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.

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.

Parkinson’s disease-associated VPS35 mutant reduces mitochondrial membrane potential and impairs PINK1/Parkinmediated mitophagy

Background:Mitochondrial dysfunction plays a prominent role in the pathogenesis of Parkinson’s disease(PD),and several genes linked to familial PD,including PINK1(encoding PTEN-induced putative kinase 1[PINK1])and PARK2(encoding the E3 ubiquitin ligase Parkin),are directly involved in processes such as mitophagy that maintain mitochondrial health.The dominant p.D620N variant of vacuolar protein sorting 35 ortholog(VPS35)gene is also associated with familial PD but has not been functionally connected to PINK1 and PARK2.Methods:To better mimic and study the patient situation,we used CRISPR-Cas9 to generate heterozygous human SH-SY5Y cells carrying the PD-associated D620N variant of VPS35.These cells were treated with a protonophore carbonyl cyanide m-chlorophenylhydrazone(CCCP)to induce the PINK1/Parkin-mediated mitophagy,which was assessed using biochemical and microscopy approaches.Results:Mitochondria in the VPS35-D620N cells exhibited reduced mitochondrial membrane potential and appeared to already be damaged at steady state.As a result,the mitochondria of these cells were desensitized to the CCCPinduced collapse in mitochondrial potential,as they displayed altered fragmentation and were unable to accumulate PINK1 at their surface upon this insult.Consequently,Parkin recruitment to the cell surface was inhibited and initiation of the PINK1/Parkin-dependent mitophagy was impaired.Conclusion:Our findings extend the pool of evidence that the p.D620N mutation of VPS35 causes mitochondrial dysfunction and suggest a converging pathogenic mechanism among VPS35,PINK1 and Parkin in PD.

Correlations of Sperm Mitochondrial Membrane Potential with Semen Parameters and Male Obesity

Background:To investigate the correlations of sperm mitochondrial membrane potential(MMP)with semen parameters and body mass index(BMI)in males with obesity.Methods:Semen samples were obtained by masturbation after 3-7 days of sexual abstinence from males who visited semen collect room of Shanghai Ninth People’s Hospital.Conventional semen analyses were performed by computer-aided sperm analysis(CASA),and sperm morphology was analyzed by modified Papanicolaou staining.Spermatozoa were stained by JC-1 to evaluate MMP through flow cytometry.Results:Sperm MMP of asthenozoospermia group(41.24%±9.71%)was significantly lower than that in control group(56.68%±11.13%).MMP was negatively correlated with BMI(r=−0.25,P<0.01),but positively correlated with total sperm motility(r=0.63,P<0.01),motility of progressive sperm(r=0.64,P<0.01),and normal sperm morphology rate(r=0.37,P<0.01).In addition,MMP showed no significant correlations with age,volume of semen,sperm concentration,sperm count,and other indexes.Conclusions:Sperm MMP is an important index in the evaluation of sperm function,and detection of MMP may provide references for the diagnosis and treatment of male infertility.

Rabbit Annulus Fibrosus Cell Apoptosis Induced by Mechanical Overload via a Mitochondrial Apoptotic Pathway

In order to investigate the apoptotic pathway of rabbit annulus fibrosus(AF) cells induced by mechanical overload,an experimental air-pressure model was established in this study to pressurize the rabbit AF cells in vitro.Cells were randomly divided into five groups in which the cells were exposed to a continuous pressure of 1.1 MPa for different lengths of time(0,5,12,24 and 36 h).The cell proliferation and apoptosis were detected by cell counting kit-8(CCK-8) assay and flow cytometry;the alterations in mitochondrial membrane potential were measured by fluorescence microscopy and fluorescence spectrophotometer;the activities of caspase-8 and 9 were determined by spectrophotometry.The results showed that after the cells were subjected to the pressure for 24 or 36 h,the cell proliferation was inhibited;the ratio of cell apoptosis was increased;the mitochondrial membrane potential was decreased;the activity of caspase-9 was enhanced;no activity changes were observed in caspase-8.The results suggested that treatment with a pressure of 1.1 MPa for more than 24 h can lead to the proliferation inhibition and the apoptosis of rabbit AF cells in vitro,and the mitochondrial-dependent pathway is implicated in the pressure-induced AF cell apoptosis.

Evaluation of sperm mitochondrial function using rh123/PI dual fluorescent staining in asthenospermia and oligoasthenozoospermia

Objective：The recent advent of flow cytometry（FCM）,coupled with fluorescent dyes,has been successfully applied to assess mitochondrial function.The aim of this study was to investigate the feasibility and clinical significance of detecting sperm mitochondrial function and to evaluate sperm mitochondrial function by using Rhodamine 123/propidium（Rh123/PI）dual fluorescent staining and FCM in asthenospermia and oligoasthenozoospermia.Methods：Twenty-five fertile men（with normal sperm parameters）and 230 infertile patients were examined.Fifty-five patients of the above 230 patients were selected for idiopathic infertility samples and were divided into two groups：asthenospermia（n=30）and oligoasthenozoospermia（n=25）.Rh123/PI dual fluorescent staining and FCM were carried out to examine sperm mitochondrial function.Results：Significant differences were found between the normal and abnormal semen samples（P0.05）when Rh123＋/PI-,Rh123-/PI＋and Rh123-/PI-sperm were examined by FCM,but there was no significant difference between the asthenospermia（P=0.469） and oligoasthenozoospermia group（P=0.950）when Rh123＋/PI-and Rh123-/PI＋sperm were then examined;however,a significant difference was found between the 2 groups（P=0.003）when Rh123-/PI-sperm were examined.There was no correlation between Rh123-/PI-sperm and semen parameters in the normal group,but there was a significant negative correlation between the sperm concentration and Rh123-/PI-sperm in asthenospermia and oligoasthenozoospermia patients（r=-0.509,-0.660;P=0.018,0.038）.Conclusion：Rh123/PI dual fluorescent staining and FCM can provide reliable information to assess the quality of sperm and reveal differences in mitochondrial membrane potential in asthenospermia and oligoasthenozoospermia.

Protection of melatonin against damage of sperm mitochondrial function induced by reactive oxygen species

Aim: To study the mitochondrial function damage of sperm induced by reactive oxygen species (ROS) and the protection of melatonin (MLT) against the damage. Methods: Normal function spermatozoa were selected from semen samples by Percoll gradient centrifugation technique. The ROS generated by the hypoxan-thine xanthine oxidase system was incubated with the normal spermatozoa in the presence or absence of MLT (6 mmol/L) for 30 and 60 minutes. After incubation, the activity of succinate dehydroge-nase (SDH) in the mitochondria of spermatozoa was assessed by histochemical method and spermatozoa were labeled with specific fluorescent probe of Rhodamine 123 to measure the mitochondrial membrane potential (MMP) by flow cytometry. Results: After the normal spermatozoa were incubated with ROS, The sperm MMP was significantly decreased and the SDH activity of almost decreased to zero. MLT reduced the mitochondrial damage induced by ROS. Conclusion: ROS damage the mitochondrial function of sperm by affecting sperm MMP and SDH activity of. MLT protects sperm mitochondria from the damage induced by ROS through its effective antioxidative potential.

Pigment epithelium-derived factor protects retinal ganglion cells from hypoxia-induced apoptosis by preventing mitochondrial dysfunction

AIM： To investigate the potential of pigment epitheliumderived factor（PEDF） to protect the immortalized rat retinal ganglion cells-5（RGC-5） exposed to Co Cl2-induced chemical hypoxia. METHODS： After being differentiated with staurosporine（SS）, RGC-5 cells were cultured in four conditions： control group cells cultured in Dulbecco ＇s modified eagle medium（DMEM） supplemented with 10% fetal bovine serum, 100 μmol/m L streptomycin and penicillin（named as normal conditions）; hypoxia group cells cultured in DMEM containing 300 μmol/m L Co Cl2; cells in the group protected by PEDF were first pretreated with 100 ng/m L PEDF for 2h and then cultured in the same condition as hypoxia group cells; and PEDF group cells that were cultured in the presence of 100 ng/m L PEDF under normal conditions. The cell viability was assessed by MTT assay, the percentage of apoptotic cells was quantified using Annexin V-FITC apoptosis kit, and intra-cellar reactive oxygen species（ROS） was measured by dichloro-dihydro-fluorescein diacetate（DCFH-DA） probe. The mitochondria-mediated apoptosis was also examined to further study the underlying mechanism of the protective effect of PEDF. The opening of mitochondrial permeability transition pores（m PTPs） and membrane potential（Δψm） were tested as cellular adenosine triphosphate（ATP） level and glutathione（GSH）. Also, the expression and distribution of Cyt C and apoptosis inducing factor（AIF） were observed.RESULTS： SS induced differentiation of RGC-5 cells resulting in elongation of their neurites and establishing contacts between outgrowths. Exposure to 300 μmol/m L Co Cl2 triggered death of 30% of the total cells in cultures within 24 h. At the same time, pretreatment with 100 ng/m L PEDF significantly suppressed the cell death induced by hypoxia（P〈0.05）. The apoptosis induced by treatment of Co Cl2 was that induced cell death accompanied with increasing intracellar ROS and decreasing GSH and ATP level. PEDF pretreatment suppressed these effects（P〈0.05）. Additionally, PEDF treatment inhibited the opening of m PTPs and suppressed decreasing of Δψm in RGC-5 cells, resulting in blocking of the mitochondrial apoptotic pathway.CONCLUSION： Pretreatment of RGC-5 cells with 100 ng/m L PEDF significantly decreases the extent of apoptosis. PEDF inhibits the opening of m PTPs and suppresses decreasing of Δψm. Moreover, PEDF also reduces ROS production and inhibits cellular ATP level＇s reduction. Cyt C and AIF activation in PEDF-pretreated cultures are also reduced. These results demonstrate the potential for PEDF to protect RGCs against hypoxic damage in vitro by preventing mitochondrial dysfunction.

3′-Daidzein sulfonate sodium improves mitochondrial functions after cerebral ischemia/reperfusion injury

3′-Daidzein sulfonate sodium is a new synthetic water-soluble compound derived from daidzein（an active ingredient of the kudzu vine root）. It has been shown to have a protective effect on cerebral ischemia/reperfusion injury in rats. We plan to study the mechanism of its protective effect. 3′-Daidzein sulfonate sodium was injected in rats after cerebral ischemia/reperfusion injury. Results showed that 3′-daidzein sulfonate sodium significantly reduced mitochondrial swelling, significantly elevated the mitochondrial membrane potential, increased mitochondrial superoxide dismutase and glutathione peroxidase activities, and decreased mitochondrial malondialdehyde levels. 3′-Daidzein sulfonate sodium improved the structural integrity of the blood-brain barrier and reduced blood-brain barrier permeability. These findings confirmed that 3′-daidzein sulfonate sodium has a protective effect on mitochondrial functions after cerebral ischemia/reperfusion injury, improves brain energy metabolism, and provides protection against blood-brain barrier damage.

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.

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.

Astragaloside Ⅳ protects RGC-5 cells against oxidative stress

Astragaloside Ⅳ is the main active compound of Astragalus membranaceus. Astragaloside Ⅳ has strong anti-oxidative activities and protective effects against progression of peripheral neuropathy. In this study, we determined whether astragaloside Ⅳ protects retinal ganglion cells（RGC） from oxidative stress injury using the rat RGC-5 cell line. Hydrogen peroxide（H_2O_2） was used to induce oxidative stress injury, with the protective effect of astragaloside Ⅳ examined. Cell Counting Kit-8 and 4′,6-diamidino-2-phenylindole staining showed that astragaloside Ⅳ increased cell survival rate and decreased apoptotic cell number. Flow cytometry showed that astragaloside Ⅳ decreased H_2O_2-induced reactive oxygen species levels. While laser confocal microscopy showed that astragaloside Ⅳ inhibited the H_2O_2-induced decrease of mitochondrial membrane potential. Western blot assay showed that astragaloside Ⅳ reduced cytochrome c release induced by H_2O_2, inhibited Bax and caspase-3 expression, and increased Bcl-2 expression. Altogether, these results indicate that astragaloside Ⅳ has potential protective effects against H_2O_2-induced oxidative stress in retinal ganglion cells.

The pathways by which mild hypothermia inhibits neuronal apoptosis following ischemia/reperfusion injury

Several studies have demonstrated that mild hypothermia exhibits a neuroprotective role and it can inhibit endothelial cell apoptosis following ischemia/reperfusion injury by decreasing casp- ase-3 expression, It is hypothesized that mild hypothermia exhibits neuroprotective effects on neurons exposed to ischemia/reperfusion condition produced by oxygen-glucose deprivation. Mild hypothermia significantly reduced the number of apoptotic neurons, decreased the expres- sion of pro-apoptotic protein Bax and increased mitochondrial membrane potential, with the peak of anti-apoptotic effect appearing between 6 and 12 hours after the injury. These findings indicate that mild hypothermia inhibits neuronal apoptosis following ischemia/reperfusion injury by protecting the mitochondria and that the effective time window is 6-12 hours after ischemia/reperfusion injury.

Protective effects of curcumin against human immunodeficiency virus 1 gp120 V3 loop-induced neuronal injury in rats

Curcumin improves the learning and memory deficits in rats induced by the gp120 V3 loop. The present study cultured rat hippocampal neurons with 1 nM gp120 V3 loop and 1 μM curcumin for 24 hours. The results showed that curcumin inhibited the gp120 V3 loop-induced mitochondrial membrane potential decrease, reduced the mRNA expression of the pro-apoptotic gene caspase-3, and attenuated hippocampal neuronal injury.

Autophagy Attenuates MnCl2-induced Apoptosis in Human Bronchial Epithelial Cells

Objective To investigate the role of autophagy in MnC l2-induced apoptosis in human bronchial epithelial 16 HBE cells.Methods Cell proliferation was measured by MTT assay.Mitochondrial membrane potential（MMP） and apoptosis were measured by flow cytometry.Autophagic vacuoles were detected by fluorescence microscopy.Cellular levels of apoptosis and autophagy-related proteins were measured by western blotting.Results 16 HBE cell proliferation was inhibited by Mn Cl2 in a dose-and time-dependent manner.Mn Cl2-induced 16 HBE cell growth inhibition was related to MMP depolarization prior to the induction of apoptosis.Our data revealed that Mn Cl2-induced apoptosis in 16 HBE cells was mediated by decreased expression of Bcl-2 and increased levels of cleaved caspase-3.It was observed that when we exposed 16 HBE cells to MnCl2 in a dose-dependent manner,the formation of autophagic vacuoles and the levels of LC-3B-II were elevated.RNA interference of LC3 B in these Mn Cl2-exposed cells demonstrated that MMP loss and apoptosis were enhanced.Additionally,the pan-caspase inhibitor Z-VAD-FMK increased the cellular levels of Bcl-2 and decreased apoptosis,but did not affect the cellular levels of LC3 B in Mn Cl2-treated 16 HBE cells.Conclusion Mn Cl2 dose-and time-dependently inhibits 16 HBE cell proliferation and induces MMP loss and apoptosis.Autophagy acts in a protective role against Mn Cl2-induced apoptosis in 16 HBE cells.

Oligomeric Procyanidins Induce Generation of Reactive Oxygen Species and Collapse of Mitochchondrial Membrane Potential in Glioblastoma Cell Lines

Objective The aim of the present study was to clarify the mechanism underlying glioma cell death upon oligomeric procyanidins (F2) exposure. Methods The cytotoxicity of F2 on U87 (human malignant glioblastoma cell line) and C6 (rat glioma cell line) cancer cells was evaluated, and changes of mitochondrial membrane potential (MMP) and production of reactive oxygen species (ROS) in drug-treated cells were monitored. Moreover, morphological changes associated with F2-induced cells death were examined. Results F2 induced a concentration-dependent increase in ROS production and decrease in MMP. Furthermore, pre-incubation with N-acetylcysteine (NAC) and rotenone (Rt), resulted in partial inhibition of F2-induced ROS generation and marked attenuation of cell death and the cytoplasmic vacuolization induced by F2. In addition, pretreatment with Rt markedly attenuated the MMP loss in F2-treated cells. However, pretreatment with NAC only markedly attenuated the MMP loss in F2-treated C6 cells. Conclusion The increase in ROS level is at least one of mechanisms associated with F2-induced glioma cell death as well as the cytoplasmic vacuolization formation that contribute to the cytotoxicity of F2 in glioma cells.

S14G-humanin restored cellular homeostasis disturbed by amyloid-beta protein

Humanin is a potential therapeutic agent for Alzheimer’s disease, and its derivative, S14G-humanin, is 1 000-fold stronger in its neuroprotective effect against Alzheimer’s disease-relevant insults. Alt-hough effective, the detailed molecular mechanism through which S14G-humanin exerts its effects remains unclear. Data from this study showed that fibril ar amyloid-beta 40 disturbed cel ular ho-meostasis through the cel membrane, increasing intracel ular calcium, generating reactive oxygen species, and decreasing the mitochondrial membrane potential. S14G-humanin restored these re-sponses. The results suggested that S14G-humanin blocked the effects of amyloid-beta 40 on the neuronal cel membrane, and restored the disturbed cel ular homeostasis, thereby exerting a neuroprotective effect on hippocampal neurons.

Hydrogen sulfide inhibits beta-amyloid peptide-induced apoptosis in PC12 cells and the underlying mechanisms

BACKGROUND：Studies have demonstrated that hydrogen sulfide（H2S） levels are 55% lower in brains of Alzheimer＇s disease（AD） patients than in age-matched normal individuals,which suggests that H2S might be involved in some aspects of AD pathogenesis.OBJECTIVE：To observe the protective mechanisms of varied concentrations of H2S against β-amyloid-peptide（Aβ） induced apoptosis in pheochromoytoma（PC12） cells,and to analyze the pathway of action.DESIGN,TIME AND SETTING：A controlled,observational,in vitro experiment was performed at Neurophysiology Laboratory in Zhongshan Medical School,Sun Yat-sen University between July 2006 and May 2007.MATERIALS：PC12 cells were provided by the Animal Experimental Center of Medical School of Sun Yat-sen University.Glybenclamide,rhodamine123,and dihydrorhodamine123 were purchased from Sigma（USA）.METHODS：PC12 cells were incubated at 37 ℃ in a 5% CO2-enriched incubator with RPMI-1640 medium,supplemented with 5% horse-serum and 10% fetal bovine serum.Cells in logarithmic growth curves received different treatment：The PC12 cells were maintains at 37 ℃ with the original medium,then incubated in Aβ25-35,sodium hydrosulfide（NaHS）,glybenclamide,NaHS＋ Aβ25-35,or pretreated with glybenclamide 30 minutes prior to administration of and Aβ25-35,respectively.MAIN OUTCOME MEASURES：（1） The survival rate of PC12 cells was detected by MTT assay and Hoechst staining.（2） The apoptosis rate of PC12 cells was detected utilizing flow cytometry with propidium iodide staining,and morphological changes of apoptotic cells were observed.（3） The mitochondrial membrane potential was detected by Rhodamine123-combined flow cytometry.（4） The intracellular reactive oxygen species content was detected by dihydrorhodamine123-combined flow cytometry.RESULTS：Aβ25-35 induced significantly decreased viability and increased percentage of apoptosis in PC12 cells,as well as dissipated mitochondrial membrane potential expression and an overproduction of reactive oxygen species.When PC12 cells were co-treated with NaHS and Aβ25-35,the decreased cell viability induced by 20 μmol/L Aβ25-35 was concentration-dependently blocked by NaHS（50,100,and 200 μmol/L）.NaHS（100 μmol/L） obviously reduced the percentage of apoptotic PC12 cells induced by 20 μmol/L Aβ25-35.In addition,100 μmol/L NaHS inhibited mitochondrial membrane potential dissipation and reactive oxygen species overproduction.When the ATP-sensitive K channel（KATP） inhibitor,glybenclamide,was administered 30 minutes prior to NaHS and Aβ25-35 treatment,the NaHS-dependent cellular protection was partly blocked.This resulted in reduced PC12 cell viability and increased the percentage of apoptosis,as well as significantly blocked mitochondrial membrane potential preservation and inhibited reactive oxygen species overproduction due to NaHS treatment.CONCLUSION：NaHS protected PC12 cells against Aβ25-35-induced damage.NaHS-dependent cellular protection was associated with mitochondrial membrane potential preservation and inhibition of reactive oxygen species overproduction.The KATP channel inhibitor,glybenclamide,significantly blocked the cellular protective effects of NaHS,indicating that KATP channel activation plays an important role in NaHS-induced protection of PC12 cells to Aβ25-35-induced damage.

N-hexane Alters the Maturation of Oocytes and Induces Apoptosis in Mice

Objective This study was aimed to determine the effects of n-hexane on the maturation of mouse oocytes. Methods Cell culture was used to observe the maturation of mouse oocytes and CLSM was employed to determine their apoptosis. Results Germinal vesicle breakdown （GVBD） and extrusion of the first polar body in mouse oocytes were significantly inhibited by n-hexane. After fertilization, the number of eggs in the mouse was significantly reduced by n-hexane. Mitochondrial membrane potentials （A^Um） were altered in mouse oocytes that were leading to apoptosis of the oocytes. Conclusion N-hexane might have affected the maturation of oocytes, causing alteration of ~qJm and leading to apoptosis which maybe one of the most imn（~rt^nt rnpch^ni^nn~