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Objective:To observe the effect of CoCl2on the cisplatin sensitivity of human ovarian cancer SKOV3cells, and to clarify the possible mechanism.Methods:The SKOV3cells were cultured in vitro and randomly divided into control group, CoCl2 group, cisplatin (DDP) group and CoCl2 combined with DDP (combination) group.The cells in CoCl2group were cultured in normal cell medium for 20hafter cultured in 200μmol·L-1 CoCl2for 4h, the cells in DDP group were cultured in normal cell medium containing 10mg·L-1 DDP for 24h, and the cells in combination group were cultured in 10mg·L-1 DDP for 20hafter cultured in 200μmol·L-1 CoCl2for 4h.The survival rates of SKOV3cells in various groups were detected by MTT method, and the positive expression intensities of hypoxia-inducible factor-1α (HIF-1α) and inducible nitric oxide synthase (iNOS) in the cells in various groups were detected by immunofluorescence method.Rhod 2-AM fluorescence probe was used to observe the levels of Ca2+in mitochondria in the cells in various groups.Western blotting method was used to observe the expression levels of cytochrome C (cyto C) , cysteinyl aspartase 3 (caspase 3) and cleaved cysteinyl aspartase 3 (cleaved caspase 3) .Muse○R apoptosis assay kit was used to detect the apoptotic rates of cells in various groups.Results:Compared with control group, the survival rate of the cells in CoCl2group had no significant change (P＞0.05) , and the survival rates of the cells in DDP and combination groups were decreased (P＜0.05) ;the survival rate in combination group was higher than that in DDP group (P＜0.05) .Compared with control group, the positive expression intensities of HIF-1αin CoCl2and combination groups were increased (P＜0.05) .Compared with control group, the positive expressions of iNOS in DDP and combination groups were increased (P＜0.05) .The Ca2+levels in the cells in DDP group and combination groups were higher than that in control group (P＜0.05) and the Ca2+level in DDP group was higher than that in combination group (P＜0.05) .Compared with control group, the expression levels of cyto C, caspase 3and cleaved caspase 3proteins in the SKOV3cells in CoCl2group had no significant changes (P＞0.05) , and the expression levels of cyto C, caspase 3and cleaved caspase 3in DDP group were increased significantly (P＜0.05) ;compared with DDP group, they were lower than those in combination group (P＜0.05) .Compared with control group, the apoptotic rate of SKOV3cells in DDP group was increased significantly (P＜0.05) ;the apoptotic rate of SKOV3cells in combination group was lower than that in DDP group (P＜0.05) .Conclusion:CoCl2can redece the mitochondrial apoptosis of human ovarian cancer SKOV3cells by inhibiting the DDP-induced enhancement of iNOS expression and decrease the sensitivity of SKOV3cells to cisplatin.
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Objective:To observe the effects of myricetin on autophagy and mitochondrial fission in the human ovarian cancer SKOV3 cells,and to explore its induction effect on autophagy and promoting effect on mitochondrial fission.Methods: The SKOV3 cells were cultured in vitro.0,20,40,and 60 g·L-1 myricetin were used in control group and low, middle, high doses of myricetin groups for 12 h.The changes of mitochondrial membrane potential were detected by flow cytometry;the morphology of mitochondria was observed by MitoTracker Red;the expression levels of dynamin related protein1 (Drp1) and fission 1 (Fis1) in various groups were detected by Western blotting method;and the expression levels of autophagy related protein LC3 were detected by both Western blotting method and immunofluorescence method.Results:Compared with control group, the ratios of decreased mitochondria in different doses of myricetin groups were significantly increased(t=3.27, t=6.85, t=5.49,P<0.05).Compared with control group, the numbers of mitochondria in different doses of myricetin groups were increased, and the mitochondria looked more like gravel.Compared with control group,the expression levels of Drp1 in different doses of myricetin groups were significantly increased (t=4.35, t=3.28, t=6.17,P<0.05), and the expression levels of Fis1 were increased also(t=8.32, t=6.74, t=9.27).The immunofluorescence results showed that the expression levels of LC3 in different doses of myricetin groups were significantly increased with the increase of myricetin dose compared with control group.The Western blotting results showed that the ratios of LC3-Ⅱ/LC3-Ⅰ in middle and high doses of myricetin groups were significantly increased compared with control group(t=3.28, t=4.21,P<0.05).Conclusion: Myricetin can induce the autophagy of SKOV3 cells, and it can also promote the mitochondrial fission.
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Objective To observe protective effects of allicin on heart and lung in aging mice induced by D-galactose, and try to explore the possible mechanism from the aspect of oxidative stress. Methods Fifty male Kunming mice were randomly divided into 5 groups:normal control group ( group C) ,model control group ( group D) and high-,middle-and low-dose of allicin groups ( H,M,L group) . The rats in group D,L,M and H were injected with D-galactose. At the same time,the rats in group L,M and H were injected with allicin. All of the injection lasted for 6 weeks. After that,athletic ability of the mice was evaluated by behavioral experiments. Malondialdehyde ( MDA ) content, superoxide dismutase ( SOD ) activity and total antioxidant capacity ( T-AOC) in the homogenate of heart and lung tissues were measured,and the expression levels of Bax and Bcl-2 were tested in heart and lung. Results Behavioral experiment showed that standing time on Rota Rod System and continuous running time on mice treadmill were significantly shortened in group D as compared with group C ( P<0.05) ,but those were prolonged in the allicin groups. Compared with group C,MDA content in homogenate of myocardium and lung tissues was significantly increased in group D (P<0.05),the activity of SOD was decreased (P<0.05) and T-AOC was decreased (P<0.05) . As compared with group D,MDA content in allicin groups was significantly decreased ( P<0.05) ,SOD activity increased (P<0.05) and A-TOC increased (P<0.05) in a dose-dependent manner. The results of immunohistochemistry showed that the expression of Bax was significantly increased in group D as compared with group C,and the expression of Bcl-2 was decreased. After the intervention of allicin,expression of Bax was decreased in group L,M and H as compared with group D, and expression of Bcl-2 was increased as compared with group D. Expression levels of Bax and Bcl-2 in lung tissues showed the same changing trend. Conclusion Allicin has a potential protect role on heart and lung in D-galactose-induced aging mice by increasing athletic ability,decreasing oxidative stress and decreasing cell apoptosis of myocardium and lung tissues.
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OBJECTIVE To observe the effect of the autophagy and endoplasmic reticulum stress (ERS) in vascular smooth muscle cells (VSMCs) with hydrogen peroxide (H2O2). METHODS The VSMCs were incubated with different concentrations of H2O2(50, 100, 200, 400, 600, 800, 1200 and 1600 μmol · L-1)for 12 and 24 h. The cell viability was determined by MTT assay. The cell morphology was observed under an inverted phase contrast microscope. The expression of autophagy related protein ubiquitin binding protein p62 and microtubule-associated protein light chain 3(LC3)was detected by indirect immunofluorescence. Western blotting was used to detect autophagy related protein human coiled-coil myosin-like BCL2-interacting protein (Beclin-1),LC3 and mammalian target of sirolimus Rapamycin(mTOR),as well as the expression of endoplasmic reticulum stress(ERS)related protein glucose regulated proteins 78 ku(GRP78)and C/EBP homologous protein(CHOP). RESULTS MTT results showed that H2O2 inhibited the growth of VSMCs cells. The half inhibitory concentration (IC50) was 597.2±2.3 and(447.4±1.7)μmol·L-1 at 12 and 24 h,respectively. The results of the inverted phase contrast microscope showed that VSMCs in H2O2 group shrinked and turned into smaller round cells. The cell survival rate declined from(97.5 ± 0.1)% in normal control group to(74.4 ± 1.0)% and(56.8 ± 0.9)% in H2O2 400μmol·L-1 at 12 and 24 h, respectively. The results of the laser scanning confocal micro?scope showed that H2O2 400 μmol · L- 1 increased the expression of LC3 and p62 protein in a cytoplasmic time-dependent manner, and increased the colocalization of LC3 and p62,especially at 8 h. The results of Western blotting demonstrated that H2O2 increased the expression of ERS related protein GRP78 and CHOP,autophagy related protein Beclin-1 and LC3Ⅱ/LC3Ⅰ(P<0.01),and decreased mTOR(P<0.01)in VSMCs. CONCLUSION H2O2 induces autophagy through ERS in VSMCs.
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Objective To investigate the effects of 2.45 GHz microwave on the apoptosis of HeLa cells and explore the underlying mechanism.Methods HeLa cells were divided into five groups randomly:control group,sham-irradiation group,and three irradiation groups exposed with 10,15 and 20 mW/cm2 2.45 GHz microwave for 10 min.Cell survival was tested by MTT assay,cell nucleus fashion was observed by a laser scanning confocal microscope,cell cycle distribution was measured by flow cytometry,and cell protein expressions were detected by Western blot.Results The cell survival in control group,shamirradiation group and low dose irradiation group had no difference but it became lower in the middle and high dose irradiated group(t =-16.47,-14.23,P ＜ 0.05).The abnormal rates of nuclear fashion in the low,middle and high dose irradiation groups were obviously higher compared with control group (t =9.37,11.25,8.47,P ＜ 0.05),while high dose irradiation group was higher than the low and middle dose group (t =15.32,10.25,P ＜0.05).The rate of HeLa cells in sub-G1 in the irradiation groups were higher than that in control group (t =15.24,22.31,10.69,P ＜ 0.05).The results of immunohistochemistry showed that the expression of Bax and Caspase-3 in irradiation groups was significantly increased,and the expression of Bcl-2 was decreased.Conclusions 2.45 GHz microwave can induce HeLa cells apoptosis with a positive correlation with microwave dose.
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<p><b>OBJECTIVE</b>To observe the effects of schizandrins on the learning and memory disorder in mice, and explore its mechanism.</p><p><b>METHOD</b>The memory impairment model was established by using the pentobarbital sodium (20 mg x kg(-1)) intraperitoneally injected in mice. Schizandrins (0.5, 1.0, 2.0 g x kg(-1)) were administered through intragavage for consecutive 14 days. Morris Water Maze test was used to evaluate the impairment of learning and memory. The energy of superoxide dismutase (SOD), nitric oxide (NO) and catalase (CAT) of brain tissue were measured. And the positive expression of nuclear transcription factor-kappaB p65 (NF-kappaB p65), caspase-3 in the hippocampus CA1 region were determined by immunohistochemical analysis. At the cellular level, 24 h after schizandrins (0.062 5, 0.125, 0.25 g x L(-1)) were pre-administered, the apoptosis model of PC12 cell was induced by H2O2, and activity of PC12 cell was detected by MTT colorimetric assay, the energy of NO in cell serum were measured. The expression of Bcl-2 was determined by the combination of immunocytochemical staining and image analysis software.</p><p><b>RESULT</b>Morris Water Maze test showed that the model group mice took shorter searching time and distance on the previous flat area than those in the control group (P < 0.05), which could be prolonged after schizandrins treatment (P < 0.05, P < 0.01). Compared with the control group, the level of NO increased while the activity of SOD, CAT decreased in the model group (both P < 0.01). After treated with schizandrins, the level of NO significantly decreased (P < 0.01), while the activity of SOD increased (P < 0.01). Immunohistochemistry analysis showed that the protein expression of NF-kappaB p65, Caspase-3 in the hippocampal CA1 region significantly increased after modeling, while schizandrins (1.0 g x kg(-1)) can significantly inhibit the protein expression of NF-kappaB p65, Caspase-3 (P < 0.05, P < 0.01). Compared with the H2O2, model group, schizandrins (0.125, 0.25 g x L(-1)) can significantly increased PC12 cell activity and decreased the NO level (P < 0.05, P < 0.01), the expression of Bcl-2 in the schizandrins group (0.125, 0.25 g x L(-1)) was up-regulated.</p><p><b>CONCLUSION</b>Schizandrins could improve the learning-memory dysfunction induced by the sodium pentobarbital in mice, and its protective mechanism is related to the lowering oxidative damage and inhibiting the cell apoptosis through up-regulating the expression of Bcl-2.</p>
Subject(s)
Animals , Female , Male , Mice , Rats , Apoptosis , Behavior, Animal , CA1 Region, Hippocampal , Metabolism , Caspase 3 , Metabolism , Cell Line , Cyclooctanes , Pharmacology , Therapeutic Uses , Disease Models, Animal , Drugs, Chinese Herbal , Pharmacology , Therapeutic Uses , Learning Disabilities , Drug Therapy , Metabolism , Lignans , Pharmacology , Therapeutic Uses , Memory Disorders , Drug Therapy , Metabolism , Nitric Oxide , Metabolism , Oxidative Stress , PC12 Cells , Polycyclic Compounds , Pharmacology , Therapeutic Uses , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Superoxide Dismutase , Metabolism , Transcription Factor RelA , MetabolismABSTRACT
Objective To examine the expression of Cath D,SP-A and GLUT-1 in patients with bronchiogenic carcinoma.Methods A total of 41 patients were included in the study,10 of whom received the histological diagnosis of small cell lung cancer(SCLC).The other 28 were squamous cell carcinoma(SC) and 3 were inflammation.And all the samples were taken from the patients′ tunica mucosa bronchiorum through bronchofibroscope,then we detected the cathepsin D,SP-A and GLUT-1 following SP immunohistochemistry.Results ①Cath D: 6 samples(60%) in group SCLC were negtive expression(-6),4(40%)were moderately positive(++4),4(14%) were negative(-4),2(7%) were positive(+2),8(28%) were moderately positive(++8) and 14(50%) were intensive positive(+++14) in the other group.SCLC was significant different from SC in expressing cathepsin D(P