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.

Mitochondrial carnitine palmitoyltransferase-Ⅱ dysfunction: A possible novel mechanism for nonalcoholic fatty liver disease in hepatocarcinogenesis

Nonalcoholic fatty liver disease(NAFLD)or metabolic-associated fatty liver disease has been characterized by the lipid accumulation with injury of hepatocytes and has become one of the most common chronic liver diseases in the world.The complex mechanisms of NAFLD formation are still under identification.Carnitine palmitoyltransferase-Ⅱ(CPT-Ⅱ)on inner mitochondrial membrane(IMM)regulates long chain fatty acidβ-oxidation,and its abnormality has had more and more attention paid to it by basic and clinical research in NAFLD.The sequences of its peptide chain and DNA nucleotides have been identified,and the catalytic activity of CPT-Ⅱ is affected on its gene mutations,deficiency,enzymatic thermal instability,circulating carnitine level and so on.Recently,the CPT-Ⅱ dysfunction has been discovered in models of liver lipid accumulation.Meanwhile,the malignant transformation of hepatocyte-related CD44^(+) stem T cell activation,high levels of tumor-related biomarkers(AFP,GPC3)and abnormal activation of Wnt3a expression as a key signal molecule of the Wnt/β-catenin pathway run parallel to the alterations of hepatocyte pathology.This review focuses on some of the progress of CPT-Ⅱ inactivity on IMM with liver fatty accumulation as a possible novel pathogenesis for NAFLD in hepatocarcinogenesis.

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.

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.

Mitochondrial membrane protein Bcl-xL, a regulator of adult neuronal growth and synaptic plasticity: multiple functions beyond apoptosis

The B-cell lymphoma 2 （Bcl2） family of proteins participates in cell death or survival through a mitochondrial pathway. The pro-apoptotic members of the Bcl2 family such as Bim, Bid, Bax and Bak trigger cell death by contributing to the enhancement of mitochondrial outer membrane permeabil- ity to pro-apoptotic factors such as cytochrome c, with the subsequent activation of caspases. The anti-apoptotic mem- bers, such as B-cell lymphoma-extra large （Bd-xL）, block the pro-apoptotic Bcl2 members and prevent cell death. Bcl-xL is abundantly expressed during development and in mature neurons, suggesting that it plays a role in protection from death from untoward events occurring in adult life such as ischemia, inflammation or trauma. When these neurotoxic in- sults occur, Bcl-xL translocates to mitochondria and prevents activation and homo-oligomerization of pro-apoptotic family members such Bax and Bak. Numerous studies have shown pro-survival roles for Bcl-xL in adult neurons using various models; nevertheless, the role of Bcl-xL outside of the field of neuronal death, i.e., in adult neuronal growth, excitability or synaptic plasticity, has not been studied in depth.

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.

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.

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.

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.

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.

Novel Pt(IV) complex OAP2 induces STING activation and pyroptosis via mitochondrial membrane remodeling for synergistic chemo-immunotherapy

Mitochondrial membrane remodeling can trigger the release of mitochondrial DNA (mtDNA), leading to the activation of cellular oxidative stress and immune responses. While the role of mitochondrial membrane remodeling in promoting inflammation in hepatocytes is well-established, its effects on tumors have remained unclear. In this study, we designed a novel Pt(IV) complex, OAP2, which is composed of oxaliplatin (Oxa) and acetaminophen (APAP), to enhance its anti-tumor effects and amplify the immune response. Our findings demonstrate that OAP2 induces nuclear DNA damage, resulting in the production of nuclear DNA. Additionally, OAP2 downregulates the expression of mitochondrial Sam50, to promote mitochondrial membrane remodeling and trigger mtDNA secretion, leading to double-stranded DNA accumulation and ultimately synergistically activating the intracellular cGAS-STING pathway. The mitochondrial membrane remodeling induced by OAP2 overcomes the limitations of Oxa in activating the STING pathway and simultaneously promotes gasdermin-D-mediated cell pyroptosis. OAP2 also promotes dendritic cell maturation and enhances the quantity and efficacy of cytotoxic T cells, thereby inhibiting cancer cell proliferation and metastasis. Briefly, our study introduces the first novel small-molecule inhibitor that regulates mitochondrial membrane remodeling for active immunotherapy in anti-tumor research, which may provide a creative idea for targeting organelle in anti-tumor therapy.

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.

S100A8/A9 induces autophagy and apoptosis via ROS-mediated cross-talk between mitochondria and lysosomes that involves BNIP3

The complex formed by two members of the S100 calcium-binding protein family, S100A8/A9, exerts apoptosisinducing activity in various cells of different origins. Here, we present evidence that the underlying molecular mechanisms involve both programmed cell death I （PCD I, apoptosis） and PCD II （autophagy）-like death. Treatment of cells with S100A8/A9 caused the increase of Beclin-1 expression as well as Atgl2-Atg5 formation. S100A8/A9-induced cell death was partially inhibited by the specific PI3-kinase class Ⅲ inhibitor, 3-methyladenine （3-MA）, and by the vacuole H＋-ATPase inhibitor, bafilomycin-A1 （Baf-A1）. S100A8/A9 provoked the translocation of BNIP3, a BH3 only pro-apoptotic Bcl2 family member, to mitochondria. Consistent with this finding, ATM-BNIP3 overexpression partially inhibited S100A8/A9-induced cell death, decreased reactive oxygen species （ROS） generation, and partially pro- tected against the decrease in mitochondrial transmembrane potential in S100A8/A9-treated ceils. In addition, either ATM-BNIP3 overexpression or N-acetyl-L-cysteine co-treatment decreased lysosomal activation in cells treated with S100A8/A9. Our data indicate that S100A8/A9-promoted cell death occurs through the cross-talk of mitochondria and lysosomes via ROS and the process involves BNIP3.

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.

Inhibitory Effect of Isorhapontigenin on Copper-Mediated Peroxidation of Human Low-Density Lipoprotein in vitro

Aim To study the effect of Isorhapontigenin (Iso) on copper-mediatedperoxidation of human low-density lipoprotein (LDL) and on the toxicity of oxidized LDL (ox-LDL) tomouse macrophages in vitro. Methods Human LDL from sera df normal lipidemic donors was separated bysequential ultracentrifugation. The separated human IDL 1 mg·mL^(-1) in phosphate buffer saline, pH7.4, was incubated with cupric sulfate (10 μmol·L^(-1) ) at 37℃ for 10 h in the presence orabsence of various concentrations of Iso. Malondialdehyde (MDA) formation, vitamin E consumption,electrophoretic mobility of LDL, mitochondria] membrane potential of mouse peritoneal macrophages,phagocytosis of neutral red, and release of nitric oxide (NO) from macrophages were determined byvarious methods. Results Iso 1 - 100 μmol·L^(-1) significantly inhibited the increase of MDAformation, vitamin E consumption and electrophoretic mobility of LDL induced by Cu^(2+) in aconcentration-dependent manner. The injury of the mitochondrial membrane potential of mouseperitoneal macrophages due to incubation with ox-LDL (0.1 mg·mL^(-1)) at 37℃ for 12 h was markedlyprotected by 10 μmol·L^(-1) Iso. After pretreat-ment of the macrophages with 10 μmol · L^(-1)of Iso and then exposure to ox-LDL for 4 h, the reduction of phagocytosis of neutral red and releaseof NO in response to lipopolysaccharide (IPS) stimulation were significantly prevented. ConclusionIso has protective action against Cu^(2+) - mediated LDL peroxidation and ox-LDL induced toxicity tomacrophages in vitro.

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.

Damaging Effect of Cigarette Smoke Extract on PrimaryCultured Human Umbilical Vein Endothelial Cells and Its Mechanism

Objective To investigate the cellular effects of cigarette smoke extract (CSE) on primarily cultured human umbilical vein endothelial cells (HUVEC). Methods The effects of CSE (5%-20%) and nicotine (10-4 mol/L) on HUVEC viability, proliferation, angiogenesis and apoptosis were observed. Results CSE decreased HUVEC survival rate and angiogenesis after 24 h as well as its proliferation after 48 h in a dose-dependent manner. Moreover, CSE induced apoptosis of HUVEC as indicated in condensation of nuclear chromatin and the presence of hypodiploid DNA. HUVEC incubated with CSE for 24 h gave a significant decrease in the expression of Bcl-2 as well as the decline in the Bcl-2/Bax ratio accompanied with the loss of mitochondrial membrane potential and excess cytosolic calcium. Our study also observed that p53 protein level decreased, rather than increased in cells treated with CSE. Nicotine had no discernible inhibitory effects on the above indices of HUVEC. Conclusion Exposure to CSE other than nicotine causes inhibition of viability, proliferation and differentiation of HUVEC. CSE-induced HUVEC injury is mediated in part through accelerated apoptosis but independent of p53 pathway. It appears that mitochondria have played a key role in the apoptosis of HUVEC induced by CSE.