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...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.展开更多
OBJECTIVE Despite the status of cisplatin(DDP) as a classical chemotherapeutic agent in the treatment of cancer,the development of multidrug resistance often leads to a failure of DDP therapy.Traditional Chinese medic...OBJECTIVE Despite the status of cisplatin(DDP) as a classical chemotherapeutic agent in the treatment of cancer,the development of multidrug resistance often leads to a failure of DDP therapy.Traditional Chinese medicine(TCM) as adjuvant chemotherapy of cancer drugs in China has been widely used in cancer treatment.ZuoJin WAN(ZJW),a TCM formula,was proved reversing drug resistance in gastric cancer,but its exact mechanism was still unclear.METHODS CCK-8 assay was used to detect the cell viability.The levels of proteins and mRNA were evaluated using Western blot and q-PCR.Mitochondrial membrane potential was measured by flow cytometry.Depolymerisa.tion of F-actin and translocation of G-actin(gamma-actin) from the cytoplasm to the mitochondria was detected using an immuno fl uorescence assay.RESULTS phosphorylated coflin-1(p-coflin-1) was overexpressed in the DDP-resistant human gastric cancer cell lines SGC7901/DDP and BGC823/DDP,relative to the respective parent cell lines(SGC7901 and BGC823),and DDP induced the dephosphory.lation of p-coflin-1 in both parent lines but not in the DDP-resistant lines.However,ZJW could induce the dephosphorylation of pcoflin-1 and promote coflin-1 translocation from the cytoplasm into the mito.chondria in both SGC7901/DDP and BGC823/DDP cells.This mitochondrial translocation of coflin-1 was found to induce the conversion of flamentous actin to globular-actin,activate mitochondrial dam.age and calcium overloading,and induce the mitochondrial apoptosis pathway.These effects of ZJW on DDP-resistant human gastric cancer cell lines could be reversed via transfection with coflin-1-specifc siRNA,or treatment with a PP1 and PP2A inhibitor.CONCLUSION ZJW can be used as an inhibitor of chemoresistance in gastric cancer,which may partly be due to dephosphorylation of p-coflin-1 via the activation of PP1 and PP2A.展开更多
Current pharmacological therapies used in clinical practice for individuals with cardiac, hepatic, renal or pulmonary fibrosis do not show desirable effects; therefore, new targets of therapy are urgently required. Mi...Current pharmacological therapies used in clinical practice for individuals with cardiac, hepatic, renal or pulmonary fibrosis do not show desirable effects; therefore, new targets of therapy are urgently required. Mitochon- dria play pivotal role in energy production, metabolism, serving as storage tanks of calcium ions and cell fate deter- mination. Recent results of our experiments and other authors＇ studies suggest that the electron transport chain dys- function, electron leakage increasing, the accumulation of fragmented mitochondria, and the impairment in the mi- tophagy system contribute to pathogenesis of tissue fibrosis. Mitochondria targeting compounds exert promise in trea- ring and attenuating the progress of these diseases, indicating potential strategies to target mitochondria in the treat- ment of tissue fibrosis.展开更多
The appearance and hair color between these two subspecies Rattus rattus Sladeni and R.r. Hainanicus are similar to each other. Their most major distinctive characteristic is the length ratio of tail to body. However,...The appearance and hair color between these two subspecies Rattus rattus Sladeni and R.r. Hainanicus are similar to each other. Their most major distinctive characteristic is the length ratio of tail to body. However, this characteristic was unstable in some measuring records. In Guangdong, R.r. Hainanicus is restrictedly distributed in the west region, and R.r. Sladeni is widely distributed in the other regions of this province. In this study, we detected the sequences of mitochondrial 12S rRNA gene fragments of 9 samples from R.r. Hainanicus and R.r. Sladeni (Longmen and Hong Kong populations). These 385 nucleotide positions of 12S rRNA gene fragment include 26 variable sites and 14 parsimony-informative sites. 3 insertion/deletion sites are observed. The phylogenetic relationships among these samples were constructed by Neighbor-joining and Maximum parsimony methods. The analysis shows that R.r. Hainanicus is more closely relative to the Longmen population of R.r. Sladeni than to the Hong Kong population of R.r. Sladeni. The sequencing analysis of 12S rRNA gene fragments is not agreement on the classification of subspecies R. r. Hainanicus inferred from the morphology and geographical distribution. The morphological variation of R.r. Hainanicus should result from the natural selection, which causes local adaptation and geographic isolation.展开更多
There are many kinds of Chinese patent medicine used to fight against influenza efficiently in clinical practice. However, little experimental data confirmed the anti-influenza the activities since non-suitable animal...There are many kinds of Chinese patent medicine used to fight against influenza efficiently in clinical practice. However, little experimental data confirmed the anti-influenza the activities since non-suitable animal model and in vitro antiviral experiments. This paradox can be explained by host factors are important in the patho- genesis and outcome of influenza infection. Accordingly, we set up a mouse model by using restraint stress plus vi- ral infection, which is more conducive to simulate the clinical features of susceptible population and evaluate the activities of Chinese herbal medicine. Our results demonstrated that stress-induced corticosterone （CORT） , a stres- sor sensor, increased the morbidity and the mortality of virus infected mice loaded with restraint stress. CORT also increased expression of Mfn2, and accordingly decreased mitochondrial antiviral signaling （MAVS） aggregates in the host cells. Mfn2 overexpression increased NP and decreased IFN-β and IFITM3 protein expressions in influenza virus infected A549 cells. These findings suggested that the mechanism of restraint stress increased the susceptibili- ty due to CORT induces activation of Mfn2 mediated MAVS pathway.展开更多
OBJECTIVE To elucidate the molecular mechanism and the anti-breast cancer effect of polyphyllinⅠ,which is a natural compound extracted from Rhizoma of Paris polyphyllin.METHODS Human breast cancer cells were treated ...OBJECTIVE To elucidate the molecular mechanism and the anti-breast cancer effect of polyphyllinⅠ,which is a natural compound extracted from Rhizoma of Paris polyphyllin.METHODS Human breast cancer cells were treated with polyphyllinⅠ,after which DRP1-dependent mitochondrial fission and apoptosis,mitophagy and PINK1/PARK2 pathway were evaluated.A genetic approach was employed to determine how knockdown of PINK1 with sh RNA regulates polyphyllinⅠ-induced mitophagy and apoptosis.The inhibitory effect of polyphyllinⅠon tumor growth in a breast cancer cell xenograft mouse model was also examined.RESULTS PolyphyllinⅠenhanced the stabilization of full-length PINK1at the mitochondrial surface,leading to PARK2 recruitment to mitochondria,and culminating in mitophagy.PolyphyllinⅠalso induced dephosphorylation of DRP1 at Ser637 and mitochondrial translocation of DRP1,leading to mitochondrial fission and apoptosis.Knockdown of PINK1 evidently suppressed mitophagy stimulated by polyphyllinⅠ,and markedly enhanced DRP1-dependent mitochondrial fission and apoptosis induced by polyphyl inⅠ.Furthermore,suppression of DRP1 by mdivi-1 or sh RNA inhibits PINK1 knockdown-mediated mitochondrial fragmentation and apoptosis in response to polyphyllinⅠtreatment,suggesting that depletion of PINK1 lead to mitochondrial fragmentation due to excessive fission.Our in vivo study also showed that knockdown of PINK1potentiated polyphyllinⅠ-mediated inhibition of tumor growth in a breast cancer cell xenograft mouse model.CONCLUSION Our study provides a mechanism to support the role of PINK1 in the regulation of polyphyl inⅠ-induced mitophagy and apoptosis,and suggest polyphylinⅠas a potential drug for treatment of breast cancer.展开更多
OBJECTIVE Refractory wounds in diabetic patients constitute a serious complication that often leads to amputation with limited treatment regimens.Recent studies have shown that the imbalance of mitochondrial dynamics ...OBJECTIVE Refractory wounds in diabetic patients constitute a serious complication that often leads to amputation with limited treatment regimens.Recent studies have shown that the imbalance of mitochondrial dynamics was associated with the increased reactive oxygen species(ROS)production in endothelial cells,which is a significant contributor to the microvascular complications of diabetes.The present study was designed to determine the involvement of transcription factor FOXO1in diabetic wound healing and investigate underlying mechanisms.METHODS&RESULTS Impaired mitochondrial networks and increased phosphorylation of dynaminrelated protein-1(Drp1)at ser616,a protein required for mitochondrial fission,were observed in human umbilical vein endothelial cells(HUVECs)24 h after exposure to high concentrations of glucose.Inhibition of FOXO1 by si RNA or by FOXO1 selective inhibitor AS1842856 abrogated high glucos-induced alterations in mitochondrial networks and phosphorylation of Drp1.Treatment with AS1842856 or si RNA of FOXO1 could significantly increase the mitochondrial membrane potential and suppress the overproduction of ROS induced by high glucose.Addition of AS1842856 inhibited glucoseinduced apoptosis,ameliorated capillary tube formation in HUVECs.In vivo,AS1842856 dose-dependently rescued the delay of wound closure in diabetic mice,and5 mg·kg-1of AS1842856 treatment significantly increased the mean perfusion rate.CONCLUSION These findings suggested that FOXO1 is critical to preserve mitochondrial quantity and function in endothelial cells,inhibition of FOXO1 rescued the delayed wound healing and improved wound angiogenesis in diabetic mice.展开更多
OBJECTIVE Shenmai Injection(SMI)is widely used in the treatment of cardiovascular diseases,such as heart failure and myocardial ischemia.In clinic,SMI showed protective effects on doxorubicin(Dox)-induced cardiac toxi...OBJECTIVE Shenmai Injection(SMI)is widely used in the treatment of cardiovascular diseases,such as heart failure and myocardial ischemia.In clinic,SMI showed protective effects on doxorubicin(Dox)-induced cardiac toxicity.In current study,we investigate the mitochondrial protective mechanisms of SMI on Dox-induced myocardial injury.METHODS C57BL/6 mice were divided into four groups:①control group;②Dox injury group;③SMI+Dox group and dexrazoxane(DRZ)+Dox group.Dex was a positive control.Myocardial injury was evaluated by echocardiography,HE and TUNEL staining,myocardial markers measurement.H9C2 cardiomyocytes pretreatment with SMI for 24 h were exposed to Dox.Cell viability and apoptosis were measured by CCK8,Hoechst33342 staining,and Annexin V/PI staining.MitoSOX,mitochondrial membrane potential,and mitochondrial respiratory function were measured to evaluate mito⁃chondrial function.RESULTS SMI decreased mortality rate of Dox-injected mice,serum CK and CK-MB levels in vivo.SMI significantly prevented Dox-induced cardiac dysfunction and apoptosis and increased expression level of PI3K,p-Akt,and p-GSK-3β.Moreover,SMI significantly inhibited Dox-induced apoptosis,mitochondrial ROS production,and reduction of mitochondrial membrane potential in H9C2 cells.Mechanismly,the cardio-protective effect of SMI was suppressed by PI3K inhibitor LY294002.CONCLUSION SMI prevents Dox-induced cardiotoxicity and mitochondrial damage through activation of PI3K/Akt signaling pathway.展开更多
OBJECTIVE Peroxisome proliferator activated receptor alpha(PPARα)is an important protective factor in neurovascular diseases such as ischemic stroke.Although PPARαexpression is higher in neurons than astrocytes and ...OBJECTIVE Peroxisome proliferator activated receptor alpha(PPARα)is an important protective factor in neurovascular diseases such as ischemic stroke.Although PPARαexpression is higher in neurons than astrocytes and microglia,the pathophysiological functions of neuronal specific-PPARαin isch⁃emic stroke remains unknown.Here,we report that neuronal PPARαdeficiency is a key factor of neuronal injury.PPARαexpression markedly decreased in neurons after ischemic stroke.METHODS AND RESULTS Neuronal-specific PPARαknockout(NCKO)exacerbates neuronal damage and brain ischemic injury.PPARαdefi⁃ciency disrupts axonal microtubule organization and mitochondrial transport by decreasing the expression of dynein light chain Tctex-type 1(Dynlt1),which is implicated in cytoprotective role with damaged neurons.Furthermore,resto⁃ration of Dynlt1 expression in neurons of NCKO mice rescue mitochondrial transport disorder,cognitive deficits and brain ischemic injury asso⁃ciated with PPARαdeletion.CONCLUSION These results reveal a critical role for neuronal PPARαin ischemic brain injury by modulating axonal mitochondrial transportation.展开更多
Isolation of high-quality mitochondrial DNA（mtDNA） is an important premise for researching molecular mechanisms in cytoplasmic male sterility of cabbage（Brassica oleracea L.var.capitata）. An efficient protocol for...Isolation of high-quality mitochondrial DNA（mtDNA） is an important premise for researching molecular mechanisms in cytoplasmic male sterility of cabbage（Brassica oleracea L.var.capitata）. An efficient protocol for separation and purification of mitochondria and extraction of mitochondrial DNA（mtDNA） from etiolated tissues of cabbage was developed. We took a method combined mannitol density gradient with differential centrifugation, selected appropriate rotational speed, extended DNase I treating time and changed mitochondria cracking condition. The results showed that the extracted mitochondria in this protocol had complete structure, appeared to ellipsoid and had not been contaminated with other impurities under the Jannus Green B staining. The isolated mitochondrial DNA had high purity and yield through detecting the optical density, nuclear specific primer PCR and agarose gel electrophoresis. The results indicated that mitochondrial DNA extracted by this protocol had high quality and enabled to be used in futher genetic studies.展开更多
The genetic differences of 15 mitten crab populations from 6 river systems in China's Mainland and 1 population from Russia were studied based on RFLP analysis of mitochondrial cytochrome oxidase subunit I （CO I...The genetic differences of 15 mitten crab populations from 6 river systems in China's Mainland and 1 population from Russia were studied based on RFLP analysis of mitochondrial cytochrome oxidase subunit I （CO I）. The results showed that Tas I-RFLP pattern could be used as a genetic marker to distinguish Eriocheir hepuensis from Eriocheir sinensis, Eriocheirjaponica and Eriocheir leptognathus; genetic distances among 13 populations of Eriocheir sinensis range from 0 to 0.015, indicating that they were different geographic strains; the subspecies status of Eriocheir sinensis and Eriocheir hepuensis （population from Nanliujiang） were considered owning to their genetic distances of 0.02-0.044, indicating that genetic divergence between them was low; Eriocheir leptognathus （population from Nanpaihe, Tianjin） was the most distant taxon with genetic distances value of 0.147-0.195, which could be defined as genetic distances between species in genus Eriocheir.展开更多
Mitochondrial dysfunction is an early prominent feature in susceptible neurons in the brain of patients with Alzheimer′s disease which likely plays a critical role in the pathogenesis of disease. Mitochondria are dyn...Mitochondrial dysfunction is an early prominent feature in susceptible neurons in the brain of patients with Alzheimer′s disease which likely plays a critical role in the pathogenesis of disease. Mitochondria are dynamic organelles and the balance of mitochondrial fission and fusion determines Our initial studies revealed an imbalance in mitochondrial fission and fusion in fibroblasts from sporadic AD patients compared with normal healthy fibroblasts from age-matched control patients. Later it was demonstrated that overexpression of familial Alzheimer disease(FAD)-causing AβPP mutant or exposure to soluble Aβ oligomers led to mitochondrial fragmentation and redistribution in neuronal cells along with altered expression of mitochondrial fission/fusion proteins. Marked mitochondrial fragmentation and abnormal mitochondrial distribution in the pyramidal neurons along with mitochondrial dysfunction in the brain of AD mouse model CRND8 as early as three months of age before the accumulation of amyloid pathology. Importantly,we demonstrate significant changes in the expression and distribution of mitochondrial fission and fusion proteins in vivo in AD in consistent with a shifted mitochondrial dynamics towards excessive fission. Most recently,we demonstrated that genetic and pharmaceutical methods to rescue mitochondrial morphology and distribution could effectively restore Aβ-induced mitochondrial function and alleviate synaptic dysfunction both in vitro and in vivo,suggesting a causal involvement of mitochondrial dynamics in mediating Aβ-induced mitochondrial dysfunction. Taken together,we suggest that such a fundamental shift in mitochondrial dynamics negatively impacts all aspect of mitochondrial function such as impaired bioenergetics,increased structural damage and ROS production and loss of mt DNA integrity which causes synaptic dysfunction and neuronal dysfunction that is critical to AD pathogenesis. Therefore,strategies to modify abnormal mitochondrial dynamics may be an attractive therapeutic intervention target for AD.展开更多
The patterns of cleavage of mtDNA by restriction endonucleases was analysed for six fowl breeds and of. Hyline White, Hyline Brown, ISA Brown, Hisex Brown, Lohmann White, Abor Acres and mtDNA polymorphisms were detect...The patterns of cleavage of mtDNA by restriction endonucleases was analysed for six fowl breeds and of. Hyline White, Hyline Brown, ISA Brown, Hisex Brown, Lohmann White, Abor Acres and mtDNA polymorphisms were detected in the restriction patterns with the following eight enzymes, Ava Ⅰ, Ava Ⅱ, EcoR Ⅰ, Hind Ⅲ, Bam HI, Pvu Ⅱ, Pst Ⅰ, Hinc Ⅱ. The restriction cleavage patterns were identical among these breeds. Hyline White, Hyline Brown, ISA Brown, Hisex Brown, Lohmann White—A type. The patterns of Abor Acres were B type. Based on their mtDNA restriction types, all the breeds were classified into two groups. Genetic distances among these groups were calculated in order to define their phylogenetic relationships. The relationship among five egg line breeds is close, while Abor Acres (Broiler fowl) is relatively far from them. The results suggest that the difference of mtDNA could result from the different origins. The polymorphic sites in mtDNA of Hyline White has been located on a restriction map.展开更多
OBJECTIVE The cause of Parkinson disease (PD) is generally not clear, but it is considered to be related to excessive oxidative damage. Therefore, the identification of therapeutic targets and compounds with antioxida...OBJECTIVE The cause of Parkinson disease (PD) is generally not clear, but it is considered to be related to excessive oxidative damage. Therefore, the identification of therapeutic targets and compounds with antioxidant damage is a reasonable strategy to slow down the progress of PD. FCPR16 is a novel phosphodiesterase4 inhibitor with little emetic potential. Our previous studies showed that FCPR16 was an effective compound for blocking 1-methyl-4-phenylpyridine(MPP+)-induced oxidative damage in SH-SY5Y cells and neurons. However, the detailed mechanisms underlying its protective effect have not been investigated. The level of oxidative stress in neurons is closely related to the balance of mitochondria mass, while autophagy strongly regulates mitochondrial activity in neurons. Our previous study indicated that inhibition of PDE4 or PDE4 knockdown enhanced the activation of autophagy in microglial cells. While whether PDE4 inhibition mediates autophagy in neurons is largely unknown. As described above, autophagy plays a pivotal role in maintaining redox and mitochondrial homeostasis. We were interested in exploring the impact of PDE4 inhibition on autophagy in neurons. METHODS SH-SY5Y cells and neurons was induced with MPP+to mimic PD cell injury in vitro, and MTT assay was used to investigate the viability effects of FCPR16 (50μmol·L-1) with or without different autophagy inhibitors on MPP+-injured SH-SY5Y cells. Detection of apoptosis was performed by PI staining fluorescence. Lysosomes are essential in autophagy, so LYT Red Stain was used to detect lysosomes in SY5Y cells and neurons. Cells were exposed to Cell ROX Deep Red Reagent to detect intracellular reactive oxygen species (ROS). Mitochondrial membrane potential (Δψm)measurement was executed by 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazolyl-carbocyanineiodide (JC-1).To better detect intracellular autophagy, we used the CYTO-ID Autophagy detection kit to detect the autophagic vacuoles and monitor autophagic flux. The expression of autophagy related proteins and other related signal molecules were demonstrated by Western blot. As relevant indicators of oxidative stress, 3-nitrotyrosine (3-NT) and highly toxic peroxide4-hydroxynonenal (4-HNE) were detected with 3-NT and 4-HNE ELISA kits. RESULTS FCPR16 could significantly decrease the expression of p62, an autophagy substrate, at 6 and 12 h, while FCPR16 enhanced the level of LC3-Ⅱ. Similarly, FCPR16 increased the lysosomes fluorescence and CYTO-ID signal in cells and neurons, while it could be blockby 3-methyladenine (3-MA) and hydroxychloroquine sulfate (HCQ). Simultaneously, Treatment of SH-SY5Y cells with FCPR16 prevented MPP+-induced production of ROS and the decline ofΔψm. Importantly, we also found that FCPR16 phosphorylated and thus activated AMPK in SH-SY5Y cells treated with MPP+. In contrast, blockade of the AMPK pathway with compound C blocked the role of FCPR16 in autophagy enhancement. MPP+-induced a significant increase in PI-positive cells, while FCPR16 decreased the ratio of PI positive cells and 3-MA and compound C could block the protective effect. Additionally, FCPR16 reduced MPP+-induced decline of cell viability, and 3-MA and compound C could block the protective effect. CONCLUSION Deficits in autophagy have been proven to participate the pathology of PD and targeting autophagic function has been viewed as a potential therapeutic strategy for the clearance of toxic proteins (such asα-synuclein) and of impaired mitochondria. this is the first time that PDE4 inhibition has been shown to induce autophagic enhancement both in SH-SY5Y cells and in primary cultured neurons. In addition, our findings indicate that inhibition of PDE4 by FCPR16 protects against MPP+-induced oxidative stress and cellular injury in SH-SY5Y cells and neurons through the activation of AMPK-dependent autophagy. Taken together, these results show that PDE4 is a promising target for developing novel drugs against neuronal apoptosis and FCPR16 may be a potential compound for the prevention and treatment of PD.展开更多
Previous studies have shown that the bcl-2protooncogene encodes a mitochondrial protein thatpromotes cell survival by blocking programmed celldeath. High levels of bcl-2 expression were found inmurine myeloid leukemic...Previous studies have shown that the bcl-2protooncogene encodes a mitochondrial protein thatpromotes cell survival by blocking programmed celldeath. High levels of bcl-2 expression were found inmurine myeloid leukemic cell lines resistant to apoptosisinduced by variety of agents. In addition artificialelevation of bcl-2 expression in a human pre-B leukemiccell line resulted in enhanced resistance to chemo-therapcutic drugs. We reported here on the effect ofbcl-2 antisense phosphorothiate展开更多
The present study is to investigate the role of Puma in cardiomyocyte death induced by hypoxia/reoxygenation(H/R).We found that H/R increased the level of Puma mRNA and protein accompanied by the elevation of cardiomy...The present study is to investigate the role of Puma in cardiomyocyte death induced by hypoxia/reoxygenation(H/R).We found that H/R increased the level of Puma mRNA and protein accompanied by the elevation of cardiomyocyte death.Inhibition of endogenous Puma by siRNA attenuated H/R-induced cell death.Puma stimulated caspase-8 activation.展开更多
北京大学系统生物医学研究所尹玉新课题组的研究取得突破，揭示了真核生物中新的蛋白编码机制，并由此发现PTEN家族的新蛋白PTENα，进一步证实其定位于线粒体，参与调控细胞能量代谢过程。该研究成果于2014年5月以封面文章形式发表于C...北京大学系统生物医学研究所尹玉新课题组的研究取得突破，揭示了真核生物中新的蛋白编码机制，并由此发现PTEN家族的新蛋白PTENα，进一步证实其定位于线粒体，参与调控细胞能量代谢过程。该研究成果于2014年5月以封面文章形式发表于Cell的子刊Cell Metabolism上（ Liang H， He S， Yang J， et al．PTENα， a PTEN isoform translated through alternative initiation ， regulates mitochondrial function and energy metabolism．2014，19（5）：836-848），这一工作获得了Nature Reviews Molecular Cell Biology的述评。展开更多
PCR has been a general preferred method for biological research in fish,and previous research have enabled us to extract and purify PCR-quality DNA templates in laboratories [1-4].The same problem among these procedur...PCR has been a general preferred method for biological research in fish,and previous research have enabled us to extract and purify PCR-quality DNA templates in laboratories [1-4].The same problem among these procedures is waiting for tissue digesting for a long time.The overabundance time spent on PCR-quality DNA extraction restricts the efficiency of PCR assay,especially in large-scale PCR amplification,such as SSR-based genetic-mapping construction [5,6],identification of germ plasm resource[7,8] and evolution research [9,10],etc.In this study,a stable and rapid PCR-quality DNA extraction method was explored,using a modified alkaline lysis protocol.Extracting DNA for PCR only takes approximately 25 minutes.This stable and rapid DNA extraction method could save much laboratory time and promotes.展开更多
基金NationalNatural Science Foundation of China (81773698)Funding from Guangzhou Science and Technology Department (2015B020211007,201604020112).
文摘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.
基金supported by National Natural Science Foundation of China(81473481)Construct Program of the Key Discipline of State Administration of Traditional Chinese Medicine of People′s Republic of Chinathe Science Foundation for Shanghai Municipal Health and Family Planning Commission(20124065)
文摘OBJECTIVE Despite the status of cisplatin(DDP) as a classical chemotherapeutic agent in the treatment of cancer,the development of multidrug resistance often leads to a failure of DDP therapy.Traditional Chinese medicine(TCM) as adjuvant chemotherapy of cancer drugs in China has been widely used in cancer treatment.ZuoJin WAN(ZJW),a TCM formula,was proved reversing drug resistance in gastric cancer,but its exact mechanism was still unclear.METHODS CCK-8 assay was used to detect the cell viability.The levels of proteins and mRNA were evaluated using Western blot and q-PCR.Mitochondrial membrane potential was measured by flow cytometry.Depolymerisa.tion of F-actin and translocation of G-actin(gamma-actin) from the cytoplasm to the mitochondria was detected using an immuno fl uorescence assay.RESULTS phosphorylated coflin-1(p-coflin-1) was overexpressed in the DDP-resistant human gastric cancer cell lines SGC7901/DDP and BGC823/DDP,relative to the respective parent cell lines(SGC7901 and BGC823),and DDP induced the dephosphory.lation of p-coflin-1 in both parent lines but not in the DDP-resistant lines.However,ZJW could induce the dephosphorylation of pcoflin-1 and promote coflin-1 translocation from the cytoplasm into the mito.chondria in both SGC7901/DDP and BGC823/DDP cells.This mitochondrial translocation of coflin-1 was found to induce the conversion of flamentous actin to globular-actin,activate mitochondrial dam.age and calcium overloading,and induce the mitochondrial apoptosis pathway.These effects of ZJW on DDP-resistant human gastric cancer cell lines could be reversed via transfection with coflin-1-specifc siRNA,or treatment with a PP1 and PP2A inhibitor.CONCLUSION ZJW can be used as an inhibitor of chemoresistance in gastric cancer,which may partly be due to dephosphorylation of p-coflin-1 via the activation of PP1 and PP2A.
文摘Current pharmacological therapies used in clinical practice for individuals with cardiac, hepatic, renal or pulmonary fibrosis do not show desirable effects; therefore, new targets of therapy are urgently required. Mitochon- dria play pivotal role in energy production, metabolism, serving as storage tanks of calcium ions and cell fate deter- mination. Recent results of our experiments and other authors＇ studies suggest that the electron transport chain dys- function, electron leakage increasing, the accumulation of fragmented mitochondria, and the impairment in the mi- tophagy system contribute to pathogenesis of tissue fibrosis. Mitochondria targeting compounds exert promise in trea- ring and attenuating the progress of these diseases, indicating potential strategies to target mitochondria in the treat- ment of tissue fibrosis.
文摘The appearance and hair color between these two subspecies Rattus rattus Sladeni and R.r. Hainanicus are similar to each other. Their most major distinctive characteristic is the length ratio of tail to body. However, this characteristic was unstable in some measuring records. In Guangdong, R.r. Hainanicus is restrictedly distributed in the west region, and R.r. Sladeni is widely distributed in the other regions of this province. In this study, we detected the sequences of mitochondrial 12S rRNA gene fragments of 9 samples from R.r. Hainanicus and R.r. Sladeni (Longmen and Hong Kong populations). These 385 nucleotide positions of 12S rRNA gene fragment include 26 variable sites and 14 parsimony-informative sites. 3 insertion/deletion sites are observed. The phylogenetic relationships among these samples were constructed by Neighbor-joining and Maximum parsimony methods. The analysis shows that R.r. Hainanicus is more closely relative to the Longmen population of R.r. Sladeni than to the Hong Kong population of R.r. Sladeni. The sequencing analysis of 12S rRNA gene fragments is not agreement on the classification of subspecies R. r. Hainanicus inferred from the morphology and geographical distribution. The morphological variation of R.r. Hainanicus should result from the natural selection, which causes local adaptation and geographic isolation.
文摘There are many kinds of Chinese patent medicine used to fight against influenza efficiently in clinical practice. However, little experimental data confirmed the anti-influenza the activities since non-suitable animal model and in vitro antiviral experiments. This paradox can be explained by host factors are important in the patho- genesis and outcome of influenza infection. Accordingly, we set up a mouse model by using restraint stress plus vi- ral infection, which is more conducive to simulate the clinical features of susceptible population and evaluate the activities of Chinese herbal medicine. Our results demonstrated that stress-induced corticosterone （CORT） , a stres- sor sensor, increased the morbidity and the mortality of virus infected mice loaded with restraint stress. CORT also increased expression of Mfn2, and accordingly decreased mitochondrial antiviral signaling （MAVS） aggregates in the host cells. Mfn2 overexpression increased NP and decreased IFN-β and IFITM3 protein expressions in influenza virus infected A549 cells. These findings suggested that the mechanism of restraint stress increased the susceptibili- ty due to CORT induces activation of Mfn2 mediated MAVS pathway.
基金The project supported by National Natural Science Foundation of China(81402970,81402202,81402013,81202869)
文摘OBJECTIVE To elucidate the molecular mechanism and the anti-breast cancer effect of polyphyllinⅠ,which is a natural compound extracted from Rhizoma of Paris polyphyllin.METHODS Human breast cancer cells were treated with polyphyllinⅠ,after which DRP1-dependent mitochondrial fission and apoptosis,mitophagy and PINK1/PARK2 pathway were evaluated.A genetic approach was employed to determine how knockdown of PINK1 with sh RNA regulates polyphyllinⅠ-induced mitophagy and apoptosis.The inhibitory effect of polyphyllinⅠon tumor growth in a breast cancer cell xenograft mouse model was also examined.RESULTS PolyphyllinⅠenhanced the stabilization of full-length PINK1at the mitochondrial surface,leading to PARK2 recruitment to mitochondria,and culminating in mitophagy.PolyphyllinⅠalso induced dephosphorylation of DRP1 at Ser637 and mitochondrial translocation of DRP1,leading to mitochondrial fission and apoptosis.Knockdown of PINK1 evidently suppressed mitophagy stimulated by polyphyllinⅠ,and markedly enhanced DRP1-dependent mitochondrial fission and apoptosis induced by polyphyl inⅠ.Furthermore,suppression of DRP1 by mdivi-1 or sh RNA inhibits PINK1 knockdown-mediated mitochondrial fragmentation and apoptosis in response to polyphyllinⅠtreatment,suggesting that depletion of PINK1 lead to mitochondrial fragmentation due to excessive fission.Our in vivo study also showed that knockdown of PINK1potentiated polyphyllinⅠ-mediated inhibition of tumor growth in a breast cancer cell xenograft mouse model.CONCLUSION Our study provides a mechanism to support the role of PINK1 in the regulation of polyphyl inⅠ-induced mitophagy and apoptosis,and suggest polyphylinⅠas a potential drug for treatment of breast cancer.
基金The project supported by National Natural Science Foundation of China(81373405,30901803)Beijing Higher Education Young Elite Teacher Project(YETP0053)
文摘OBJECTIVE Refractory wounds in diabetic patients constitute a serious complication that often leads to amputation with limited treatment regimens.Recent studies have shown that the imbalance of mitochondrial dynamics was associated with the increased reactive oxygen species(ROS)production in endothelial cells,which is a significant contributor to the microvascular complications of diabetes.The present study was designed to determine the involvement of transcription factor FOXO1in diabetic wound healing and investigate underlying mechanisms.METHODS&RESULTS Impaired mitochondrial networks and increased phosphorylation of dynaminrelated protein-1(Drp1)at ser616,a protein required for mitochondrial fission,were observed in human umbilical vein endothelial cells(HUVECs)24 h after exposure to high concentrations of glucose.Inhibition of FOXO1 by si RNA or by FOXO1 selective inhibitor AS1842856 abrogated high glucos-induced alterations in mitochondrial networks and phosphorylation of Drp1.Treatment with AS1842856 or si RNA of FOXO1 could significantly increase the mitochondrial membrane potential and suppress the overproduction of ROS induced by high glucose.Addition of AS1842856 inhibited glucoseinduced apoptosis,ameliorated capillary tube formation in HUVECs.In vivo,AS1842856 dose-dependently rescued the delay of wound closure in diabetic mice,and5 mg·kg-1of AS1842856 treatment significantly increased the mean perfusion rate.CONCLUSION These findings suggested that FOXO1 is critical to preserve mitochondrial quantity and function in endothelial cells,inhibition of FOXO1 rescued the delayed wound healing and improved wound angiogenesis in diabetic mice.
基金National Natural Science Foundation of China(8177401781202779+2 种基金81973624)Natural Science Foundation of Tianjin City(19JCYBJC28200)the Scientific Research Project of Tianjin Education Commission(2017KJ140)
文摘OBJECTIVE Shenmai Injection(SMI)is widely used in the treatment of cardiovascular diseases,such as heart failure and myocardial ischemia.In clinic,SMI showed protective effects on doxorubicin(Dox)-induced cardiac toxicity.In current study,we investigate the mitochondrial protective mechanisms of SMI on Dox-induced myocardial injury.METHODS C57BL/6 mice were divided into four groups:①control group;②Dox injury group;③SMI+Dox group and dexrazoxane(DRZ)+Dox group.Dex was a positive control.Myocardial injury was evaluated by echocardiography,HE and TUNEL staining,myocardial markers measurement.H9C2 cardiomyocytes pretreatment with SMI for 24 h were exposed to Dox.Cell viability and apoptosis were measured by CCK8,Hoechst33342 staining,and Annexin V/PI staining.MitoSOX,mitochondrial membrane potential,and mitochondrial respiratory function were measured to evaluate mito⁃chondrial function.RESULTS SMI decreased mortality rate of Dox-injected mice,serum CK and CK-MB levels in vivo.SMI significantly prevented Dox-induced cardiac dysfunction and apoptosis and increased expression level of PI3K,p-Akt,and p-GSK-3β.Moreover,SMI significantly inhibited Dox-induced apoptosis,mitochondrial ROS production,and reduction of mitochondrial membrane potential in H9C2 cells.Mechanismly,the cardio-protective effect of SMI was suppressed by PI3K inhibitor LY294002.CONCLUSION SMI prevents Dox-induced cardiotoxicity and mitochondrial damage through activation of PI3K/Akt signaling pathway.
文摘OBJECTIVE Peroxisome proliferator activated receptor alpha(PPARα)is an important protective factor in neurovascular diseases such as ischemic stroke.Although PPARαexpression is higher in neurons than astrocytes and microglia,the pathophysiological functions of neuronal specific-PPARαin isch⁃emic stroke remains unknown.Here,we report that neuronal PPARαdeficiency is a key factor of neuronal injury.PPARαexpression markedly decreased in neurons after ischemic stroke.METHODS AND RESULTS Neuronal-specific PPARαknockout(NCKO)exacerbates neuronal damage and brain ischemic injury.PPARαdefi⁃ciency disrupts axonal microtubule organization and mitochondrial transport by decreasing the expression of dynein light chain Tctex-type 1(Dynlt1),which is implicated in cytoprotective role with damaged neurons.Furthermore,resto⁃ration of Dynlt1 expression in neurons of NCKO mice rescue mitochondrial transport disorder,cognitive deficits and brain ischemic injury asso⁃ciated with PPARαdeletion.CONCLUSION These results reveal a critical role for neuronal PPARαin ischemic brain injury by modulating axonal mitochondrial transportation.
基金Supported by Funding of Utilization of Heterosis and Breeding of New Variety in Brassicaceous Vegetable(2012BAD02B01)
文摘Isolation of high-quality mitochondrial DNA（mtDNA） is an important premise for researching molecular mechanisms in cytoplasmic male sterility of cabbage（Brassica oleracea L.var.capitata）. An efficient protocol for separation and purification of mitochondria and extraction of mitochondrial DNA（mtDNA） from etiolated tissues of cabbage was developed. We took a method combined mannitol density gradient with differential centrifugation, selected appropriate rotational speed, extended DNase I treating time and changed mitochondria cracking condition. The results showed that the extracted mitochondria in this protocol had complete structure, appeared to ellipsoid and had not been contaminated with other impurities under the Jannus Green B staining. The isolated mitochondrial DNA had high purity and yield through detecting the optical density, nuclear specific primer PCR and agarose gel electrophoresis. The results indicated that mitochondrial DNA extracted by this protocol had high quality and enabled to be used in futher genetic studies.
基金Tianjin Development Foundation of Science and Technology and International Cooperation Foundation(033803511,033803511G)Tianjin Higher Educational Development Foundation(2004BA31)
文摘The genetic differences of 15 mitten crab populations from 6 river systems in China's Mainland and 1 population from Russia were studied based on RFLP analysis of mitochondrial cytochrome oxidase subunit I （CO I）. The results showed that Tas I-RFLP pattern could be used as a genetic marker to distinguish Eriocheir hepuensis from Eriocheir sinensis, Eriocheirjaponica and Eriocheir leptognathus; genetic distances among 13 populations of Eriocheir sinensis range from 0 to 0.015, indicating that they were different geographic strains; the subspecies status of Eriocheir sinensis and Eriocheir hepuensis （population from Nanliujiang） were considered owning to their genetic distances of 0.02-0.044, indicating that genetic divergence between them was low; Eriocheir leptognathus （population from Nanpaihe, Tianjin） was the most distant taxon with genetic distances value of 0.147-0.195, which could be defined as genetic distances between species in genus Eriocheir.
文摘Mitochondrial dysfunction is an early prominent feature in susceptible neurons in the brain of patients with Alzheimer′s disease which likely plays a critical role in the pathogenesis of disease. Mitochondria are dynamic organelles and the balance of mitochondrial fission and fusion determines Our initial studies revealed an imbalance in mitochondrial fission and fusion in fibroblasts from sporadic AD patients compared with normal healthy fibroblasts from age-matched control patients. Later it was demonstrated that overexpression of familial Alzheimer disease(FAD)-causing AβPP mutant or exposure to soluble Aβ oligomers led to mitochondrial fragmentation and redistribution in neuronal cells along with altered expression of mitochondrial fission/fusion proteins. Marked mitochondrial fragmentation and abnormal mitochondrial distribution in the pyramidal neurons along with mitochondrial dysfunction in the brain of AD mouse model CRND8 as early as three months of age before the accumulation of amyloid pathology. Importantly,we demonstrate significant changes in the expression and distribution of mitochondrial fission and fusion proteins in vivo in AD in consistent with a shifted mitochondrial dynamics towards excessive fission. Most recently,we demonstrated that genetic and pharmaceutical methods to rescue mitochondrial morphology and distribution could effectively restore Aβ-induced mitochondrial function and alleviate synaptic dysfunction both in vitro and in vivo,suggesting a causal involvement of mitochondrial dynamics in mediating Aβ-induced mitochondrial dysfunction. Taken together,we suggest that such a fundamental shift in mitochondrial dynamics negatively impacts all aspect of mitochondrial function such as impaired bioenergetics,increased structural damage and ROS production and loss of mt DNA integrity which causes synaptic dysfunction and neuronal dysfunction that is critical to AD pathogenesis. Therefore,strategies to modify abnormal mitochondrial dynamics may be an attractive therapeutic intervention target for AD.
文摘The patterns of cleavage of mtDNA by restriction endonucleases was analysed for six fowl breeds and of. Hyline White, Hyline Brown, ISA Brown, Hisex Brown, Lohmann White, Abor Acres and mtDNA polymorphisms were detected in the restriction patterns with the following eight enzymes, Ava Ⅰ, Ava Ⅱ, EcoR Ⅰ, Hind Ⅲ, Bam HI, Pvu Ⅱ, Pst Ⅰ, Hinc Ⅱ. The restriction cleavage patterns were identical among these breeds. Hyline White, Hyline Brown, ISA Brown, Hisex Brown, Lohmann White—A type. The patterns of Abor Acres were B type. Based on their mtDNA restriction types, all the breeds were classified into two groups. Genetic distances among these groups were calculated in order to define their phylogenetic relationships. The relationship among five egg line breeds is close, while Abor Acres (Broiler fowl) is relatively far from them. The results suggest that the difference of mtDNA could result from the different origins. The polymorphic sites in mtDNA of Hyline White has been located on a restriction map.
基金National Natural Science Foundation of China(81773698)Guangzhou Science and Technology Department(2015B020211007 and 201604020112)
文摘OBJECTIVE The cause of Parkinson disease (PD) is generally not clear, but it is considered to be related to excessive oxidative damage. Therefore, the identification of therapeutic targets and compounds with antioxidant damage is a reasonable strategy to slow down the progress of PD. FCPR16 is a novel phosphodiesterase4 inhibitor with little emetic potential. Our previous studies showed that FCPR16 was an effective compound for blocking 1-methyl-4-phenylpyridine(MPP+)-induced oxidative damage in SH-SY5Y cells and neurons. However, the detailed mechanisms underlying its protective effect have not been investigated. The level of oxidative stress in neurons is closely related to the balance of mitochondria mass, while autophagy strongly regulates mitochondrial activity in neurons. Our previous study indicated that inhibition of PDE4 or PDE4 knockdown enhanced the activation of autophagy in microglial cells. While whether PDE4 inhibition mediates autophagy in neurons is largely unknown. As described above, autophagy plays a pivotal role in maintaining redox and mitochondrial homeostasis. We were interested in exploring the impact of PDE4 inhibition on autophagy in neurons. METHODS SH-SY5Y cells and neurons was induced with MPP+to mimic PD cell injury in vitro, and MTT assay was used to investigate the viability effects of FCPR16 (50μmol·L-1) with or without different autophagy inhibitors on MPP+-injured SH-SY5Y cells. Detection of apoptosis was performed by PI staining fluorescence. Lysosomes are essential in autophagy, so LYT Red Stain was used to detect lysosomes in SY5Y cells and neurons. Cells were exposed to Cell ROX Deep Red Reagent to detect intracellular reactive oxygen species (ROS). Mitochondrial membrane potential (Δψm)measurement was executed by 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazolyl-carbocyanineiodide (JC-1).To better detect intracellular autophagy, we used the CYTO-ID Autophagy detection kit to detect the autophagic vacuoles and monitor autophagic flux. The expression of autophagy related proteins and other related signal molecules were demonstrated by Western blot. As relevant indicators of oxidative stress, 3-nitrotyrosine (3-NT) and highly toxic peroxide4-hydroxynonenal (4-HNE) were detected with 3-NT and 4-HNE ELISA kits. RESULTS FCPR16 could significantly decrease the expression of p62, an autophagy substrate, at 6 and 12 h, while FCPR16 enhanced the level of LC3-Ⅱ. Similarly, FCPR16 increased the lysosomes fluorescence and CYTO-ID signal in cells and neurons, while it could be blockby 3-methyladenine (3-MA) and hydroxychloroquine sulfate (HCQ). Simultaneously, Treatment of SH-SY5Y cells with FCPR16 prevented MPP+-induced production of ROS and the decline ofΔψm. Importantly, we also found that FCPR16 phosphorylated and thus activated AMPK in SH-SY5Y cells treated with MPP+. In contrast, blockade of the AMPK pathway with compound C blocked the role of FCPR16 in autophagy enhancement. MPP+-induced a significant increase in PI-positive cells, while FCPR16 decreased the ratio of PI positive cells and 3-MA and compound C could block the protective effect. Additionally, FCPR16 reduced MPP+-induced decline of cell viability, and 3-MA and compound C could block the protective effect. CONCLUSION Deficits in autophagy have been proven to participate the pathology of PD and targeting autophagic function has been viewed as a potential therapeutic strategy for the clearance of toxic proteins (such asα-synuclein) and of impaired mitochondria. this is the first time that PDE4 inhibition has been shown to induce autophagic enhancement both in SH-SY5Y cells and in primary cultured neurons. In addition, our findings indicate that inhibition of PDE4 by FCPR16 protects against MPP+-induced oxidative stress and cellular injury in SH-SY5Y cells and neurons through the activation of AMPK-dependent autophagy. Taken together, these results show that PDE4 is a promising target for developing novel drugs against neuronal apoptosis and FCPR16 may be a potential compound for the prevention and treatment of PD.
文摘Previous studies have shown that the bcl-2protooncogene encodes a mitochondrial protein thatpromotes cell survival by blocking programmed celldeath. High levels of bcl-2 expression were found inmurine myeloid leukemic cell lines resistant to apoptosisinduced by variety of agents. In addition artificialelevation of bcl-2 expression in a human pre-B leukemiccell line resulted in enhanced resistance to chemo-therapcutic drugs. We reported here on the effect ofbcl-2 antisense phosphorothiate
文摘The present study is to investigate the role of Puma in cardiomyocyte death induced by hypoxia/reoxygenation(H/R).We found that H/R increased the level of Puma mRNA and protein accompanied by the elevation of cardiomyocyte death.Inhibition of endogenous Puma by siRNA attenuated H/R-induced cell death.Puma stimulated caspase-8 activation.
文摘北京大学系统生物医学研究所尹玉新课题组的研究取得突破，揭示了真核生物中新的蛋白编码机制，并由此发现PTEN家族的新蛋白PTENα，进一步证实其定位于线粒体，参与调控细胞能量代谢过程。该研究成果于2014年5月以封面文章形式发表于Cell的子刊Cell Metabolism上（ Liang H， He S， Yang J， et al．PTENα， a PTEN isoform translated through alternative initiation ， regulates mitochondrial function and energy metabolism．2014，19（5）：836-848），这一工作获得了Nature Reviews Molecular Cell Biology的述评。
基金Special Fund for Agro-scientific Research in the Public Interest:200903045Natural Science Foundation of China:31101894
文摘PCR has been a general preferred method for biological research in fish,and previous research have enabled us to extract and purify PCR-quality DNA templates in laboratories [1-4].The same problem among these procedures is waiting for tissue digesting for a long time.The overabundance time spent on PCR-quality DNA extraction restricts the efficiency of PCR assay,especially in large-scale PCR amplification,such as SSR-based genetic-mapping construction [5,6],identification of germ plasm resource[7,8] and evolution research [9,10],etc.In this study,a stable and rapid PCR-quality DNA extraction method was explored,using a modified alkaline lysis protocol.Extracting DNA for PCR only takes approximately 25 minutes.This stable and rapid DNA extraction method could save much laboratory time and promotes.
