showed that the fluorescence intensity was703.2±32.0, and261.7±24.6compared with blank control group, with significant difference (P<0.01), group VCR fluorescence intensity was1137.2±137.2, and blank control group compared to261.7±24.6, with significant difference (P<0.01), fluorescence intensity NE+VER group was280.1±21.9, compared with the NE group, with significant difference (P<0.01), NE+fluorescence intensity of shikonin group was311.9±26.1, compared with the NE group, with significant difference (P<0.01), shikonin VCR group the fluorescence intensity was290.3±62.8, compared with the VCR group, with significant difference (P<0.01).4. Analysis showed that shikonin can eliminate the chemotherapy drug VCR mitotic arrest of cells by flow cytometry, the percentage of G2cells returned to normal,can improve the normal proportion of cells in S phase when single drug.5. Optical microscope was used to observe the morphological changes of shikonin on apoptosis of Hela cells, Hela cells pyknosis, cell rounding, narrow, nuclear chromatin dense concentration, medium level floating dead cells.6. MTT assay results showed that, shikonin significantly inhibited the growth of Hela cells, and increased with the extension of concentration and action time, showed a time-and dose-dependent manner.7. Determination of Caspase-3,-8,-9showed the vitality, different concentrations of shikonin in Hela cells compared with the control group, Caspase-3,-8,-9activity significantly increased, and with the increase in dose of shikonin, absorbance value also increased, leading to apoptosis,1,5,10μg-ml-1from group caspase-3absorbance values were0.284±0.002,0.313±0.006,0.372±0.005, and0.213±0.006compared with blank control group, with statistical significance (P<0.05),15,20μg·ml-1from group caspase-3value was0.416±0.007,0.490±0.03, and blank control group0.213±0.006, significant differences (P<0.01);1,5μg·ml-1from group caspase-8value was0.349±0.004,0.402±0.003, and0.213±0.006compared with blank control group, with statistical significance (P<0.05),10,15,20μg·ml-1from group caspase-8absorbance values were0.495±0.006,0.565±0.002,0.696±0.003, and0.213±0.006compared with blank control group, with significant difference (P<0.01);1,5μg·ml-1from group caspase-9value was0.384±0.002,0.472±0.007, and the blank control group0.213±0.006compared, with statistical significance (P<0.05),10,15,20μg·ml-1from group caspase-9value was0.561±0.004,0.620±0.006,0.748±0.005, and0.213±0.006compared with blank control group, with significant difference (P<0.01).8. Analysis shows that with the increase of the concentration of Lithospermum flow cytometry, of cells in S phase was increased, and G1phase cells decreased, most of the cells arrest in S phase of cell cycle. CONCLUSION:1Shikonin induced vascular smooth muscle cells of chemotherapeutic drugs VCR damage has the protective and repairing effects of shikonin, with5μg-ml-1,0.5,1h effect. Shikonin can increase LDH of vascular smooth muscle cells induced by VCR reversal of chemotherapy drugs, and then through the protected the integrity of cell membrane and protect and repair cells. Shikonin repair the damage of cell and cell membrane Ca2+channel, has inhibitory effect on injury caused by strong flow of drugs within the Ca2+, combined with calcium channel blockers such as Vera Pammy can also prevent the influx of Ca2+, play a protective role. Shikonin can eliminate the chemotherapy drug VCR mitotic arrest effect on cells, the percentage of G2cells returned to normal, the prevention of single drug use had no obvious effect on cell cycle, when treatment is the percentage of cells in S phase increased significantly.2Shikonin significantly inhibited the growth of Hela cells, and increased with the extension of concentration and action time, showed a time-and dose-dependent manner. Shikonin induced Hela cell apoptosis may be through the activation of Caspase-3,-8, and-9. Shikonin make Hela cell cycle arrest in S phase, G1phase cells decreased, in a dose-dependent manner. |
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