Inhibition of phosphodiesterase 4 by FCPR16 protects SH-SY5Y cells against MPP^+ -induced decline of mitochondrial membrane potential and oxidative stress

Parkinson disease(PD) is a chronic neurodegenerative disorder caused by progressive dopaminergic neuronal death in the substantia nigra pars compacta within the midbrain.There still is no cure,effective treatments for PD,available therapies are only capable of offering temporary and symptomatic relief to the patients.There are certain patents that claim phosphodiesterase(PDE) inhibitors as possible anti-PD drugs,PDE4 is a promising target for the treatment of PD and the underlying mechanism has not yet been well elucidated.PDE4 is an enzyme that specifically hydrolyzes intracellular cyclic adenosine monophosphate(cAMP)throughout the body,including the brain.Most of the available PDE4 inhibitors exert unpleasant and serious side effects,such as emesis and nausea,which hinder its clinical application.Therefore,more efforts are needed before PDE4 inhibitors with high therapeutic indices are available for treatment of PD.FCPR16 is a novel PDE4 inhibitor with little emetic potential,which exhibits excellent enzyme inhibition activity(IC50=90 nmol·L^(-1)).METHODS SH-SY5 Y cell was induced with 1-methyl-4-phenylpyridinium(MPP+)to mimic PD cell injury in vitro,and CCK-8 assay was used to investigate the viability effects of different concentration of FCPR16(3.1-50 μmol·L^(-1)) on MPP+-injured SH-SY5 Y cells.Detection of apoptosis was performed by flow cytometry.The level of ntracellular reactive oxygen species was detected with the fluorescent probe DCFH-DA,and the mitochondrial membrane potential of cells in different experimental groups was detected with the JC-1 fluorescent probe.AO staining and Lysotracker Red staining were used to detect the intracellular antophagy changes.The expression of apoptosis related proteins,autophagy and other related signal molecules were demonstrated by Western blotting.Different cellular signaling pathway inhibitors were used to invesitigate the specific cellular mechanisms of FCPR16 protecting MPP+-induced cell injury.RESULTS FCPR16(12.5-50 μmol·L^(-1)) dose-dependently reduced MPP+-induced decline of cell viability,accompanied by reductions in nuclear condensation and lactate dehydrogenase release.The level of cleaved caspase 3 and the ratio of Bax/Bcl-2 were also decreased after treatment with FCPR16 in MPP+-treated cells.Furthermore,FCPR16(25 μmol·L^(-1)) significantly suppressed the accumulation of reactive oxygen species(ROS),prevented the decline of mitochondrial membrane potential(Δψm) and attenuated the expression of malonaldehyde level.Further studies disclosed that FCPR16 enhanced the levels of cA MP and the exchange protein directly activated by cA MP(Epac) in SHSY5 Y cel s.Western blotting analysis revealed that FCPR16 increased the phosphorylation of c AMP response element-binding protein(CREB) and protein kinase B(Akt)down-regulated by MPP+in SHSY5 Y cells.Moreover,the inhibitory effects of FCPR16 on the production of ROS and Δψm loss could be blocked by PKA inhibitor H-89 and Akt inhibitor KRX-0401.CONCLUSION The novel PDE4 inhibitor FCPR16 can protect against damaging pathways including oxidative stress,mitochondrial dysfunction and apoptosis in SH-SY5 Y cells.FCPR16 preventes MPP+-induced neurotoxicity through activation of cAMP/PKA/CREB and Epac/Akt signaling pathways.These may lead to develop mechanism based therapeutics and improved pharmacotherapy for PD.It is reasonable to assume that FCPR16 is a potential candidate for the prevention and treatment of PD.

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

Objective:To investigate the effect of berbamine 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(?ψm) 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 acti-vation.

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.

YB-1 downregulation attenuates UQCRC1 protein expression level in H9C2 cells and decreases the mitochondrial membrane potential

UQCRC1 is one of the 10 mitochondrial complex III subunits,this protein has a role in energy metabolism,myocardial protection,and neurological diseases.The upstream mechanism of the UQCRC1 protective effect on cardiomyocytes is currently unavailable.In order to explore the upstream molecules of UQCRC1 and elucidate the protective mechanism of UQCRC1 on cardiomyocytes in more detail,we focused on the nuclease-sensitive elementbinding protein 1(YB-1).We hypothesized YB-1 acts as an upstream regulatory molecule of UQCRC1.This study found that YB-1 RNAi significantly reduces the expression of the UQCRC1 protein level(p<0.05)and obviously decreases the mitochondrial membrane potential(p<0.05),and that YB-1 interacts with UQCRC1 protein in vivo,but YB-1 RNAi has little effect on the UQCRC1 gene transcription.

Cannabidiol (CBD) Prevents Palmitic Acid-Induced Drop in Mitochondrial Membrane Potential

Exposure of macrophages and microglia cells to the saturated palmitic acid (PA) leads to reduction in the mitochondrial membrane potential (), shrinkage of the cells and apoptosis. Here we show that the Cannabis component Cannabidiol (CBD) rescues both macrophages and microglia cells from the detrimental effects of PA. CBD prevents the shrinkage in cell size and the reduction incaused by PA. The protective effect of CBD on the macrophage mitochondria is important for sustaining the macrophage population even under the immunosuppressed conditions caused by this drug. To a similar extent, the antagonistic effect of CBD on PA-mediated microglia cytotoxicity is important for its role in neuroprotection.

Transfer of mitochondria from mesenchymal stem cells derived from induced pluripotent stem cells attenuates hypoxia-ischemia-induced mitochondrial dysfunction in PC12 cells

Mitochondrial dysfunction in neurons has been implicated in hypoxia-ischemia-induced brain injury.Although mesenchymal stem cell therapy has emerged as a novel treatment for this pathology,the mechanisms are not fully understood.To address this issue,we first co-cultured 1.5×10^5 PC12 cells with mesenchymal stem cells that were derived from induced pluripotent stem cells at a ratio of 1:1,and then intervened with cobalt chloride(CoCl2)for 24 hours.Reactive oxygen species in PC12 cells was measured by Mito-sox.Mitochondrial membrane potential(ΔΨm)in PC12 cells was determined by JC-1 staining.Apoptosis of PC12 cells was detected by terminal deoxynucleotidal transferase-mediated dUTP nick end-labeling staining.Mitochondrial morphology in PC12 cells was examined by transmission electron microscopy.Transfer of mitochondria from the mesenchymal stem cells derived from induced pluripotent stem cells to damaged PC12 cells was measured by flow cytometry.Mesenchymal stem cells were induced from pluripotent stem cells by lentivirus infection containing green fluorescent protein in mitochondria.Then they were co-cultured with PC12 cells in Transwell chambers and treated with CoCl2 for 24 hours to detect adenosine triphosphate level in PC12 cells.CoCl2-induced PC12 cell damage was dose-dependent.Co-culture with mesenchymal stem cells significantly reduced apoptosis and restoredΔΨm in the injured PC12 cells under CoCl2 challenge.Co-culture with mesenchymal stem cells ameliorated mitochondrial swelling,the disappearance of cristae,and chromatin margination in the injured PC12 cells.After direct co-culture,mitochondrial transfer from the mesenchymal stem cells stem cells to PC12 cells was detected via formed tunneling nanotubes between these two types of cells.The transfer efficiency was greatly enhanced in the presence of CoCl2.More importantly,inhibition of tunneling nanotubes partially abrogated the beneficial effects of mesenchymal stem cells on CoCl2-induced PC12 cell injury.Mesenchymal stem cells reduced CoCl2-induced PC12 cell injury and these effects were in part due to efficacious mitochondrial transfer.

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.

circRNA MYLK基因干扰对胰腺癌PANC-1细胞线粒体膜电位、氧化损伤和微管形成的影响

目的探讨circRNA肌球蛋白轻链激酶(myosin light chain kinase,MYLK)基因干扰对胰腺癌PANC-1细胞线粒体膜电位、氧化损伤和微管形成的影响。方法将对数生长期PANC-1细胞分为空白对照组、shRNA-NC组和circMYLK-shRNA组。转染后,采用细胞克隆形成实验检测各组PANC-1细胞的生长。试剂盒检测各组细胞上清液中超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malondialdehyde,MDA)水平。流式细胞仪分析细胞线粒体膜电位。显微镜下观察各组细胞微管结节数。Western blot检测各组细胞Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)/B细胞淋巴瘤2基因(B-cell lymphoma 2,Bcl-2)、原癌基因c-Myc、血管内皮生长因子(vascular endothelial growth factor,VEGF)和波形蛋白(Vimentin)表达。结果与shRNA-NC组相比,circMYLK-shRNA1组PANC-1细胞的克隆形成率、JC-1红色荧光所占百分比、微管结节数明显降低(P均<0.05);细胞上清中SOD水平明显降低,MDA水平明显升高(P均<0.05);细胞内Bax/Bcl-2蛋白表达明显升高(P<0.05),c-Myc、VEGF和Vimentin蛋白表达明显降低(P均<0.05)。结论circRNA MYLK基因沉默可以抑制PANC-1细胞增殖能力,降低PANC-1细胞的线粒体膜电位,诱导细胞氧化损伤,抑制微管的形成。

Betanodavirus: Mitochondrial disruption and necrotic cell death

Betanodaviruses cause viral nervous necrosis, an infectious neuropathological condition in fish that is characterized by necrosis of the central nervous system, including the brain and retina. This disease can cause mass mortality in larval and juvenile populations of several teleost species and is of global economic importance. The mechanism of brain and retina damage during betanodavirus infection is poorly understood. In this review, we will focus recent results that highlight betanodavirus infection-induced molecular death mechanisms in vitro. Betanodavirus can induce host cellular death and post-apoptotic necrosis in fish cells. Betanodavirus-induced necrotic cell death is also correlated with loss of mitochondrial membrane potential in fish cells, as this necrotic cell death is blocked by the mitochondrial membrane permeability transition pore inhibitor bongkrekic acid and the expression of the antiapoptotic Bcl-2 family member zf Bcl-x L. Moreover, this mitochondria-mediated necrotic cell death may require a caspase-independent pathway. A possible cellular death pathway involving mitochondrial function and the modulator zf Bcl-xs is discussed which may provide new insights into the necrotic pathogenesis of betanodavirus.

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.

基因干预MST2的施万细胞稳转株建立及其在线粒体膜电位检测的应用

目的构建稳定敲低及过表达哺乳动物Ste20样激酶2(mammalian ste20-like kinase 2, MST2)的大鼠施万细胞株,并初步探讨MST2对线粒体膜电位的调控作用。方法 大鼠施万细胞株(RSC96)分为正常对照组(对照组)、氧糖剥夺(oxygen and glucose deprivation, OGD)模型组、MST2敲低对照组、MST2敲低组、MST2过表达对照组、MST2过表达组。逆转录PCR法扩增大鼠Mst2基因全长序列,构建过表达Mst2基因的慢病毒表达载体重组质粒并鉴定。使用第二代慢病毒包装系统获取Mst2基因敲低及过表达的慢病毒毒液,并分别感染RSC96细胞株,建立基因干预MST2的稳转株。实时荧光定量PCR结合western blot检测基因干预效率,光镜结合S100荧光染色观察稳转株形态学变化。使用OGD模型处理细胞,以线粒体膜电位检测试剂盒(JC-1)检测细胞线粒体膜电位水平(mitochondrial membrane potential, MMP)。结果 经测序分析,成功构建Mst2慢病毒表达载体重组质粒。施万细胞稳转株具有施万细胞的形态特征,并且表达施万细胞特异性标志物S100。在慢病毒感染的施万细胞,MST2表达在mRNA及蛋白水平上均较对照组有稳定且显著的差异,敲低效率超过90%,过表达约为对照组的40倍。稳转株在OGD模型中,对于MST2蛋白的基因干预仍然维持稳定。OGD可显著增加MST2蛋白表达,并降低施万细胞MMP,提示线粒体功能受损;而敲低MST2则显著改善MMP。结论 本研究成功构建了稳定敲低及过表达MST2的大鼠施万细胞株,并且在OGD模型中,敲低MST2可改善线粒体膜电位,提示其对线粒体具有保护作用。

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.

新型抗菌肽Mt-22S3对白念珠菌细胞的作用及机制

目的探讨家蝇抗真菌肽-1A(MAF-1A)突变体-22S3(Mt-22S3)对白念珠菌(C.albicans)细胞的作用及机制。方法取对数生长期的C.albicans菌悬液,分别与0、125、250及500 mg/L Mt-22S3作用12 h,采用荧光显微技术及荧光检测法观测Mt-22S3作用C.albicans后细胞内活性氧(ROS)的变化;以荧光检测法测定Mt-22S3作用C.albicans后细胞内线粒体膜电位水平的变化;通过Annexin V-FITC/PI双染法,观察Mt-22S3对C.albicans细胞凋亡的影响;以琼脂糖凝胶电泳阻滞实验分析Mt-22S3对C.albicans细胞DNA的影响。结果经Mt-22S3作用的C.albicans细胞内ROS增高,且随着Mt-22S3浓度升高,细胞内ROS上升(P<0.05);线粒体膜电位检测发现,经Mt-22S3作用的C.albicans线粒体膜电位下降(P<0.01);Mt-22S3可引起C.albicans细胞凋亡及细胞坏死的发生,且Mt-22S3浓度越高,细胞凋亡及细胞坏死现象越明显;凝胶阻滞实验结果显示,Mt-22S3与C.albicans DNA孵育后产生明显的凝胶阻滞现象。结论Mt-22S3可进入C.albicans细胞内发挥抗菌作用,其机制与诱导细胞内ROS积累、去极化线粒体膜、诱导细胞凋亡和死亡、结合DNA等有关。

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.

Polydatin prevents the induction of secondary brain injury after traumatic brain injury by protecting neuronal mitochondria

Polydatin is thought to protect mitochondria in different cell types in various diseases.Mitochondrial dysfunction is a major contributing factor in secondary brain injury resulting from traumatic brain injury.To investigate the protective effect of polydatin after traumatic brain injury,a rat brain injury model of lateral fluid percussion was established to mimic traumatic brain injury insults.Rat models were intraperitoneally injected with polydatin(30 mg/kg)or the SIRT1 activator SRT1720(20 mg/kg,as a positive control to polydatin).At 6 hours post-traumatic brain injury insults,western blot assay was used to detect the expression of SIRT1,endoplasmic reticulum stress related proteins and p38 phosphorylation in cerebral cortex on the injured side.Flow cytometry was used to analyze neuronal mitochondrial superoxide,mitochondrial membrane potential and mitochondrial permeability transition pore opened.Ultrastructural damage in neuronal mitochondria was measured by transmission electron microscopy.Our results showed that after treatment with polydatin,release of reactive oxygen species in neuronal mitochondria was markedly reduced;swelling of mitochondria was alleviated;mitochondrial membrane potential was maintained;mitochondrial permeability transition pore opened.Also endoplasmic reticulum stress related proteins were inhibited,including the activation of p-PERK,spliced XBP-1 and cleaved ATF6.SIRT1 expression and activity were increased;p38 phosphorylation and cleaved caspase-9/3 activation were inhibited.Neurological scores of treated rats were increased and the mortality was reduced compared with the rats only subjected to traumatic brain injury.These results indicated that polydatin protectrd rats from the consequences of traumatic brain injury and exerted a protective effect on neuronal mitochondria.The mechanisms may be linked to increased SIRT1 expression and activity,which inhibits the p38 phosphorylation-mediated mitochondrial apoptotic pathway.This study was approved by the Animal Care and Use Committee of the Southern Medical University,China(approval number:L2016113)on January 1,2016.