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Objective To investigate the lethal effect of melittin on human hepatocellular carcinoma cell line SMMC-7721 and its possible mechanism. Methods MTT assay was used to investigate the killing effect of different concentrations melittin on human hepatocellular carcinoma cells SMMC-7721. Meanwhile, the inhibitory effect of specific programmed necrosis inhibitors necrostain-1(Nec-1) on melittin killing SMMC-7721 cells was detected. Programmed cell necrosis observed by Hoechst 33342 and PI double staining. The necrotosis rate was detected by flow cytometry. Ultrastructural changes of cell was detected by transmission electron microscopy. The expression of receptor interacting protein 1(RIP1) was detected by Western blotting. Results Compared with the control group, the proliferation activity of SMMC-7721 cells was significantly decreased after treatment with different concentrations of melittin for 24 hours (P < 0. 05) . Cells stained in dark blue and red. Cell membrane integrity was destroyed, organelle swelling, organelle membrane was destroy, that demonstrates cell was necrosis. Westen blotting result showed an increased proportion of RIP1 expression in SMMC-7721 cells. Compared with the melittin group, cell proliferation activity was significantly increased, cell necrotosis rate was decreased, and intracellular RIP1 expression was down-regulated in the Nec-1 pretreatment group. Conclusion Melittin induces cell death in SMMC-7721 cells through the RIP1-mediated programmed necrosis pathway.
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AIM:To investigate the effect of reactive oxygen species(ROS)on the apoptosis of HepG2 cells induced by artesunate.METHODS:The effect of artesunate on the viability of HepG 2 cells was measured by MTT assay. The morphological changes of the apoptotic cells were observed by the method of Hoechst 33258 fluorescence staining.The apoptosis of HepG2 cells was analyzed by flow cytometry.DCFH-DA was used to detect the changes of ROS generation dur-ing the process of apoptosis.The protein levels of Bax ,Bcl-2,cleaved caspase-3 and cytochrome C(Cyt C)were deter-mined by Western blot.HepG2 cells were pretreated with apocynin and then Western blot was used to detect the expression of p47phox and p22phox,and ROS changes were analyzed by flow cytometry.RESULTS:Compare with control group,the cell viability was obviously inhibited after treatment with artesunate for 24 h(P<0.05).The nuclei were densely stained ,and the proportion of apoptotic cells was increased(P<0.05).ROS was increased significantly(P<0.05).The results of Western blot demonstrated that the expression level of Bax was increased ,Bcl-2 was decreased ,the ratio of Bax/Bcl-2 was increased ,and the protein levels of cleaved caspase-3 and Cyt C were increased.Pretreatment with apocynin reduced the expression of p47phox and p22phox and the generation of ROS in the artesunate treatment group.CONCLUSION:Artesu-nate induces the apoptosis of HepG 2 cells.The possible mechanism may be related to the increase in the generation of ROS.
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OBJECTIVE: To investigate the apoptosis effect of human hepatocellular carcinoma cell line SMMC-7721 induced by dihydroartemisinin in vitro and the possible mechanism. METHODS: After treatment with 25, 50, 100, 200, and 400 μmol•L-1 dihydroartemisinin for 24 h. The proliferation inhibitory effect of dihydroartemisinin on SMMC-7721 cell was detected by MTT assay. Cell cycle and apoptosis were detected by flow cytometry. The change of apoptotic morphology was detected by confocal laser scanning microscopy. Rho 123 staining method was used to detect the changes of mitochondrial membrane potential. Western blot was used to detect expression of Bcl-2, Bax, Cleaved Caspase-3, Cleaved Caspase-9 and Cyto C. RESULTS: MTT results showed that 25-400 μmol•L -1 dihydroartemisinin can inhibit the proliferation of SMMC-7721 cells obviously. The cell cycle detection results of flow cytometry showed that dihydroartemisinin could block SMMC-7721 cell cycle in G2/M phase. The results of Hochest 333258 staining showed that the nuclei were heterogeneous, condensed and fragmented in the DHA treatment group. The cell apoptosis detection results of flow cytometry showed that the apoptosis rate of dihydroartemisinin treated groups were increased obviously (P < 0.01). The results of Rho 123 staining showed that the mitochondrial membrane potential was decreased significantly (P < 0.01). Western blot results showed that the expression of Bcl-2 was down-regulated, expression of Bax was up-regulated, the ration of Bax /Bcl-2 was increased and the expression of Cleaved Caspase-3, Cleaved Caspase-9 and Cyto C were up-regulated. CONCLUSION: Dihydroartemisinin can induce apoptosis of SMMC-7721 cells, on the mechanism of apoptosis may be related to mitochondrial pathway.
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OBJECTIVE: To investigate the protective effects of broccoli polypeptide component on vascular endothelial cells injury induced by hydrogen peroxide (H2O2). METHODS: The broccoli polypeptide was isolated by methods of alcohol precipitation and sephadex G-25 gel filtration chromatography. The EVC-304 cells were cultured in logarithmic growth phase and incubated with 100 μmol·L-1 H2O2 for another 12 h after pretreatment with broccoli polypeptide component (1, 2, 4, 8 μg·mL-1) for 24 h. Cell viability was determined by MTT assay, the change of cell apoptosis and mitochondrial membrane potential were measured with cytoanalyzer, the activity of SOD and the level of MDA were detected with the kit and the expression levels of Cleaved Caspase-3, Cyto C, Bax and Bcl-2 were detected by Western blotting. RESULTS: Four main elution peaks were obtained after Sephadex G-25 gel filtration chromatography and the broccoli polypeptide components II was selected subsequent experiments. The experimental results demonstrate that compared with the normal control group, the survival rate of H2O2-treated cells was decreased and the apoptosis ratio was increased significantly (P<0.01), mitochondrial membrane potential depolarization ratio was increased significantly (P<0.01), mitochondrial membrane potential depolarization ratio decreased (P<0.01), intracellular SOD activity decreased and MDA content increased (P<0.01), the proportion of cleaved caspase-3, Cyto C and Bax/Bcl-2 were increased (P<0.01). Compared with H2O2-treated group, the survival rate of cells in broccoli polypeptide component II- pretreated groups was increased significantly (P<0.01), the apoptosis rate was decreased significantly (P<0.01). The SOD activity increased and MDA concentration decreased (P<0.01). The expression of intracellular cleaved caspase-3, Cyto C and Bax/Bcl-2 were decreased (P<0.01). CONCLUSION: Broccoli polypeptide component II has protective effect on hydrogen peroxide-induced vascular endothelial cells apoptosis and the possible mechanism may be associated with protection the function of mitochondrion.
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To investigate the effect of dihydroartemisinin on apoptosis of human pancreatic cancer cell line JF-305 and the role of reactive oxygen species(ROS) in the apoptosis of JF-305 cells induced by dihydroartemisinin. MTT assays were used to detect effect of different concentrations of dihydroartemisinin on cells proliferation of JF-305 lines. Cell cycle was detected by flow cytometry, and the apoptotic morphology was observed by Hoechst 333258 fluorescence staining. Annexin V fluorescence staining was used to detect the apoptosis changes of JF-305 cells, while DCFH-DA was used to detect the changes of ROS during apoptosis process. Western blot was used to detect the protein expression changes of Bax, Bcl-2, Cleaved caspase-3, Cleaved caspase-9 and Cyto C. As compared with the control group, the JF-305 cells proliferation was inhibited significantly(P<0.05) after treatment with different concentrations of dihydroartemisimin for 48 h; cell cycle was blocked in the G2/M phase; apoptotic morphology of nuclear condensation, aggregation, and fragmentation was found, and the apoptosis ratio was increased(P<0.05). DCFH-DA detection showed that the cell ROS was increased significantly after dihydroartemisinin treatment(P<0.05). Western blot results showed that the expression of Bcl-2 protein was down-regulated; the expression of Bax protein was up-regulated; the ration of Bax/Bcl-2 was increased and the protein expression levels of Cleaved caspase-3, Cleaved caspase-9 and Cyto C were increased after dihydroartemisinin treatment. Therefore, dihydroartemisinin could induce apoptosis of JF-305 cells, and the possible mechanism may be related to the formation and increasing of ROS.
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<p><b>OBJECTIVE</b>To explore the protection of high intensity microwave radiation on hypothalamo-pituitary-adrenal axis (HPAA) activity and hippocampal CA1 structure in rats and the protectiveeffect of Qindan Granule (QG) on radiation injured rats.</p><p><b>METHODS</b>Totally 48 Wistar rats were randomlydivided into 8 groups, i.e., the normal control group, post-radiation day 1, 7, and 10 groups, 7 and 10days prevention groups, day 7 and 10 treatment groups, 6 in each group. Rats in prevention groups wererespectively administered with QG liquid (1 mL/100 g, 4. 75 g crude drugs) for 7 days and 10 days bygastrogavage and then microwave radiation. Then preventive effect for radiation injury was statisticallycalculated with the normal control group and the post-radiation day 1 group. Rats in treatment groupswere firstly irradiated, and then administered with QG liquid (1 mL/100 g, 4.75 g crude drugs). Finally preventive effect for radiation injury was statistically calculated with the normal control group, post-radiation day 7 and 10 groups. Contents of corticotrophin releasing hormone (CRH), beta endorphin (beta-EP), adrenocorticotropic hormone (ACTH), and heat shock protein 70 (HSP70) were detected. Morphological changes and structure of hippocampal CA1 region were observed under light microscope.</p><p><b>RESULTS</b>Compared with the normal control group, contents of CRH and beta-EP significantly decreased in each radiation group. Serum contents of ACTH and beta-EP significantly increased in post-radiation day 1 and 7 groups (P < 0.05). Compared with radiation groups, beta-EP content in serum and pituitary significantly increased, and serum ACTH content significantly decreased in prevention groups (P < 0.05). Pituitary contents of CRH and beta-EP significantly increased in prevention groups. Serum contents of ACTH, beta-EP, and HSP70 were significantly lower in day 7 treatment group than post-radiation day 7 group (P < 0.05). Morphological results showed that pyramidal neurons in the hippocampal CA1 region arranged in disorder, with swollen cells, shrunken and condensed nucleus, dark dyeing cytoplasm, unclear structure. Vessels in partial regions were dilated with static blood; tissues were swollen and sparse. In prevention and treatment groups pathological damage of hippocampal CA1 region was obviously attenuated; neurons were arranged more regularly; swollen, pycnotic, or deleted neuron number were decreased; vascular dilatation and congestion was lessened.</p><p><b>CONCLUSION</b>QG could affect HPAA function and activity of high intensity microwave radiated rats, showing certain preventive and therapeutic effects of microwave radiated rats by adjusting synthesis and release of partial bioactive peptides and hormones in HPAA, improving pathological injury in hippocampal CA1 region.</p>