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Glial cells have been implicated in temporal lobe epilepsy in humans and in its models. Astrocytes are lost in several brain regions after acute seizures induced by pilocarpine and may suffer hyperplasia at subsequent time points. This study investigated the effect of N-methyl-(2S,4R)-trans-4-hydroxy-L-proline (NMP) on astrocytes exposed to cytotoxic concentrations of pilocarpine. Astrocytes were incubated with pilocarpine (half maximal inhibitory concentration (IC50)=31.86 mM) for 24 h. Afterwards, they were treated with NMP at concentrations ranging from 3.12 to 100 μg/mL for 24 h. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytoplasmic reactive oxygen species (ROS) and mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry using 2',7'-dichlorofluorescein diacetate (DCFH-DA) and rhodamine-123 (Rho123), respectively. Expression of glial fibrillary acidic protein (GFAP) and voltage-dependent anion channel-1 (VDAC-1) were measured by western blot. Pilocarpine significantly decreased cell viability and mitochondrial potential and increased ROS concentration significantly by 6.7 times compared to the control. NMP concentrations ≥25 µg/mL protected astrocytes against pilocarpine-induced injury in a concentration-dependent manner. Concomitantly, NMP reduced cytoplasmic ROS accumulation to 27.3, 24.8, and 12.3% in the groups treated with 25, 50, and 100 µg/mL NMP, respectively. NMP also protected mitochondria from pilocarpine-induced depolarization. These effects were associated with improvement of pilocarpine-induced GFAP and VDAC-1 overexpression, which are important biomarkers of astrocyte dysfunction. In conclusion, the improvement of ROS accumulation, VDAC-1 overexpression, and mitochondrial depolarization are possible mechanisms of the NMP protective action on reactive astrocytes.
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OBJECTIVE：To investigate the effects and its mechanism of pseudostrychnine on the apoptosis of human colon can - cer HT- 29 cells. METHODS ：Human colon cancer HT- 29 cells were randomly divided into blank group ，pseudostrychnine low-dose，medium-dose and high-dose groups （125，250，500 μmol/L）. They were cultured with culture medium without medicine or relevant concentration of pseudostrychnine for 48 h. The cell apoptosis and mitochondrial transmembrane potential were detected by flow cytometry. Western blotting assay was employed to detect the protein expression of P 53，Caspase-3，Caspase-9，DNA re - pair enzyme from rabbit （c-PARP）and Bcl- 2. RESULTS ：Compared with blank group ，apoptotic rate of cells was increased signifi - cantly in pseudostrychnine low-dose ，medium-dose and high-dose groups ，while mitochondrial transmembrane potential was de - creased significantly （P＜0.01），in concentration-dependent manner. The protein expression levels of P 53，Caspase-3，Caspase-9 and c-PARP were increased in pseudostrychnine medium-dose and high-dose groups ，compared with blank group ；while those of Bcl-2 were decreased significantly in pseudostrychnine low-dose ，medium-dose and high-dose groups （P＜0.05 or P＜0.01）. CON - CLUSIONS：Pseudostrychnine may change mitochondrial membrane potential by up-regulating the protein expression of P 53 and down-regulating the protein expression of Bcl- 2，and activate the expression of Caspase- 3，c-PARP and Caspase- 9，so as to acti - vate endogenous mitochondrial pathway and promote the apoptosis of HT- 29 cells.
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OBJECTIVE： To observe the inhibitory effects and possible mechanism of new small molecular kinase inhibitors Ibr-7 [Irutinil（Ibr） derivatives] on human pancreatic cancer Capan-2 cells. METHODS： Taking Capan-2 cells as objects， CCK-8 method was used to determine the proliferation of cells after treated with 1， 2， 4， 8 μmol/L Ibr/Ibr-7 for 48 h. The survival rates of cells were calculated. Sensitization effects of 1 μmol/L Ibr/Ibr-7 on different doses of gemcitabine/paclitaxel （0.062 5， 0.125， 0.25， 0.5， 1 μmol/L） were detected. Clone formation test was used to detect the situation of cell clone formation after treated with 1， 2， 4 μmol/L Ibr/Ibr-7 for 48 h. The number of cell colony formation was recorded. Flow cytometry or JC-1 method was used to detect the apoptosis of cells after treated with 2， 4， 8 μmol/L Ibr-7 for 24 or 16 h and the changes of mitochondrial transmembrane potential； total apoptotic rate and the percentage of mitochondrial membrane potential decrease were calculated. Western blotting was used to detect the expression of related apoptotic protein （PARP， Noxa， Bcl-2， Bax， Mcl-1， Bcl-xL）. RESULTS： After treated with 1， 2， 4， 8 μmol/L Ibr/Ibr-7 for 48 h， the survival rates of cells were decreased significantly； those of Ibr-7 groups were significantly lower than those of same-dose Ibr groups； IC50 of Ibr-7 was significantly lower than that of Ibr （P＜0.05 or P＜0.01）. After combined with Ibr/Ibr-7， the survival rate of cells was significantly lower than that of same-dose gemcitabine/paclitaxel alone group， and the Ibr-7 combination group was significantly lower than same-dose Ibr combination group （P＜0.05 or P＜0.01）. After treated with 2， 4 μmol/L Ibr and 1， 2， 4 μmol/L Ibr-7 for 48 h， the number of cell clone formation was decreased significantly， while Ibr-7 groups were significantly lower than same-dose Ibr groups （P＜0.01）. After treated with different doses of Ibr-7 for 24 or 16 h， total apoptosis rate of cells （2， 4， 8 μmol/L）， the proportion of cell mitochondrial membrane potential decrease （8 μmol/L）， the relative protein expression of Noxa （2， 4， 8 μmol/L） and Bax （8 μmol/L） were increased significantly， while the protein expression of PARP （8 μmol/L）， Bcl-2 （4 μmol/L）， Mcl-1 （2， 4， 8 μmol/L） were decreased significantly； above indexes （except for relative expression of PARP and Bcl-2） of 8 μmol/L Ibr-7 group were significantly better than same-dose Ibr group （P＜0.05 or P＜0.01）. There was no statistical significance in protein expression of Bcl-xL among those groups （P＞0.05）. CONCLUSIONS： Compared with Ibr， Ibr-7 has better inhibitory and apoptotic effects on human pancreatic cancer Capan-2 cells in vitro， and has stronger chemotherapeutic drug sensitization activity， the mechanism of which may be associated with reducing mitochondrial transmembrane potential， down-regulating the protein expression of PARP， Bcl-2 and Mcl-1 and up-regulating the protein expression of Noxa and Bax.
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OBJECTIVE:To study the mechanism of apoptosis of human liver cancer SMMC7721 cells induced by tetrandrine (Tet). METHODS:MTT assay was used to test the cells activity and calculate the inhibition rate after the cells were cultured by 4, 6,8 and 10 mg/L Tet for 24,48 and 72 h. Flow cytometry was used to test the cells apoptotic ratio after the cells were cultured by 6 mg/L Tet for 24,48 and 72 h;Mitochondrial membrane potential (JC-1) was examined under an inverted fluorescence micro-scope;colorimetric method was used to detect the Caspase-3 activity;the protein expressions of Cyto-C and Pro-caspase-3 were ex-amined by Western blot. All of the tests were operated with blank control (normal culture medium or cultivated for 0 h). RE-SULTS:There was an obvious inhibition on the proliferation of SMMC7721 cells in a time- and dose-dependent manner after cells were cultured by 4,6,8 and 10 mg/L Tet for 24,48 and 72 h. Compared with the blank control,after cells were cultured by 6 mg/L Tet for 24,48 and 72 h,the cells apoptotic ratio was increased,JC-1 was decreased in a dose-dependent manner. There was an in-crease in the Caspase-3 activity after cells were cultured by 6 mg/L Tet for 24 and 48 h. After cells were cultured by 6 mg/L Tet for 24 h,the Cyto-C protein expression was strengthened,and Pro-caspase-3 protein expression was decreased,with statistical differ-ences (P<0.05). CONCLUSIONS:Tet treatment can induce the apoptosis of SMMC7721 cells by a mechanism that is related to the activation of mitochondrial apoptotic pathway.
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Objective To observe the process of Vibrio vulnificus inducing dendritic cell strain apoptosis.Methods We established the mixed culture model of mouse dendritic cell ( DC 2.4 strain) and Vibrio vulnificus( Vv1.1758 strain ),analyzed morphological characteristics of cell apoptosis by DAPI fluorescence staining,detect DNA fragmentation level of apoptosis cells by DNA Ladder assay,analyze DC2.4 apoptosis rate by Annexin V FITC/PI staining,determine activities of caspase-3 and caspase-8 by means of spectrophotometric method and detect changes of mitochondrial transmembrane potential ( △ Ψm ) by JC-1 fluorescence labeling.Results After Vv1.1758 strain and DC2.4 cell were mixed and cultured for 4 h,DAPI fluorescence staining showed typical apoptosis characteristics-chromatin condensation and marginalization; DNA agarose gel electrophoresis showed apoptosis band; apoptosis rates at 2,4 and 6 h were respectively (37.8±9.8) ％,(54.3 ± 12.7 ) ％ and ( 68.2± 14.6 ) ％ ; Mitochondrial transmembrane potentials (△Ψm) at 1 h,2 h and 4 h reduced by 7.1％,16.1％ and 46.7％ respectively; caspase-8 activity increased at 1.5 h and reached the peak at 2 h [ (2.48±0.19) U/μg],while caspase-3 activity started to increase at 3h and reached the peak at 4 h [ ( 1.91 ±0.16) U/μg ].Conclusion Vibrio vulnificus could induce dendritic cells by two pathways: reducing mitochondrial transmembrane potential and activating caspase-8 promoter and finally activate effector caspase-3 to promote apoptosis.
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An atisane diterpene was isolated from Xylopia langsdorfiana St. Hilaire & Tulasne, Annonaceae, leaves, ent-atisane-7α,16α-diol (xylodiol). Preliminary study showed that xylodiol was cytotoxic and induced differentiation on human leukemia cell lines. However, the molecular mechanisms of xylodiol-mediated cytotoxicity have not been fully defined. Thus, we investigated the anti-tumor effect of xylodiol in human leukemia HL60 cell line. Xylodiol induced apoptosis and necrosis. HL60 cells treated with xylodiol showed biochemical changes characteristic of apoptosis, including caspases-8, -9 and -3 activation and loss of mitochondrial transmembrane potential (∆ Ψm). However, there was a condensation rather than swelling of mitochondria. Moreover, the formation of condensed mitochondria and the loss of ∆ Ψm occurred downstream of caspase activation. Cyclosporine A did not protect HL60 cells from the cytotoxic effects of xylodiol, suggesting that the loss of ∆ Ψm is a late event in xylodiol-induced apoptosis. Oxidative stress was involved in xylodiol-induced apoptosis. Thus, we conclude that activated caspases cleave cellular proteins resulting in mitochondrial damage leading to mitochondrial condensation, loss of ∆ Ψm and ROS release from the mitochondria. ROS can further induce and maintain a collapse of ∆ Ψm leading to cellular damage through oxidation of lipids and proteins resulting in apoptotic cell death.
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Objective To explore the effects of Jiedu Quyu Ziyin Recipe (JQZR) on the apoptosis and expressions of bcl-2 and bax mRNA of peripheral-blood lymphocyte in MRL/lpr mice. Methods 80 MRL/lpr mice were randomly divided into model group,TCM group, Western medicine group and TCM and Western medicine group,20 mice in each group, meanwhile,20 Kunming mice were selected as normal group, then intragastrically administered normal sodium, JQZR apozem, prednisone suspension and JQZR apozem and prednisone suspension, 0. 5ml every time,once daily for 12 weeks respectively. At the end of the 12th week, peripheral-blood lymphocytes of every mice purified by gradient centrifugation were cultivated for 48 hours ,then the apoptosis was detected by flow cytometry. Furthermore,the expressions of bcl-2 and bax mRNA of peripheral-blood lymphocyte were detected by RT-PCR. Results At Oh or 48h,the apoptosis ratios of PBLC in normal group, TCM group,Western medicine group and TCM and Western medicine group are higher than model group and the differences are significant(P <0. 05 ,P <0. 01 ) ,while,at Oh,which are not significant within Western medicine group, normal group and TCM and Western medicine group ( P > 0. 05) ,even if which are significant between TCM group and TCM and Western medicine group or between model group and TCM and Western medicine group( P < 0. 05 ). At 48h, the differences of apoptosis ratios of PBLC are significant between Western medicine group and TCM and Western medicine group( P < 0. 05 ), and between TCM group and TCM and Western medicine group or between model group and TCM and Western medicine group which are significant, too( P < 0. 01 ). The ratios of bcl-2/bax mRNA of normal group,TCM group and TCM and Western medicine group are significantly lower than which of model group or Western medicine group ( P < 0. 05, P < 0. 05, P <0. 01 ,respectively) ,but there is no significant difference between which of model group and Western medicine group.Conclusion JQZR has adjustable effects on the apoptosis and expressions of bcl-2 and bax mRNA of peripheralblood lymphocyte in MRL/lpr Mice.
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Objective To study the morphology and the effect of bovine lactoferricin (LfcinB) for Jurkat T leukemia cells and HFL-I cell and further validate and analyze the signal transduction passage way of LfcinB-induced apoptosis. Method The Jurkat T leukemia cells and fibroblasts stained with Hoechst 33258 after in vitro treatment with LfcinB were observed through fluorescence microscope, compared with positive and negative control groups to analyze the difference between the effect of LfcinB on Jurkat T leukemia cells and HFL-I cell with gel electrophoresis analysis of DNA fragmentation. Early apoptosis and mitochondrial membrane potential of Jurkat T leukemia cells were analyzed by flow cytometry, and the changes of caspase-3, caspase-8, caspase-9 and cytochrome C in endochylema of Jurkat T leukemia cells after exposed to LfcinB during 4, 6 and 8 h were detected by Western blotting. Results DNA Ladder was shown by gel electrophoresis analysis of DNA fragmentation in Jurkat T leukemia cells following treatment with LfcinB, which was not found in HFL-I cell. Through fluorescence microscope, we found that nucleus of LfcinB-exposed Jurkat T leukemia cells stained with Hoechst 3325 became condense, but the nucleus of HFL-I cell was not changed. The results of flow cytometry analysis indicated that apoptosis rates of Jurkat T leukemia cells exposed to LfcinB for 2, 4 h were 11.5% and 17.8% respectively, with decline of mitochondria membrane potential. Immunoblot analysis showed that LfcinB could increase the content of cytochrome C, with the activated caspase-3 and caspase-9 increase gradually in endochylema of Jurkat T leukemia cells, however there was no effect of LfcinB on caspase-8. Conclusion LfcinB induced apoptosis of Jurkat T lenkemia cells, but not affected fibroblasts. After Jurkat T leukemia cells, contacted with LfcinB for more than 2 h, the contents of caspase-3, caspase-9 and cytochrome C in the cells were cumulatively increasing, which further validated that LfcinB induced Jurkat apoptosis by intra-cellular signal pathway depending on caspase family.
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Objective:To investigate the mechanism of ~(131)I-GM-CSF inducing apoptosis of HL-60/ADM cells.Methods:An in vitro radioimmunotherapy(RAIT) model was employed.Data were collected from transmission electrion microscopy,flow cytometric analysis and immunocytochemistry assay.Results:After exposure to ~(131)I-GM-CSF,HL-60/ADM cells were induced to apoptosis.The cell mitochondrial transmembrane potential(MTP) reduced and HL-60/ADM cells appeared the classical apoptotic morphology.The Bax was up regulated,while Bcl-2 and Bcl-xl was decreased.Conclusion:~(131)I-GM-CSF can induce HL-60/ADM apoptosis through opening the mitochondrial permeability transition pore and reducing MTP,which suggests that ~(131)I-GM-CSF may be available in therapy of AML.
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Objective To observe the effects of different doses of rotenone on reactive oxygen species, mitochondrial transemembrane potential as well as mitochondrial ultrastructure in order to investigate the possible mechanism of mitochondrial dysfunction in cultured pheochromaffinoma cells. Methods ROS production induced by rotenone in cells were measured using flow cytometry. Mitochondrial transmembrane potential was detected by laser scanning confocal microscope and mitochondrial ultrastructures were observed under transmission electron microscope. Results Treatment with 0.1, 1.0, 2.0 and 3.0 ?mol/L concentrations of rotenone could increase the generation of ROS in PC12 cells as 1.55?0.17, 2.16?0.10, 1.77?0.20 and 1.40?0.12, and disrupt the mitochondrial transmembrane potential about 93.86?10.12, 119.43?7.09, 102.71?9.36 and 83.14?10.70. There were significant differences as compared with the control group (P
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AIM: To investigate the possible role of the mitochondrial transmembrane potential (Δψm) and caspase 3 in arsenic trioxide (As2O3)-induced apoptosis of cancer cells. METHOD: Namalwa, SGC7901 and Bcap37 cell lines were used as in vitro models. Apoptosis was confirmed by sub-G1, cells content as well as phosphatidylserine (PS) externalization. The Δψm was detected on flow cytometry through double staining of Rhodamine 123 (Rh123) and popidium iodide(PI). In addition, the effect of DEVD-CHO, a selective inhibitor of Caspase 3, on As2O3-induced apoptosis was studied. RESULT: The As2O3-induced apoptosis closely associated with the externalization of the Δψm and the activation of Caspase 3. As2O3 induced cells necrosis when Caspase 3 was inhibited. CONCLUSION: As2O3 may selectively activates Caspase 3 after it induces externalization of Δψm, which causes cancer cells apoptosis.
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AIM To investigate the possible role of the mitochondrial transmembrane potential (??m) and caspase 3 in arsenic trioxide(As 2O 3)-induced apoptosis of cancer cells. METHOD Namalwa, SGC7901 and Bcap37 cell lines were used as in vitro models. Apoptosis was confirmed by sub-G 1 cells content as well as phosphatidylserine(PS) externalization. The ??m was detected on flow cytometry through double staining of Rhodamine 123(Rh123) and popidium iodide(PI). In addition, the effect of DEVD-CHO, a selective inhibitor of Caspase 3, on As 2O 3-induced apoptosis was studied. RESULT The As 2O 3-induced apoptosis closely associated with the externalization of the ??m and the activation of Caspase 3. As 2O 3 induced cells necrosis when Caspase 3 was inhibited. CONCLUSION As 2O 3 may selectively activates Caspase 3 after it induces externalization of ??m, which causes cancer cells apoptosis.