Mitochondrial protection effect of sirtuin2 inhibitor AGK2 on thioacetamide-induced L02 cells / 中国医师杂志

Objective:To explore the protective effect of AGK2, a selective inhibitor of sirtuin 2 (SIRT2), on the mitochondria of L02 hepatocytes induced by thioacetamide (TAA) and its related mechanism.Methods:Human-derived hepatocyte line L02 cells were cultured in vitro. Different concentrations of SIRT2 inhibitor AGK2 were used as intervention drugs. Cell counting kit-8 (CCK8) was used to detect the effects of different concentrations of AGK2 on the activity of L02 cells, and the appropriate concentration was selected as the AGK2 intervention group. The normal group was not given any drug intervention. The model group was given 90 mmol/L TAA for modeling. Low, medium and high dose AGK2 groups were added with 1, 2 and 4 μmol/L AGK2, respectively 2 h before modeling. CCK8 was used to detect cell activity in each group. Morphological changes of cells were observed under inverted light microscope. The relative protein expression levels of isocitrate dehydrogenase (IDH1), malate dehydrogenase (MDH1), SIRT2 and fission protein 1 homologue (FIS1) were detected by Western blot. The expression of SIRT2 in cells of each group was observed by confocal laser scanning microscope. The mitochondrial membrane potential of cells in each group was observed under a fluorescence microscope. Results:When AGK2 concentration was 1, 2 and 4 μmol/L, the survival rate of cells were 98.05%, 95.76% and 91.65%, respectively, with no statistical significance compared with normal group (all P>0.05). When AGK2 concentration was 8, 16, 32, 64, 128 μmol/L, the cell survival rate was significantly decreased compared with normal group (all P<0.05). Compared with the model group, the L02 cells in low, medium and high AGK2 groups had better activity and adherence, and the floating cells were significantly reduced. The higher the concentration of AGK2, the better the cell activity and adherence, and the less floating cells. Compared with the model group, the red fluorescence of L02 cells in AGK2 group was enhanced, while the green fluorescence was weakened. The higher the AGK2 concentration was, the stronger the red fluorescence was, and the weaker the green fluorescence was. Compared with the model group, the fluorescence of SIRT2 in L02 cells of low, medium and high AGK2 groups was weakened, and the higher the concentration of AGK2, the weaker the fluorescence of SIRT2. The protein expressions of IDH1 and MDH1 in L02 cells of low, medium and high AGK2 groups were significantly higher than those of model group (all P<0.05), and were positively correlated with the concentration of AGK2 ( r=0.818, P<0.05; r=0.960, P<0.05); the protein expressions of SIRT2 and FIS1 were significantly lower than those of the model group (all P<0.05), and were negatively correlated with the concentration of AGK2 ( r=-0.992, P<0.05; r=-0.998, P<0.05). Conclusions:AGK2 can reduce the mitochondrial membrane potential stimulated by TAA in L02 cells, increase the protein expression of IDH1 and MDH1, and inhibit the protein expression of SIRT2 and FIS1 in L02 cells in a dose-dependent manner.

Herbal formula Renshenwuweizi decoction induces p53-mediated cell cycle arrest and apoptosis in A549 cells.

OBJECTIVE: To investigate the effect of Renshenwuweizi decoction (RSWWZ decoction) on the growth of non-small cell lung cancer cells in vitro. METHODS: A549 non-small cell lung cancer cells were divided into two groups: control and RSWWZ decoction treatment groups. Cell Counting Kit-8 was used to measure the inhibitory effect of RSWWZ decoction on the growth of A549 cells. 4', 6-diamidino-2-phenylindole staining and Annexin V-fluorescein isothiocyanate/propidium iodide double staining were used to investigate apoptosis in A549 cells following RSWWZ decoction treatment, and the mitochondrial membrane potential of treated cells was detected with Rhodamine 123. Cell cycle progression was analyzed by flow cytometry. The mRNA levels of p53, Bax, B-cell lymphoma-2 (Bcl-2) and p21 were measured by quantitative real-time reverse transcription polymerase chain reaction. The protein expressions of p53, Bax, Bcl-2, p21, cyclin-dependent kinases 2 (CDK2), and cyclin A were detected by Western blot. RESULTS: RSWWZ decoction reduced the viability of A549 cells in a dose-dependent manner by inducing apoptosis and decreased mitochondrial membrane potential. RSWWZ decoction increased p53 and Bax expression and decreased Bcl-2 expression in a dose-dependent manner. RSWWZ decoction also induced an S-phase cell cycle arrest by increasing p21 and decreasing cyclin A and CDK2 expression. CONCLUSION: In vitro experiments revealed that the Renshenwuweizi decoction-induced decrease in A549 cell proliferation was achieved by inducing apoptosis and S-phase cell cycle arrest via the p53 pathway. These findings provide the experimental basis for Renshenwuweizi decoction treatment of lung cancer.

[Effects and mechanism of pyrroloquinoline quinine on mitochondrial function and cell survival of rat bone marrow mesenchymal stem cells under oxidative stress].

Objective: To observe the effects of pyrroloquinoline quinine (PQQ) on the mitochondrial function and cell survival of rat bone marrow mesenchymal stem cells (BMSCs) under oxidative stress, and to explore its mechanism. Methods: BMSCs of rats were cultured in vitro with Dulbecco's minimum essential medium/F12 medium containing fetal bovine serum in the volume fraction of 10% (hereinafter referred to as normal medium). The rat BMSCs of third to fifth passages in logarithmic growth phase were selected for the following experiments. (1) The cells were divided into normal control group, normal control+ PQQ group, hydrogen peroxide (H(2)O(2)) alone group, and H(2)O(2)+ PQQ group. The cells in normal control group were cultured in normal medium for 24 hours; the cells in normal control+ PQQ group were cultured in normal medium containing 100 µmol/L PQQ for 24 hours; the cells in H(2)O(2) alone group were cultured in normal medium containing 200 µmol/L H(2)O(2) for 24 hours; the cells in H(2)O(2)+ PQQ group were pre-incubated with normal medium containing 100 µmol/L PQQ for 2 hours, and then with H(2)O(2) added to the concentration of 200 µmol/L and cultured for 24 hours. The cell morphology of each group was observed under the inverted phase contrast microscope, and the cell survival rate was detected by cell count kit 8 method. (2) Five batches of cells were collected, and the cells of each batch were divided into normal control group, H(2)O(2) alone group, and H(2)O(2)+ PQQ group. The cells in each group received the same treatment as that in the corresponding group of experiment (1). After 24 hours of culture, one batch of cells was collected for apoptosis detection by flow cytometry, and the apoptosis rate was calculated. One batch of cells was subjected to mitochondrial membrane potential assay and JC-1 fluorescent staining observation using the JC-1 mitochondrial membrane potential detection kit and the inverted phase contrast fluorescence microscope, respectively. One batch of cells was collected for mitochondrial morphology observation under the transmission electron microscope. One batch of cells was subjected to catalase (CAT) and superoxide dismutase (SOD) activity assay by CAT activity assay kit and SOD activity assay kit, respectively. One batch of cells was subjected to Western blotting for determination of protein level of Epac1, adenine monophosphate activated protein kinase (AMPK), phosphorylated AMPK, cysteinyl aspartate-specific proteinase 3 (caspase-3), and cleaved caspase-3, and the phosphorylation level of AMPK and cleaved caspase-3/caspase-3 ratio were calculated. Six replicates were measured in each group for each index except for morphological observation. Data were statistically analyzed with one-way analysis of variance and independent sample equal variance t test. Results: (1) After 24 hours of culture, compared with those in normal control group (the cell survival rate was set to 100.0%), there was an increase in cell vacuole and a decrease in cell number in H(2)O(2) alone group, and the cell survival rate was significantly reduced to (74.3±2.9)% (t=6.39, P<0.01). Compared with those in H(2)O(2) alone group, the cell morphology of H(2)O(2)+ PQQ group was significantly improved, and the cell survival rate was significantly increased to (116.9±4.2)% (t=6.92, P<0.01); the cell survival rate in normal control+ PQQ group was (101.2±1.1)%, close to that of control group (t=1.06, P>0.05). (2) After 24 hours of culture, compared with (13.6±1.0)% in normal control group, the apoptosis rate of cells in H(2)O(2) alone group was significantly increased to (37.1±2.0)% (t=10.57, P<0.01). Compared with that in H(2)O(2) alone group, the apoptosis rate of cells in H(2)O(2)+ PQQ group was significantly declined to (17.0±0.7)% (t=9.49, P<0.01). (3) After 24 hours of culture, compared with those in normal control group, the mitochondrial membrane potential of cells in H(2)O(2) alone group was depolarized, the JC-1 fluorescent dye mainly existed in the cytoplasm in the form of monomer, which emitted green fluorescence, and a significant decrease in mitochondrial membrane potential was shown (t=4.18, P<0.01). Compared with those in H(2)O(2) alone group, the mitochondrial membrane potential of cells in H(2)O(2)+ PQQ group was increased to normal level (t=4.43, P<0.01), and the JC-1 fluorescent dye accumulated in mitochondria following the polarized mitochondrial membrane potential and emitted red fluorescence. (4) After 24 hours of culture, compared with that in normal control group, the mitochondrial structure of cells in H(2)O(2) alone group was disordered, with disappeared mitochondrial cristae and decreased mitochondrial matrix density. Compared with that in H(2)O(2) alone group, the mitochondrial structure of cells in H(2)O(2)+ PQQ group was regular and intact, with clearly visible mitochondrial cristae and increased mitochondrial matrix density. (5) After 24 hours of culture, compared with those in normal control group, the CAT activity of cells in H(2)O(2) alone group was significantly increased (t=4.54, P<0.05), and the SOD activity was significantly decreased (t=3.93, P<0.05). Compared with those in H(2)O(2) alone group, the CAT activity of cells in H(2)O(2)+ PQQ group was obviously increased (t=8.65, P<0.01), while there was no significant change in the SOD activity (t=0.72, P>0.05). (6) After 24 hours of culture, compared with those in normal control group, the protein expression of Epac1 of cells in H(2)O(2) alone group was significantly decreased (t=4.67, P<0.01), while the AMPK phosphorylation level and the cleaved caspase-3/caspase-3 ratio were significantly increased (t=7.88, 3.62, P<0.01). Compared with those in H(2)O(2) alone group, the protein expression of Epac1 and the AMPK phosphorylation level of cells in H(2)O(2)+ PQQ group were both significantly increased (t=4.34, 16.37, P<0.01), while the cleaved caspase-3/caspase-3 ratio was significantly declined (t=3.17, P<0.05). Conclusions: Pretreatment with PQQ can improve the mitochondrial function, reduce cell apoptosis rate, and enhance cell survival rate of rat BMSCs under oxidative stress, which may be related to the up-regulation of Epac1 protein expression, activation of AMPK signaling pathway, and down-regulation of cleaved caspase-3 protein level.

Effect of hydrogen on mitochondrial membrane potential during focal cerebral ischemia-reperfusion in rats / 中华麻醉学杂志

Objective To investigate the effect of hydrogen on mitochondrial membrane potential during focal cerebral ischemia-reperfusion (I/R) in rats.Methods Eighty-four healthy adult male SPF Sprague-Dawley rats,weighing 220-240 g,were divided into 3 groups (n=28 each) using a random number table:sham operation group (Sham group),I/R group and hydrogen group (H2 group).Focal cerebral I/R was produced by mid-cerebral artery occlusion in I/R and H2 groups.Hydrogen 10 ml/kg was intraperitoneally injected immediately after onset of reperfusion and at 12 h of reperfusion in group H2.Neurological deficit was scored at 24 h of reperfusion.The rats were then sacrificed and brain tissues in cortex were removed for microscopic examination of the pathological changes and for determination of cerebral infarct size (by TFC staining),nerve cell apoptosis (by TUNEL),mitochondrial membrane potential (by JC-1 staining) and expression of Bcl-2 and Bax (by Western blot).Apoptotic index (AI) was calculated.Results Compared with Sham group,the neurological deficit score,percentage of cerebral infarct size and AI were significantly increased,the mitochondrial membrane potential was decreased,Bax expression in brain tissues was up-regulated,and Bcl-2 expression in brain tissues was down-regulated in I/R and H2 groups (P＜0.05).Compared with I/R group,the neurological deficit score,percentage of cerebral infarct size and AI were significantly decreased,the mitochondrial membrane potential was increased,Bax expression in brain tissues was down-regulated,and Bcl-2 expression in brain tissues was up-regulated (P＜0.05),and the pathological changes of brain tissues were significantly attenuated in H2 group.Conclusion The mechanism by which hydrogen reduces focal cerebral I/R injury is related to decreasing dissipation of mitochondrial membrane potential and inhibiting nerve cell apoptosis in rats.

Extracellular Mitochondria in Cerebrospinal Fluid and Neurological Recovery After Subarachnoid Hemorrhage.

BACKGROUND AND PURPOSE: Recent studies suggest that extracellular mitochondria may be involved in the pathophysiology of stroke. In this study, we assessed the functional relevance of endogenous extracellular mitochondria in cerebrospinal fluid (CSF) in rats and humans after subarachnoid hemorrhage (SAH). METHODS: A standard rat model of SAH was used, where an intraluminal suture was used to perforate a cerebral artery, thus leading to blood extravasation into subarachnoid space. At 24 and 72 hours after SAH, neurological outcomes were measured, and the standard JC1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanineiodide) assay was used to quantify mitochondrial membrane potentials in the CSF. To further support the rat model experiments, CSF samples were obtained from 41 patients with SAH and 27 control subjects. Mitochondrial membrane potentials were measured with the JC1 assay, and correlations with clinical outcomes were assessed at 3 months. RESULTS: In the standard rat model of SAH, extracellular mitochondria was detected in CSF at 24 and 72 hours after injury. JC1 assays demonstrated that mitochondrial membrane potentials in CSF were decreased after SAH compared with sham-operated controls. In human CSF samples, extracellular mitochondria were also detected, and JC1 levels were also reduced after SAH. Furthermore, higher mitochondrial membrane potentials in the CSF were correlated with good clinical recovery at 3 months after SAH onset. CONCLUSIONS: This proof-of-concept study suggests that extracellular mitochondria may provide a biomarker-like glimpse into brain integrity and recovery after injury.

The role of neuroglobin in oxygen-glucose deprivation and reoxygenation-induced mitochondrial ;depolarization and reactive oxygen species production in SH-SY5Y cells / 中华内科杂志

Objective To investigate the role of neuroglobin ( NGB) in oxygen-glucose deprivation and reoxygenation ( OGD/R ) induced mitochondrial depolarization and reactive oxygen species ( ROS ) production in a human neuroblastoma cell line (SH-SY5Y).Methods SH-SY5Y cells were transfected with lentivirus to establish a stable cell line of NGB knockdown ( KD).After treated with OGD/R, cells were collected at different time points to analyze NGB mRNA and protein levels.Furthermore, cells were stained with JC-1 and DCFH-DA to evaluate mitochondrial depolarization and ROS production by inverted fluorescence microscope.Also, to determine the neurotoxicity , we measured the lactate dehydrogenase ( LDH) level in the cell culture medium.Results After the treatment of OGD/R, the NGB mRNA and protein started to elevate and peak at 4 h and 8 h (2.04 ±0.35 fold,1.69 ±0.18 fold).Compared with the vector group , NGB KD group had much more mitochondrial depolarization [ JC-1 red/green ( 1.10 ±0.10 ) vs (1.46 ±0.11),P<0.05] and ROS production [DCFH-DA fluorescence (36.30 ±5.32) vs (16.26 ± 2.97),P<0.05].Furthermore, NGB KD groups had a higher level of LDH release [(63.42 ±6.14)%vs (49.65 ±5.09 )%, P <0.05 ].Conclusions NGB plays an important role in the homeostasis of mitochondria.Knockdown of NGB results in increased mitochondrial depolarization , ROS production and neurotoxicity under hypoxia circumstances.

TNF-α induced RIP1-dependent apoptosis in L929-A cells by interrupting mitochondrial respiratory chain complex Ⅲ / 军事医学

Objective To explore the mechanism by which tumor necrosis factor alpha(TNF-α) induces RIP1 kinase-dependented apoptosis in L929-A fibroblastoma cells.Methods The sub-mitochondrial localization of receptor-interacting protein 1(RIP1),caspase-8 and Bid proteins was detected by dose-gradient trypsin digestion and Western blotting.The levels of reactive oxygen species (ROS),intracellular calcium concentration,mitochondrial membrane potential (MMP),and cellular adenosine triphosphate(ATP) content were determined by fluorescent probe labeling and flow cytometry assay.The mitochondrial respiratory chain complex Ⅰ and Ⅲ activities were detected by commercial kits.Nec-1,A RIP1 kinase specific inhibitor,and RIP1-/-or Bid-/-L929-A cells were used to detect the roles of RIP1 kinase and Bid protein in cell death.Results RIP1,caspase-8 and Bid proteins were co-located in the outer membrane of mitochondrial.TNF-α exposure for 3 h could induce Bid cleavage,inhibit mitochondrial respiratory chain complex Ⅲ activity and reduce MMP.Following these changes and after TNF-α exposure for 6-12 h,the intracellular calcium concentration and ROS were increased,whereas the ATP concentration was decreased,and the cells were killed.Inhibiting RIP1 kinase or knockdown RIP1 or Bid protein could suppress all the cytotoxic effects of TNF-α.Conclusion TNF-α treatment can result in RIP1 kinase-mediated Bid cleavage and inhibit mitochondrial respiratory chains and cell energy metabolism,which ultimately leads to the death of L929-A cells.

Effects of carbon monoxide on lipopolysaccharide induced damage in rat alveolar macrophages / 天津医药

Objective To evaluate effects of carbon monoxide (CO) on lipopolysaccharide (LPS) induced damage and possible mechanism in rat alveolar macrophages. Methods Rat alveolar macrophages were cultured in DMEM containing 10%fetal bovine serum with 5%CO2 at 37℃in Heraeus sepatech. The cells were divided into four groups using random number table (n=10): control group (group C),CO group, LPS group and LPS+CO group. The CO release molecule-2 (CORM-2) 100 μmol/L was added into CO group,LPS 10 mg/L was added into LPS group, cells were pretreated with CORM-2 100μmol/L for 1 h then LPS 10 mg/L was added into LPS+CO group, the same amount of PBS was added to group C. Proliferation was measured by MTT assay. Apoptosis and mitochondrial membrane potential were detected with flow cytometer. The content of ATP was tested by ATP content kit. Drp1 mRNA was measured by RT-PCR, and Drp1 expression was determined by Western blot assay. Results Compared with group C, the cell vitality, content of ATP and mitochondrial membrane potential were decreased in LPS group and LPS+CO group,and cell apoptosis rate, Drp1 mRNA and protein expression were increased (P<0.05). There were no significant changes were found in CO group. Compared with LPS group, the cell vitality, content of ATP and mitochondrial membrane potential were increased in LPS+CO group,and the cell apoptosis rate, Drp1 mRNA and protein expression were decreased (P<0.05). Conclusion Carbon monoxide can alleviate LPS-induced damage in rat alveolar macrophages, which is related with down-regulation of Drp1 and amelioration of mitochondrial function.

A novel histone deacetylase inhibitor induces proliferation inhibition of human colon cancer cells / 军事医学

Objective To examine the anticancer effect of a novel histone deacetylase inhibitor (HDACi), JZ004, on colon cancer cells HCT-8 and HT-29, and to investigate the molecular mechanisms of proliferation inhibition and apoptosis induction of cancer cells treated by JZ 004.Methods Colon cancer cells were treated with a series of concentrations of JZ004 .MTT assay was used to detect the proliferation of cancer cells .The cell cycle distribution and apoptosis were deter-mined by flow cytometry .Rhodamine 123 and DCFH-DA were applied to detect the mitochondrial membrane potential (ΔΨm) and reactive oxygen species ( ROS) production.The protein expressions of acetyl-histone H3, p21, cyclin-dependent kinase(CDK)4, Bcl-2, Mcl-1 and Bax were assayed by Western blotting .Results JZ004 was found to inhibit proliferation and induce apoptosis of colon cancer cells in a time-and dose-dependent manner , accompanied by a dose-dependent hyperacetylation of histone H3.JZ004 induced the cancer cell arrest in G 0/G1 phase by increasing the expres-sion level of p21 while CDK4 was downregulated .JZ004 also increased cellular ROS production and reduced ΔΨm by regu-lating the expressions of Bcl-2 family proteins .Conclusion As a novel HDACi , JZ004 effectively inhibits proliferation and increases ROS production to induce apoptosis of colon cancer cells .The results indicate that JZ004 is a potential compound to be developed as an anti-colon cancer agent for clinic application .

8-chloroadenosine 3 ', 5 '-monophosphate induces apoptosis of multiple myeloma cells catalyzed by arsenic trioxide / 白血病·淋巴瘤

Objective To investigate the response of multiple myeloma (MM) cells to 8-chloroadenosine 3',5'-monophosphate (8-Cl-cAMP) and the impact of arsenic trioxide (As2O3) on the above reaction.Methods MM-derived cell lines RPMI8226 and U266 were used as in vitro models.Cell apoptosis was evaluated according to cellular morphology and DNA content measured by flow cytometry.Meanwhile,rhodamine 123 (Rh123) staining and flow cytometry assay were used to detect the changes of mitochondrial transmembrane potentials (△ψm) in MM cells before and after the treatment.The synergic effects of 8-Cl-cAMP and As2O3 were evaluated by King' s formula.Results The 8-Cl-cAMP could induce growth inhibition of RPMI8226 and U266 cells in dose and time-related manners.The 8-Cl-cAMP could trigger apoptosis and △ψm collapse in MM cells through cellular morphology and flow cytometry analysis.As2O3 accelerated 8-Cl-cAMP-mediated apoptosis of RPMI8226 cells,but there were few synergic effects observed.Conclusion 8-Cl-cAMP could induce cell proliferation inhibition and apoptosis in MM cells.Mitochondria may be one of targets in 8-Cl-cAMP-mediated apoptosis.Furthermore,As2O3 catalyzes 8-Cl-cAMP-induced apoptosis.

Oxidative damage effect of the serum of severe preeclamptic patients on human umbilical vein endothelial cell / 中华妇产科杂志

Objective To investigate oxidative damage effect of the serum of severe preeclamptic patients on human umbilical vein endothelial cells (HUVEC).Methods (1) HUVEC were randomly divided into 4 groups according to the following:blank group as control,normal group added 20％ normal sera of pregnant women,group PE added 20％ sera of severe preeclamptic patients,and group PE + Cat added 20％ sera of severe preeclamptic patients plus 3 x 103 U/ml catalase.After cultured for 24 hours,the injury morphology and APO2.7 expression of HUVEC were detected by transmission electron microscopy and flow cytometry respectively.(2) Under the real-time scanning by laser scanning confocal microscopy,HUVEC were randomly divided into 4 groups according to the following:control group added 100 μmol/L H2O2 as positive control,normal group,group PE,and group PE + Cat.HUVEC of each group was scanned for 120 seconds to determine levels of reactive oxidative species (ROS),calcium homeostasis,and mitochondria membrane potential.Results (1) Obvious injury morphology of HUVEC was observed in group PE,and it was obviously improved by catalase in group PE + Cat.Percentage of HUVEC expressed APO2.7 was (37.8 ± 1.1) ％ in group PE,which was significantly higher than (13.4 ± 1.1) ％ in blank group or (13.5 ± 1.5) ％ in normal group,but significantly lower than (19.2 ± 1.6) ％ in group PE + Cat (all P ＜ 0.01).(2) The fluorescence intensity curves of intracellular ROS and calcium showed slowly rising in group PE,but no obvious changes in normal group and PE + Cat.The values of ROS and calcium in group PE (12.0±1.3,4.1 ±0.7) were higher than those in normal group (1.1 ±0.4,0.6 ±0.4),but lower than those in group PE + Cat (1.5 ± 0.5,0.9 ± 0.5 ; all P ＜ 0.01).Conclusion The serum of severe preeclamptic patients caused oxidative damage on HUVEC by increasing intracellular ROS generation,calcium overload,and decreasing mitochondrial membrane potential.

Photo-induced inhibitory effect of titanium dioxide nanoparticles on a human epidermal squamous cell carcinoma cell line A431 / 中华皮肤科杂志

Objective To evaluate the inhibitory effect of photocatalytic titanium dioxide (TiO2)on the growth of a human epidermal squamous cell carcinoma cell line A431 and its mechanism.Methods Cultured A431 cells were classified into various groups to remain untreated (blank control group),be treated with different concentrations (100,200,300,400,500,600 mg/L) of TiO2 nanoparticles alone or in combination with ultraviolet (UV,main wavelength 253.7 nm,power 30 W,distance 30 cm,exposure duration 15 min) irradiation.After additional culture for different durations,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate cell growth,annexin V-fluorescein isothiocyanate/propidium iodide (PI) double staining to observe cell apoptosis,and Rho123 staining to determine mitochondrial transmembrane potential.Statistical analysis was carried out using SPSS 13.0 software.Analysis of variance (AOV),t test and Student-Newman-Keuls (SNK) test were performed to assess the differences in these parameters between these groups.Results The growth of A431 cells was inhibited by pretreatment with TiO2 nanoparticles followed by UV irradiation,and the inhibitory effect was enhanced as the dose of TiO2 nanoparticles increased.As AOV and SNK test showed,there were significant differences in the growth inhibition rate among A431 cells treated with different concentrations of TiO2 nanoparticles at the three time points (24,48 and 72 hours) after UV irradiation (n =6,F =21.54,77.56,20.27,respectively,all P ＜ 0.05).No statistical inhibition was observed in the growth of A431 cells treated with TiO2 nanoparticles alone compared with untreated A431 cells (all P ＞ 0.05).Photocatalytic TiO2 nanoparticles also induced the apoptosis but decreased the mitochondrial transmembrane potential in A431 cells.In detail,the apoptosis rate was 8.86％ ± 0.22％,11.72％ ± 0.29％ and 31.24％ ± 0.78％ in A431 cells treated with TiO2nanoparticles of 100,200,400 mg/L followed by UV irradiation,respectively,compared to 2.69％ ± 0.28％ in the blank control group (n =3,F =256.61,P ＜ 0.05).Decreased mitochondrial transmembrane potential (expressed as total fluorescence intensity) was observed in A431 cells treated with TiO2 nanoparticles of 100,200,400 mg/L followed by UV irradiation compared with blank control group (758.48 ± 15.42,676.60 ± 14.35,557.71 ± 13.12vs.2943.65 ± 70.26,F =208.57,P ＜ 0.05,n =3),and SNK test also revealed statistical differences between these groups.Conclusions TiO2 nanoparticles combined with UV can inhibit the growth of but induce the apoptosis in A431 cells,which may be associated with the reduction in mitochondrial transmembrane potential in A431 cells,while TiO2 nanoparticles alone show no inhibitory effect on the growth of A431 cells.

Effect of melatonin on ketamine-induced apoptosis in hippocampal neurons of fetal rats / 中华麻醉学杂志

Objective To investigate the effect of melatonin on ketamine-induced apoptosis in hippocampal neurons of fetal rats.Methods Sixteen to eighteen day pregnant Sprague Dawley rats were anesthetized.The fetal rats were obtained under sterile condition and decapitated.The hippocampal neurons were isolated and primary cultured for 5 days.The primary cultured neurons were randomly divided into 5 groups (n =6 each):control group (group C),ketamine group (group K),and 1.0,2.5 and 5.0 mmol/L melatonin groups (groups M1-3 respectively).Ketamine with the final concentration of 1 000 μmol/L was added to the culture medium and the neurons were incubated for 3 h in group K.In groups M1-3,1.0,2.5 and 5.0 mmol/L melatonin were added to the culture medium,respectively,at 60 min before the addition of ketamine,and the neurons were incubated for 3 h.While the equal volume of normal saline was added instead in group C.The neuronal viability during the developmental phase was assessed by MTT assay.The mitochondrial membrane potential (Ψm) was measured by flow cytometry.The expression of cAMP response element binding protein phosphorylation (p-CREB (Ser133)),Bcl-2,Bax,and cytochrome C was detected by Western blot.Results Compared with group C,the neuronal viability and Ψm were significantly decreased,and the expression of p-CREB and Bcl-2 was down-regulated,while the expression of Bax and cytochrome C was up-regulated in group K (P ＜ 0.05).Compared with group K,Ψm was significantly increased in groups M2 and M3,and the neuronal viability was significantly increased,the expression of Bcl-2 was up-regulated,while the expression of Bax and cytochrome C was down-regulated in groups M1-3 (P ＜ 0.05).Conclusion Melatonin can protect the hippocampal neurons of fetal rats from apoptosis triggered by ketamine via regulating the expression of Bcl-2 and Bax,stabilizing Ψm,inhibiting the release of cytochrome C from mitoehondria,and preventing apoptosome formation.

The death way and its mechanisms of pancreatic cancer PANC1 cells induced by pharmacologic ascorbic acid concentrations / 中华胰腺病杂志

ObjectiveTo investigate the biological effects and its mechanisms of ascorbic acid on pancreatic cancer PANC1 cells. Methods PANC1 cells were treated by ascorbic acid of different concentrations (0 ～40 mmol/L) for 24,48,72 hours.The proliferation of PANC1 cells was analyzed by MTT method; cell cycle and apoptosis were assessed by flow cytometry (FCM); inverted microscopy and transmission electron microscopy were used to observe cell morphology. The membrane potential of mitochondria were mearured by with JC-1 staining and FCM.Meanwhile,the changes of cell morphology and mitochondrial membrane potential induced by ascorbic acid after pretreatment with hydrogen peroxidescavenging enzyme (catalase) and red blood cells were also detected. Results Ascorbic acid in pharmacologic concentrations selectively inhibited the proliferation of PANC1 cells in a dose and time dependent manner.PANC1 cells were arrested in G2/M phase after treatment with 5 mmol/L ascorbic acid [ (32.55 ± 7.14)％ vs (22.00 ±1.27)％,t =5.808,P＜0.05],but there was no changes on apoptosis rate [ (1.98 ± 1.80)％ vs (1.09 ±0.16)％ ].Inverted microscope and transmission electron microscopy showed that oncosislike cell death of PANC1 cells was induced after treatment with ≥5 mmol/L ascorbic acid.Mitochondrial membrane potential of PANC1 cells was significantly lower than that of the control group in a dose dependent manner.The descent of mitochondrial membrane potential was significantly inhibited by pretreatment with catalase and red blood cells,and the degree of cell oncosis was attenuated.ConclusionsAscorbic acid significantly inhibited the proliferation of pancreatic cancer PANC1 cells in vitro.Ascorbic acid induced PANC1 cell oncosis,but not apoptosis.The possible mechanisms of inducing oncosis may be related to the descent of mitochondrial membrane potential.

Comparative analysis of nuclear proteomes in mitochondrial DNA-depleted A549 cells and their parental cells / 中国药理学与毒理学杂志

OBJECTIVE To investigate the nuclear proteomes in mitochondrial DNA (mtDNA)-depleted A549 cells (Rho0 cells) and their parental cells (Rho+ cells),and to learn more about the nuclear responses to mitochondrial dysfunction.METHODS The nuclear proteomes of Rho and Rho + cells were characterized by two dimensional electrophoresis (2-DE) and SELDI-TOF ProteinChip technologies,the differentially expressed protein- spots were identified by MALDI-TOF mass spectrum (MS),the nucleophosmin and P53 expression were detected by Western blotting assay,and the mitochondrial memhrane potential (MMP) was measured by the laser scanning confocal microscope.RESULTS 2-DE results showed 11 protein-spots were down-regulated and 21 protein-spots were up-regulated in Rho0 cell nuclei.SELDI-TOF MS analysis with NP20 ProteinChips revealed 4 protein-peaks decreased in Rho0 cell nuclei.One down-regulated protein-spot was identified as elF-6,and 4 up-regulated proteinspots were identified as nucleophosmin,SFRS1,SFRS3 and hnRNP G,respectively.The increased expression of nucleophosmin in Rho0 cells was verified by Western blotting.Based on the clues from proteomic analysis,P53 expression in Rho0 cells was higher than in Rho + cells,and MMP was consistent in Rho + and Rho0 cells.CONCLUSION mtDNA-depletion induces nuclear proteome alteration.Rho0 cells can be used as a model to study the crosstalk between mitochondrion and nucleus.

Effects of iodine excess on mitochondrial superoxide production and mitochondrial membrane potential in rat thyroid cell line cells / 中国地方病学杂志

Objective To investigate the effects of iodine excess on mitochondrial superoxide production and mitoehondrial membrane potential(△ψ)changes in Fisher rat thyroid cell line(FRTL)cells.Methods FRTL cells were treated with 10-4mol/L potassium iodine(KI),10 U/L thyrotropin(TSH),10-4 mol/L KI+10 U/L TSH respectively for 24 h.Effects on cell proliferation were assayed by methyl thiazolyl tetrazolium(MTT)colorimetric method.Changes of mitochondrial superoxide production and △ψ were measured by live cell imaging and spectrofluorometer using MitoSOX and rhodamine 123(rh123)respectively.Results Absorbance(A)in the KI group (0.794±0.144)showed a significant decline compared to the control group(1.000 ±0.183,P<0.05),whereas a significant elevation was observed in the TSH group(1.215±0.156,P<0.05).No significant differences was found between the KI+TSH group(1.025±0.254)and the control group(P>0.05),but the former was marked higher than the KI group(P<0.05).Compared to the control group(9.74±3.24).MitoSOX mean fluorescence intensity (MFI)in the KI and KI+TSH groups(18.16±6.57,13.33±2.92)were significantly increased(all P<0.05),which was a significant decline in the TSH group(6.64±2.15,P<0.05).MitoSOX MFI in the KI+TSH group was lower than the KI group(P<0.05).Rh123 MFI in the KI and KI+TSH groups(210 593±31 328,295 525±34 243)showed significant decline than the control group(407 824±37 198,all P<0.05).Compared with the KI group.the KI+TSH group pronouncedly attenuated the reduction of Rh 123 MFI(P<0.05).No significant differences of Rh 123 MFI were found between the TSH group(411 187 ± 72 852) and the control group(P > 0.05). Conclusion Iodine excess (10-4 mol/L KI) may lead to peroxide damage on the mitochondria of FRTL cells, and cell proliferation is inhibited. Combining treatment with 10 U/L TSH may attenuate mitochondrial peroxide damage and inhibition of cell proliferation caused by iodine excess.

Influence of Hydrogen Peroxide on Mitochondrial Membrane Potential and Superoxide Production in FRTL Cell / 天津医药

Objective:To investigate the effects of exogenous hydrogen peroxide(H_2O_2)on mitochondrial superoxide production and mitochondrial membrane potential changes(△ψ)in fisher rat thyroid cell line(FRTL).Methods:Following 1 mmol/L H_2O_2 exposure in FRTL cells for 10 min,30 min and 24 h,mitochondrial superoxide production was measured by living cell imaging and flow cytometry using MitoSOX.The mitochondrial membrane potential was assayed by spectrofluorimeter and fluorescent microscopy using rhodamine 123(rh123).The cell viability was detected by MTT colorimetric method.Morphological changes were observed by invert microscope.Apoptosis assay was performed by acridine orange staining.Results:Quantitative measurements of the mean intensities of MitoSOX demonstrated significant increase with 1 mmol/L H_2O_2 following 10 min,30 min and 24 h treatment in FRTL cells compared with that of control.Fluorescence intensity of rh123 and optical density of MTr were significantly decreased in FRTL ceils with 1 mmol/L H_2O_2 following 30 min and 24 h treatment(P ＜ 0.01).Under light microscope and fluorescence microscope the characteristic morphological features of programmed cell death,pickuosis,karyorrhexis,and cell shrinkage were observed after acridine orange staining.Conclusion:Acute and chronic exogenous H_2O_2 exposure cause oxide stress in FRTL cells,which result in the increase of mitochondrial superoxide production,△ψdecline,cell necrosis and apoptosis.

Effects of Honokiol on Proliferation and Apoptosis on U937 Cells / 中山大学学报(医学科学版)

[Objective] To investigate the anti-proliferative and apoptosis effect induced by Honokiol (HNK) on human myeloid leukemia cell line U937 cells in vitro.[Methods] After treated with different concentration of HNK,Hoechst33342 fluorescent staining was used to detect cell apoptosis;the growth inhibition ration of U937 cells and PBMCs were analyzed by MTT assay;the apoptosis ration was detected by flow cytometry;mitochondrial membrane potential was explored by rhodamine 123 stain;Caspase3/7 protein activity kit was used to test the Caspase3/7 activity;the Caspase-3 and Caspase-7 mRNA levels were detected by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR).[Results] Honokiol could significantly inhibit the proliferation of U937 cells in terms of the indexes of IC50/U937 11.8 μg/mL and IC50/PBMCs 40.3 μg/mL,and the anti-proliferative effect was in a time and concentration dependent manner;Flow cytometry analysis manifested that Honokiol could induce U937cells apoptosis by Annexin V/PI double Annexin V/PI fluorescein stain;Honokiol significantly inhibited the mitochondrial membrane potential of U937 cells and enhanced the ability of Caspase3/7 and the mRNA expression levels,but not the PBMCs.[Conclusion] HNK can inhibit U937 cells proliferation and induce cells apoptosis via activating Caspase 3/7.

Effect of arsenic trioxide on apoptosis and mitochondrial transmembrane potential of Marek disease cancer cells / 中国地方病学杂志

Objective To investigate the mechanism of apoptosis induced by arsenic trioxide(As2O3) on MDCC-MSB1 cancer cell line in vitro. Methods MDCC-MSB1 cells were divided into 4 groups, treated with 0 (control group), 2, 4 or 8 μmoL/L of As2O3. At 48 h following the treatment, MTr assay was applied to detect the inhibitory effect of As2O3 on MDCC-MSB1. Morphological changes of apoptosis were observed by fluorescence microscopy. Apoptosis was examined by DNA Ladder. Changes of mitochondrial transmembrane potential were examined by Rhodamin 123 dye and flow cytometry. Results Inhibition ratio was 0, (5.34±3.02)%, (10.78± 0.55)% and (20.02±3.24)% respectively, along with the dosages of As2O3, the differences between the groups were statistically significant(P<0.01). Morphologic changes of apoptosis were observed by DNA ladder of agarose gel electrophoresis. Apoptosis rates were significandy increased from 3.200±0.459, 11.543±0.391, 17.206±0.636 to 21.343±0.620, and the differences between the groups were statistically significant(P<0.01). DNA ladder of experimental group was detected, Intact cell membrane, and mitochondrial transmembrane potential Pl-/Rh123- decreased apoptotic cells percentage was significantly increased from (1.06±0.14)%, (4.63±0.04)%, (9.62±0.07)% to (10.39±0.10)%, respective to doses of 0, 2,4, and 8 μmol of As2O3. The differences between the groups were statistically significant(P<0.01). Conclusions As2O3 can induce apoptosis in MDCC-MSB1 cells by decreasing the mitochondrial transmembrane potential.

Effects of telmisartan on mitochondrial membrane potential and cardiomyocyte apoptosis in type 1 diabetic rats / 解放军医学杂志

Objective To investigate the effect of telmisartan on mitochondrial membrane potential and cardiomyocyte apoptosis of rats with type 1 diabetes.Methods Type 1 diabetic rat model was reproduced by intraperitoneal injection of streptozotocin to adult male Wistar rats.Sixteen diabetic rats were randomly divided into two groups (8 each):DM group and T group (rats were treated with telmisartan).Another 8 normal rats served as control (Con group).After 12 weeks,the levels of reactive oxygen species (ROS) were detected by fluorescent probe DCFH-DA,and the changes in mitochondrial membrane potential (?m) was detected by fluorescent probe JC-1.Cardiomyocyte apoptosis was evaluated by TUNEL.Results The levels of blood glucose were higher and body weight was lower in both DM and T group than in Con group at 7th day and 12th week after streptozotocin injection (P