Membrane Potentials Across Hybrid Charged Mosaic Membrane in Organic Solutions

Membrane potentials across hybrid charged mosaic membrane in organic solutions were measured. Equilibrium swelling degree （SD） and fixed charge density in both organic solutions and water were also determined. Ethylene glycol, ethanol, n-propanol and glycerol were used as organic solutes; meanwhile 0.001mol-dm^-3 aqueous KCl solution was utilized as a strong electrolyte to measure the electrical difference. Equilibrium swelling degree indicated that it could be affected by the density of organic solutes; while it enhanced with the increasing density of these solutes. The measurement of fixed charge density showed that the membrane had the maximal absolute value in water among these solvents whether for cationic or anionic groups; the difference of dielectric constant between the water and the organic solutes might be responsible for these change trends. It was confirmed that membrane potentials increased with both the increasing concentration of the organic solutions and the elevated pH values. These results demonstrated that the characteristics of the hybrid charged mosaic membrane could be highly impacted by the properties of the organic solutes. A theoretical modal for charged membranes in ternary ion systems of weak electrolyte can be used to explain the above-mentioned phenomena.

Arsenic trioxide induces multiple myeloma cell apoptosis via disruption of mitochondrial transmembrane potentials and activation of caspase-3

OBJECTIVE: To investigate the response of multiple myeloma (MM) cells to arsenic trioxide (As2O3) and their possible mechanisms. METHODS: Two MM-derived cell lines RPMI8226 and U266 cells were used as in vitro models. Cell apoptosis was assessed by morphology, flow cytometry, and DNA gel electrophoresis. Mitochondrial transmembrane potentials (delta psi m) were evaluated by measuring cellular Rhodamine 123 staining intensity. Protein expression was analyzed using Western blot. RESULTS: Zero point one to 0.5 mumol/L As2O3 inhibited cell proliferation and 2.0 mumol/L As2O3 induced cell apoptosis, while 1.0 mumol/L As2O3 inhibited proliferation with a weak degree of apoptosis induction in RPMI8226 and U266 cell lines. As2O3-induced apoptosis was accompanied by mitochondrial transmembrane potentials (delta psi m) collapse and caspase-3 activation in the presence of intact membrane. Glutathione depleter buthionine sulfoximine enhanced, while disulfide bond-reducing agent dithiothreitol partially antagonized As2O3-induced delta psi m collapse and apoptosis in MM cells. All-trans retinoic acid (ATRA) could also induce apoptosis in RPMI8226 cells, but it did not show any cooperative effects with As2O3. CONCLUSION: As2O3 exerts apoptosis-inducing and growth-inhibiting effects on MM cells, and mitochondrium is a pivotal and common target of As2O3 for apoptosis induction.

Immp2l Mutation Induces Mitochondrial Membrane Depolarization and Complex Ⅲ Activity Suppression after Middle Cerebral Artery Occlusion in Mice

Objective We previously reported that mutations in inner mitochondrial membrane peptidase 2-like(Immp2l)increase infarct volume,enhance superoxide production,and suppress mitochondrial respiration after transient cerebral focal ischemia and reperfusion injury.The present study investigated the impact of heterozygous Immp2l mutation on mitochondria function after ischemia and reperfusion injury in mice.Methods Mice were subjected to middle cerebral artery occlusion for 1 h followed by 0,1,5,and 24 h of reperfusion.The effects of Immp2l^(+/−)on mitochondrial membrane potential,mitochondrial respiratory complex III activity,caspase-3,and apoptosis-inducing factor(AIF)translocation were examined.Results Immp2l^(+/−)increased ischemic brain damage and the number of TUNEL-positive cells compared with wild-type mice.Immp2l^(+/−)led to mitochondrial damage,mitochondrial membrane potential depolarization,mitochondrial respiratory complex III activity suppression,caspase-3 activation,and AIF nuclear translocation.Conclusion The adverse impact of Immp2l^(+/−)on the brain after ischemia and reperfusion might be related to mitochondrial damage that involves depolarization of the mitochondrial membrane potential,inhibition of the mitochondrial respiratory complex III,and activation of mitochondria-mediated cell death pathways.These results suggest that patients with stroke carrying Immp2l^(+/−)might have worse and more severe infarcts,followed by a worse prognosis than those without Immp2l mutations.

Understanding mechanisms for differential salinity tissue tolerance between quinoa and spinach:Zooming on ROS-inducible ion channels

Soil salinity is a wo rldwide issue and a major threat to global food security.Salinity tolerance is a complex mechanism that is conferred by numerous molecular,physiological,and biochemical traits.Of critical importance are plant's ability to regulate redox balance without compromising reactive oxygen species(ROS)signalling and maintain cytosolic ion homeostasis.In this study,the mechanistic basis of H^(+) retention ability in leaf mesophyll(an important but highly sensitive plant tissue)was compared between halophytic quinoa and glycophytic spinach.Phenotypic data showed quinoa outperformed spinach under 100 to 500 mmol L^(-1) NaCl salinity.The major difference behind this differential salinity sensitivity was a differential H^(+) uptake in leaf mesophyll.Electrophysiological and molecular experiments revealed that a superior ability of mesophyll H^(+) retention in quinoa was conferred by three complementary mechanisms:(ⅰ)an intrinsically lower H^(+)-ATPase activity in quinoa(potentially as an energy saving strategy);(ⅱ)reduced sensitivity of H^(+) transporters to ROS;and(ⅲ)increased sensitivity of ROS-inducible Ca^(2+)-permeable channels,Moreover,the sensitivity of H^(+)-transport systems to ROS was further examined in NaCl-acclimated quinoa and spinach plants.The key factors differentiating between H^(+) retention in acclimated leaf mesophyll was associated with the reduced sensitivity and gene expression of H^(+)-permeable outward rectifying channel(GORK),Arabidopsis potassium transporter 1(AKT1),and high affinity potassium transporter 5(HAK5)to additional NaCl and ROS stress,along with the upregulation of ROS scavenging system.Taken together,our results showed that the tissue-specific and ROS-specific regulation of H^(+) retention are important for conferring salinity tolerant at least in the halophyte quinoa.

JTE-522-induced apoptosis in human gastric adenocarinoma cell line AGS cells by caspase activation accompanying cytochrome C release,membrane translocation of Bax and loss of mitochondrial membrane potential

AIM: To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (Deltapsim). METHODS: Cell culture, cell counting, ELISA assay, TUNEL, flow cytometry, Western blot and fluorometric assay were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanism. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Caspases 8 and 9 were activated during apoptosis as judged by the appearance of cleavage products from procaspase and the caspase activities to cleave specific fluorogenic substrates. To elucidate whether the activation of caspases 8 and 9 was required for the apoptosis induction, we examined the effect of caspase-specific inhibitors on apoptosis. The results showed that caspase inhibitors significantly inhibited the apoptosis induced by JTE-522. In addition, the membrane translocation of Bax and cytosolic release of cytochrome C accompanying with the decrease of the uptake of Rhodamin 123, were detected at an early stage of apoptosis. Furthermore, Bax translocation, cytochrome C release, and caspase 9 activation were blocked by Z-VAD.fmk and Z-IETD-CHO. CONCLUSION: The present data indicate a crucial association between activation of caspases 8, 9, cytochrome C release, membrane translocation of Bax, loss of Deltapsim and JTE-522-induced apoptosis in AGS cells.

Berbamine induces apoptosis in human hepatoma cell line SMMC7721 by loss in mitochondrial transmembrane potential and caspase activation

Objective： To investigate the effect ofberbamine on human hepatoma cell line SMMC7721. Methods： The effects of 24 h and 48 h incubation with different concentrations （0-64 μg/ml） of the berbamine on SMMC7721 cells were evaluated using 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide （MTT） assay. Hoechst 33258 staining was conducted to distinguish the apoptotic cell, and the appearance of sub-G1 stage was determined by PI （propidium iodide） staining, the percentage of apoptotic cell was determined by flow cytometry following annexin V/PI staining. Flow cytometry was performed to analyze the cell cycle distribution and the mitochondrial membrane potential （△ψm）, the expression of activated caspase3 and caspase9 was analyzed by Western-blot. Results： The proliferation of SMMC7721 was decreased after treatment with berbamine in a dose- and time-dependent manner. Berbamine could induce apoptosis in SMMC7721 cells and could cause cell cycle arrest in G0/G1 phase, to induce loss of mitochondrial membrane potential （AVm） and activate caspase3 and caspase9. Berbamine-induced apoptosis could be blocked by the broad caspase inhibitor z-VAD-fmk. Conclusion： Berbamine exerts antiproliferative effects on human hepatocellular carcinoma SMMC7721 cells. The anticancer activity of berbamine could be attributed partly to its inhibition of cell proliferation and induction of apoptosis in cancer cells through loss in mitochondrial transmembrane potential and caspase activation.

Effect of solanine on the membrane potential of mitochondria in HepG_2 cells and [Ca^(2+)]i in the cells

AIM： To observe the effect of solanine on the membrane potential of mitochondria in HepG2 cells and [Ca^2＋]i in the cells, and to uncover the mechanism by which solanine induces apoptosis.METHODS： HepG2 cells were double stained with AO/EB, and morphological changes of the cells were observed using laser confocal scanning microscopy （LCSM）. HepG2 cells were stained with TMRE, and change in the membrane potential of mitochondria in the cells were observed using LCSM. HepG2 cells were double stained with Fluo-3/AM, and change of [Ca^2＋]i in the cells were observed using LCSM. HepG2 cells were double stained with TMRE and Fluo-3/AM, and both the change in membrane potential of mitochondria and that of [Ca^2＋]i in the cells were observed using LCSM.RESULTS： Cells in treated groups showed typical signs of apoptosis. Staining with TMRE showed that solanine could lower membrane potential; staining with Fluo-3/AM showed that solanine could increase the concentration of Ca^2＋ in tumor cells; and those of double staining with TMRE and Fluo-3/AM showed that solanine could increase the concentration of Ca^2＋ in the cells at the same time as it lowered the membrane potential of mitochondria.CONCLUSION： Solanine opens up the PT channels in the membrane by lowering the membrane potential, leading to Ca^2＋ being transported down its concentration gradient, which in turn leads to the rise of the concentration of Ca^2＋ in the cell, turning on the mechanism for apoptosis.

Changes of plasma membrane ATPase activity, membrane potential and transmembrane proton gradient in Kandelia candel and Avicennia marina seedlings with various salinities

The salt-secreting mangrove, Avicennia marina, and non-salt-secreting mangrove, Kandelia candel were cultivated in sand with various salinities(0‰, 10‰, 20‰, 30‰, 40‰) for 60 d. Plasma membrane vesicles of high-purity in leaves and roots of A.marina and K. candel seedlings were obtained by two-phase partitioning. The function of the plasma membranes, the activity of ATPase, membrane potential and transmembrane proton gradient, at various salinities were investigated. The results showed that within a certain range of salinity(A. marina and roots of K. candel: 0—30‰; leaves of K.candel: 0—20‰), the activity of ATPase increased with increasing salinity, while high salinity(above 30‰ or 20‰) inhibited ATPase activity. In comparison with A. marina, K. candel appeared to be more sensitive to salinity. The dynamics of membrane potential and transmembrane proton gradient in leaves and roots of A. marina and K. candel seedlings were similar to that of ATPase. When treated directly by NaCl all the indexes were inhibited markedly: there was a little increase within 0—10‰(K. candel) or 0—20‰(A. marina) followed by sharp declining. It indicated that the structure and function of plasma membrane was damaged severely.

Electrical stimulation modulates injury potentials in rats after spinal cord injury

An injury potential is the direct current potential difference between the site of spinal cord injury and the healthy nerves. Its initial amplitude is a significant indicator of the severity of spinal cord injury, and many cations, such as sodium and calcium, account for the major portion of injury potentials. This injury potential, as wel as injury current, can be modulated by direct current field stimulation;however, the appropriate parameters of the electrical field are hard to define. In this paper, injury potential is used as a parameter to adjust the intensity of electrical stimulation. Injury potential could be modulated to slightly above 0 mV （as the anode-centered group） by placing the anodes at the site of the injured spinal cord and the cathodes at the rostral and caudal sections, or around-70 mV, which is resting membrane potential （as the cathode-centered group） by reversing the polarity of electrodes in the anode-centered group. In addition, rats receiving no electrical stimulation were used as the control group. Results showed that the absolute value of the injury potentials acquired after 30 minutes of electrical stimulation was higher than the control group rats and much lower than the initial absolute value, whether the anodes or the cathodes were placed at the site of injury. This phenomenon il ustrates that by changing the polarity of the electrical field, electrical stimulation can effectively modulate the injury potentials in rats after spinal cord injury. This is also beneficial for the spontaneous repair of the cel membrane and the reduction of cation influx.

Effects of NaCI and Ca^(2+) on Membrane Potential of Epidermal Cells of Maize Roots

The effects of salt-stress on plants involve not only the water stress caused by low osmotic pressure, but also the toxicity of excess Na^＋. A large amount of Na^＋ entering cells would reduce K^＋ uptake, which leads to an imbalance of K：Na ratio in cells. One of the reasons for the reduced K^＋-uptake is the closure of K^＋-channel which is controlled by membrane potential. Calcium is usually applied to improve the growth of plants on saline soils and shows positive influence in the integrality of cell membrane. This study applied glass microelectrode technique to monitoring the NaCl-induced changes of membrane potential of root epidermal cells of maize （Zea mays L., Denghai 11） seedlings at NaCl concentrations of 0, 8, 20, 50, 100, 200 mmol L^-1, respectively. The effect of Ca^2＋ on the changes of membrane potential caused by NaCl was also studied. The results showed that： NaCl caused cell membrane depolarization. The depolarization became greater and faster with increasing of NaCl concentration. Moreover, the extent of depolarization was positively correlated with NaCl concentration. The addition of calcium postponed the depolarization, and decreased the degree of depolarization caused by NaCl. High NaCl concentration leads to depolarization of maize root cell membrane, which can partly be counteracted by calcium.

The Measurement of Membrane Potential and NO3 Activity in Root Cells Using Ion-Selective Microelectrodes

Remobilisation of nitrate in plants, especially in vacuole of plant, is mostly related to the qua- lity of agricultural products and the high nitrogen use efficiency in plants. Ion-selective microelectrodes offer a non-destructive and non-interruptive method to measure NO 3 gradients and electric potential differences across both the plasma membrane and tonoplast. Thus, a double-barrelled microelectrode backfilled with a membrane sensor for NO 3 embedded in poly vinyl chloride (PVC) can record the NO 3 activity in cytoplasm and vacuole of a cell. This paper presented how to make this kind of microelectrode and how to do the intracellular measurements on intact plants. Our result showed that nitrate activity was about 2.7 mmol L 1 in cytoplasm while 70 mmol L 1 in vacuole, which implicated that vacuole was a pool of nitrate in plants.

Interventional effect of phycocyanin on mitochondrial membrane potential and activity of PC12 cells after hypoxia/reoxygenation

BACKGROUND： Phycocyanin can relieve decrease of mitochondrial membrane potential through reducing production of active oxygen so as to protect neurons after hypoxia/reoxygenation. OBJECTIVE： To observe the effect of phycocyanin on activity of PC12 cells and mitochondrial membrane potential after hypoxia/reoxygenation. DESIGN： Randomized controlled study SETTING ： Cerebrovascular Disease Institute of Affiliated Hospital, Medical College of Qingdao University MATERIALS： The experiment was carried out at the Key Laboratory of Prevention and Cure for cerebropathia in Shandong Province from October to December 2005. PC12 cells, rat chromaffin tumor cells, were provided by Storage Center of Wuhan University; phycocyanin was provided by Ocean Institute of Academia Sinica; Thiazoyl blue tetrazolium bromide （MTT） and rhodamine 123 were purchased from Sigma Company, USA; RPMI-1640 medium, fetal bovine serum and equine serum were purchased from Gibco Company, USA. METHODS： ① Culture of PC12 cells： PC12 cells were put into RPMI-1640 medium which contained 100 g/L heat inactivation equine serum and 0.05 volume fraction of fetal bovine serum and incubated in CO2 incubator at 37℃. Number of cells was regulated to 4 × 10^5 L 1, and cells were inoculated at 96-well culture plate. The final volume was 100μL. ② Model establishing and grouping： Cultured PC12 cells were randomly divided into three groups： phycocyanin group, model control group and non-hypoxia group. At 24 hours before hypoxia, culture solution in phycocyanin group was added with phycocyanin so as to make sure the final concentration of 3 g/L , but cells in model control group did not add with phycocyanin. Cells in non-hypoxia group were also randomly divided into adding phycocyanin group （the final concentration of 3 g/L） and non-adding phycocyanin group. Cells in model control group and phycocyanin group were cultured with hypoxia for 1 hour and reoxygenation for 1, 2 and 3 hours; meanwhile, cells in non-hypoxia group were cultured with oxygen and were measured at 1 hour after hypoxia/reoxygenation. ③ Detecting items： At 1, 2 and 3 hours after reoxygenation, absorbance （A value） of PC12 cells was measured with MTT technique so as to observe activity and quantity of cells. Fluorescence intensity of PC12 cells marked by rhodamine 123 was measured with confocal microscope in order to observe changes of mitochondrial membrane potential. MAEN OUTCOME MEASURES： Comparisons between quantity and activity of PC12 cells and mitochondria membrane potential at 1, 2 and 3 hours after reoxygenation. RESULTS： ① Effect of phycocyanin on quantity and activity of PC12 cells： A value was 0.924±0.027 in adding phycocyanin group and 0.924±0.033 in non-adding phycocyanin group. A value was lower in model control group and phycocyanin group than that in non-hypoxia group at 1, 2 and 3 hours after reoxygenation （0.817±0.053, 0.838±0.037, 0.875±0.029; 0.842±0.029, 0.872±0.025, 0.906±0.023, P 〈 0.05）. A value was higher in phycocyanin group than that in model control group at 1, 2 and 3 after culture （P 〈 0.05）. With culture time being longer, A value was increased gradually in phycocyanin group and model control group after reoxygenation （P 〈 0.05）. ~ Effect of phycocyanin on mitochondrial membrane potential of PC12 cells： Fluorescence intensity was 2.967±0.253 in adding phycocyanin group and 2.962±0.294 in non-adding phycocyanin group. Fluorescence intensity was lower in model control group and phycocyanin group than that in non-hypoxia group at 1, 2 and 3 hours after hypoxia/reoxygenation （1.899±0.397, 2.119±0.414, 2.287±0.402; 2.191±0.377, 2.264±0.359, 2.436±0.471, P 〈 0.05）; but it was higher in phycocyanin group than that in model control group at 1, 2 and 3 after reoxygenation （P 〈 0.05）. With culture time being longer, fluorescence intensity was increased gradually in phycocyanin group and model control group after reoxygenation （P 〈 0.05）. CONCLUSION： Phycocyanin and reoxygenation can protect PC12 cells after hypoxia injury through increasing mitochondrial membrane potential and cellular activity, and the effect is improved gradually with prolonging time of reoxygenation.

Ammonium Effects on Nitrate Uptake by Roots of Upland and Paddy Rice Seedlings Related to Membrane Potential Differences

Nitrate uptake characteristics and ammonium effects on nitrate uptake were compared between upland rice （Brazilian upland rice） and paddy rice （Wuyujing 3 and Yangdao 6） through the glass microelectrode technique and the concentration gradient method of uptake kinetics.Results indicated that nitrate uptake by rice seedlings and ammonium effects were depending on membrane potential of root cells.And upland rice and paddy rice presented obviously different responses.For all cultivars,the nitrate treatments induced rapid depolarization and then slow repolarization of membrane potential in root epidermal cells,and even hyperpolarization was observed when nitrate concentration was low.The membrane potential of epidermal cells in Brazilian upland rice roots was larger and its response to NO3- was bigger than those of two paddy rice cultivars.Depolarization of membrane potential was amplified when ammonium was simultaneously added with nitrate into the measure medium,but repolarization was reduced,even disappeared.Brazilian upland rice seedlings had high Vmax of nitrate uptake and low Km,furthermore,Vmax and Km were little affected by ammonium,but Vmax of Wuyujing 3 was reduced significantly.Therefore,inhibition of NH4＋ differed obviously between upland rice and paddy rice.

Effects of La^(3+)on H^+ Transmembrane Gradient and Membrane Potential in Rice Seedling Roots

The effects of LaCl 3 on membrane potential and transmembrane proton gradient for rice ( Oryza sativa ) seedling roots were studied. Highly purified plasma membrane was isolated by aqueous two phase partitioning method. Both the gradient of transmembrane proton and membrane potential were stimulated by certain low concentration of LaCl 3 and depressed by high concentration of LaCl 3. The optimal concentration of La 3+ is around 40～60 μmol·L -1 for transmembrane proton gradient and membrane potential. It shows that La 3+ can influence the generations and maintenances of membrane potential and transmembrane proton gradient in rice seedling roots.

K^+ Channels and Their Effects on Membrane Potential in Rat Bronchial Smooth Muscle Cells

In order to investigate the K+ channels and their effects on resting membrane potential (Em) and excitability in rat bronchial smooth muscle cells (BSMCs), the components of outward K+ channel currents and the effects of K+ channels on Em and tension in rat bronchial smooth muscle were observed by using standard whole-cell recording of patch clamp and isometric tension recording techniques. The results showed that under resting conditions, total outward K+ channel currents in freshly isolated BSMCs were unaffected by ATP-sensitive K+ channel blocker. There were two types of K+ currents: voltage-dependent delayed rectifier K+ channel (Kv) and large conductance calcium-activated K+ channel (BKc.) currents. 1 mmol/L 4-aminopyridine (4-AP, an inhibitor of Kv) caused a significant depolarization (from -8. 7±5. 9 mV to -25. 4±3. 1 mV, n=18, P<0. 001). In contrast, 1 mmol/L tetraethylammonium (TEA, an inhibitor of BKc.) had no significant effect on Em (from -37. 6±4. 8 mV to -36. 8±4.1mV, n=12, P>0. 05). 4-AP caused a concentration-dependent contraction in resting bronchial strips. TEA had no effect on resting tension, but application of 5 mmol/L TEA resulted in a left shift with bigger pD2(the negative logarithm of the drug concentration causing 50% of maximal effect) (from 6. 27±0. 38 to 6. 89±0. 54, n= 10, P<0. 05) in the concentration-effect curve of endothine-1, and a right shift with smaller pD2(from 8. 10±0. 23 to 7. 69±0. 08, n=10, P<0. 05) in the concentration-effect curve of isoprenaline. It was suggested that in rat BSMCs there may be two types of K+ channels, Kv and BKca, which serve distinct roles. Kv participates in the control of resting Em and tension. BKca is involved in the regulation of relaxation or contraction associated with excitation.

Mitochondrial membrane stabilization by Angelica sinensis polysaccharide in murine aplastic anemia

In order to investigate the mechanism of mitochondrial membrane stabilization by Angelica sinensis polysaccharide (ASP) in murine aplastic anemia (AA).ICR mice were randomly divided into control, AA and ASP-treated groups. The AA group mice were treated with 60Coγand intraperitoneal injections of cyclophosphamide and chloramphenicol. The control animals were treated with lead shielding irradiation and saline injection. The treated AA mice were fed with ASP for 2 wk. Mitochondrial ultrastructure of the bone marrow was observed by transmission electron microscopy, and the transmembrane potential of bone marrow-nucleated cells (BMNC)was examined by fluorescence spectrophotometry. The Cox and MDH contents of the medium were also studied in the three groups.The mitochondrial number and transmembrane potential of BMNC in the bone marrow decreased in the AA group as compared to the control group, but improved in the ASP-treated group as compared to the AA group. Complete mitochondrial cleavage in the ASP-treated group was significantly delayed (P<0.05) as compared to the AA group. We conclude that ASP might improve mitochondrial membrane stabilization, and suppress the downregulation of transmembrane potential and apoptosis of BMNC in AA.

Study on the in vitro anti ovarian cancer effect and mechanism of quinazoline derivative(N111)

Objective:To study the anti-ovarian cancer effect and mechanism of Quinazoline derivative(N111)in vitro;Method:Using an online database to predict the therapeutic targets of N111 for ovarian cancer,and conducting biological functional analysis of the therapeutic targets.The experiment was divided into N111 treatment group(N111 compound group),positive control group(cisplatin group),and negative control group(DMSO group);After grouping,MTT assay was used to detect cell proliferation;Morphological observation was used to observe changes in cell morphology;JC-1 and DCFH-DA probes were used to detect the changes of mitochondrial Membrane potential and intracellular reactive oxygen species;PI,Annexin V-FITC,and DAPI staining were used to detect cell cycle arrest and apoptosis;Clone formation experiments and scratch tests were conducted to detect the cell's ability to form clones and migrate;Western blot method was used to detect the expression level of related proteins.Result:The biological function research results show that the biological function of N111 anti ovarian cancer target protein suggests that the target function aggregates human diseases,inflammation,tumors,and other aspects.Compared with the control group,N111 has a significant inhibitory effect on the proliferation of ovarian cancer cells(IC50=14.62 mmol/L)(P<0.0001);In a concentration dependent manner,it inhibited the formation and migration of single cell colonies,and induced the disorder of mitochondrial Membrane potential,ROS and cell cycle arrest in S phase(P<0.0001);As the concentration of N111 treatment increased,the expression levels of Bcl2,Caspase 3,P-AKT,and SHIP2 decreased,while the expression levels of AKT remained unchanged.The expression levels of Bax and Cleared Caspase 3 increased(P<0.0001).Conclusion:Compound N111 inhibits SHIP2,promotes ROS level disorder,weakens the activation of AKT signaling pathway,and thus inhibits the proliferation,migration,and clone formation of tumor cell A2780,inducing cell apoptosis.

Ethanol induced mitochondria injury and permeability transition pore opening: Role of mitochondria in alcoholic liver disease

AIM: To observe changes of mitochondria and investigate the effect of ethanol on mitochondrial perme- ability transition pore (PTP), mitochondrial membrane potential (MMP, ΔΨm) and intracellular calcium concentration in hepatocytes by establishing an animal model of alcoholic liver disease (ALD). METHODS: Fourty adult male Wistar rats were randomly divided into two groups, the model group (20) was administered alcohol intragastrically plus an Oliver oil diet to establish an ALD model, and the control group (20) was given an equal amount of normal saline. The ultramicrostructural changes of mitochondria were observed under electron microscopy. Mitochondria of liver was extracted, and patency of PTP, mitochondrial membrane potential (ΔΨm), mitochondrial mass and intracellular calcium concentration of isolated hepacytes were detected by flow cytometry using rhodamine123 (Rh123), Nonyl-Acridine Orange and calcium fluorescent probe Fluo-3/AM, respectively. RESULTS: Membrane and cristae were broken or disappeared in mitochondria in different shapes under electron microscopy. Some mitochondria showed U shape or megamitochondrion. In the model group, liver mitochondria PTP was broken, and mitochondria swelled, the absorbance at 450 nm, A540 decreased (0.0136 ± 0.0025 vs 0.0321 ± 0.0013, model vs control, P < 0.01); mitochondria transmembrane potential (239.4638 ± 12.7263 vs 377.5850 ± 16.8119, P < 0.01) was lowered; mitochondrial mass (17.4350 ± 1.9880 vs 31.6738 ± 3.4930, P < 0.01); and [Ca2+]i was increased in liver cells (7.0020 ± 0.5008 vs 10.2050 ± 0.4701, P < 0.01).CONCLUSION: Chronic alcohol intake might lead to broken mitochondria PTP, decreased mitochondria membrane potential and injury, and elevated intracellular Ca2+ production. Ethanol-induced chondriosome injury may be an important mechanism of alcoholic diseases.

Liriodendrin protects SH-SY5Y cells from dopamine-induced cytotoxicity

Aim To investigate the effect of liriodendrin, an extract from Fraxinus sielboldiana blume belonging to the Oleaceae family, on dopamine-induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Methods Cell viability was processed when treated with 50 μmol·L^-1 of dopamine for 24 h by MTT assay. Early apoptosis, late apoptosis/necrosis were analyzed by flow cytometry using Annexin V-FITC and propidium iodide （PI） double-staining, respectively. Generation of reactive oxygen species （ROS） was assessed by DCFH-DA, an oxidation-sensitive fluorescent probe. To evaluate mitochondrion membrane potential （Δψm） using flow cytometry with the fluorescent dye Rhodamine 123. The transcriptional level of P53 was studied using RT- PCR. Results The dopamine-induced loss of cell viability was significantly attenuated by liriodendrin treatment at the concentration of 10^-8, 10^-7, 10^-6, 10^-5 and 10^-4 mol·L^-1. The protective effects of liriodendrin （10^-7, 10^-6 and 10^-5 mol·L^-1） on dopamine-induced cytotoxicity may be ascribed to its anti-oxidative properties by reducing ROS level and anti-apoptotic effect via protection of Δψm. In addition, the effect of liriodendrin may involve the P53 pathway in apoptosis. Conclusion Liriodendrin may provide a useful therapeutic strategy for the treatment of neurodegenerative diseases such as Parkinson＇s disease （PD）

Lycium barbarum polysaccharides protects retinal ganglion cells against oxidative stress injury

The accumulation of excessive reactive oxygen species can exacerbate any injury of retinal tissue because free radicals can trigger lipid peroxidation,protein damage and DNA fragmentation.Increased oxidative stress is associated with the common pathological process of many eye diseases,such as glaucoma,diabetic retinopathy and ischemic optic neuropathy.Many studies have demonstrated that Lycium barbarum polysaccharides(LBP)protects against oxidative injury in numerous cells and tissues.For the model of hypoxia we used cultured retinal ganglion cells and induced hypoxia by incubating with 200μM cobalt chloride(CoCl2)for 24 hours.To investigate the protective effect of LBP and its mechanism of action against oxidative stress injury,the retinal tissue was pretreated with 0.5 mg/mL LBP for 24 hours.The results of flow cytometric analysis showed LBP could effectively reduce the CoCl2-induced retinal ganglion cell apoptosis,inhibited the generation of reactive oxygen species and the reduction of mitochondrial membrane potential.These findings suggested that LBP could protect retinal ganglion cells from CoCl2-induced apoptosis by reducing mitochondrial membrane potential and reactive oxygen species.