Study on regulating mechanisms of oxocrebanine obtained from Stephania hainanensis H.S.Lo et Y.Tsoong on microtubule sites and tubulin in human breast cancer MCF-7 cells

Objective:To determine the destructive ability of oxocrebanine,an anti-breast cancer active compound obtained from Stephania hainanensis H.S.Lo et Y.Tsoong,on microtubule network,and investigate the effect of oxocrebanine on microtubule network homeostasis at both molecular and cellular levels.Methods:the EBI site competition method and molecular docking method were used to determine the occupation of the microtubule site of oxocrebanine.Western Blot was used to detect the effect of oxocrebanine on microtubule-associated proteins including STAT3,PAK1,CAMK4,and PKA.Results:The results of EBI site competition assay showed that the binding of EBI toβ-Tubulin covalent fusions produced adducts that appeared in regions of lower molecular weight thanβ-tubulin(ctrl 2).Molecular docking results showed that oxocrebanine could occupy the colchicine site of microtubule proteins.As revealed by Western Blot,the expression of STAT3 protein was decreased after MCF-7 cells have been treated with low,medium,and high concentration of oxocrebanine or the positive drug taxol for 48 h(P<0.01).The expression levels of PAK1 and Camk4 proteins aslo showed significant reductions(P<0.05,or P<0.01).Oxocrebanine also decreased the PKA protein in MCF-7 cells compared to the control group(P<0.01).Conclusions:Oxocrebanine,a ligand that binds at the colchicine site of tubulin,perturbs tubulin polymerization and causes mitosis in MCF-7 cells,thus leading to MCF-7 cell death.Oxocrebanine may promote microtubule dynamics through stathmin by inhibiting the expression levels of STAT3,PAK1,Camk4,and PKA proteins in MCF-7 cells.Oxocrebanine interfers with spindle formation,and ultimately causes mitotic catastrophe in MCF-7 cells.

Two potentially specific but relevant patterns of proteomic change Response of SH-SY5Y cells to differentiation with retinoic acid followed by phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate, and susceptibility of differentiated cells to dopamine

Dopamine （DA） exposure at a dose of 100 pmol/L for 24 hours causes oxidative stress in SH-SY5Y cells with induction of neuronal differentiation by retinoic acid （RA,10 pmol/L,72 hours） followed by phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate （TPA,80 nmol/L,72 hours）. However,it remains unclear whether the alteration of phenotype observed in response to oxidative stress is associated with protein regulation in this cellular model for Parkinson＇s disease. The present study detected protein regulation affected by oxidative stress at a proteomic level：selection of differentially altered proteins using two dimensional difference in-gel electrophoresis and identification of these proteins using matrix assisted laser desorption/ionization time-of-flight mass spectrometry. The results demonstrated significant alterations in expression of six proteins in SH-SY5Y cells following the differentiation and fourteen proteins in the differentiated cells following the exposure,exemplified by an increase of tubulin alpha1 in the former but a decrease of tubulin alpha-ubiquitous chain in the latter. These results suggest that two potentially specific but relevant patterns of proteomic change may be produced in SH-SY5Y cells with the induction of differentiation by RA followed by TPA,and in the differentiated cells after DA exposure.

Protective effect of Fructus Mume total flavone against SH-SY5Y cells damage induced by MPP+and its mechanism

Objective:To investigate the neuroprotective effects of Fructus Mume total flavone(FMF)against cell apoptosis and mitochondrial injury induced by 1-methyl-4-phenylpyridinium(MPP^(+))in human neuroblastoma(SH-SY5Y)cells and explore its molecular mechanisms.Methods:MPP^(+) induced SH-SY5Y cells injury model were established in vitro cell culture,the cells were divided into 5 groups:normal control group,model group(250μmol·L^(-1) MPP^(+)),FMF low-and middle-and high-dose experimental group(10,50,100μmol·L^(-1) FMF).After 72 h administration,4’,6-diamidino-2-phenylindole(DAPI)staining was used to observe the effects of different concentrations of FMF on the morphologic changes of apoptotic cells,the ratio of cell apoptosis was measured by Annexin-FITC/PI double staining.The mitochondrial membrane electro-bit were detected by flow cytometry(FCM).The expression of Bcl-2,Bax and Caspase-3 were detected by Western blot.Results:The results of DAPI staining showed that the injury SH-SY5Y cells induced by MPP+were densely condensed,the nucleus showed nuclear shrinkage,showing an apoptotic characteristic morphology;after 72h of FMF action,the apoptotic morphology of the cells showed different degrees of improvement,and the apoptotic number of SH-SY5Y cells also decreased.Compared with that in the normal control group,the apoptotic rate and of mitochondrial membrane electrobit of SH-SY5Y cells in the model group increased significantly(P<0.01),the expression of Bax and Caspase-3 proteins increased significantly(P<0.01),Bcl-2 protein and the ratio of Bcl-2/Bax decreased significantly(P<0.01).Compared with that in the model group,the apoptotic rate and mitochondrial membrane electro-bit of SH-SY5Y cells in FMF groups(10,50,100μmol·L^(-1))were significantly lower,while Bax and Caspase-3 proteins were significantly lower(P<0.01),and Bcl-2 protein and the ratio of Bcl-2/Bax were significantly higher,with statistically significant difference in FMF middle-and high-dose experimental groups(P<0.01).The results indicated that FMF can decrease the experession level of Bax and Caspase-3 and increase the ratio of Bcl-2/Bax,inhibit MPP+induced apoptosis.Conclusion:FMF improves the damage of SH-SY5Y cells induced by MPP+,and plays a neuroprotective effect by regulating the expressions of related proteins in mitochondrial apoptosis pathway.

Effect of Alteration of Glutathione Content on Cell Viability in α-Synuclein-Transfected SH-SY5Y Cells

It is well known that α-synuclein (αS) plays an important role in the pathogenesis of Parkinson’s disease (PD). Moreover, oxidative stress is also thought to be an important factor in PD due to induction of dopaminergic neuronal cell death by free radicals and enhancement of αS fibrillation by oxidized stress. In the present study, to clarify the role of glutathione (GSH), an intracellular antioxidant, on the molecular mechanism of αS-induced cell injury, we examined the effects of L-buthionine-SR-sulfoximine (BSO), a GSH synthase inhibitor, with or without N-acetyl-L-cysteine (NAC), a source of GSH, on αS-induced cell injury in human neuroblastoma SH-SY5Y cells. Treatment with BSO significantly reduced the cell viability of both empty-vector- and αS-transfected SH-SY5Y cells in a dose-dependent manner (p < 0.01), although the ratio of αS-induced reduction of cell viability in α-syn-transfected cells was much greater than that in empty-vector-transfected cells. Moreover, BSO significantly reduced the intracellular total GSH level in both types of transformant cells. However, NAC significantly prevented BSO-induced reduction of both cell viability and GSH level in the αS-transfected cells. These findings suggest that GSH plays an important role in αS-induced cell injury by reducing cell viability.

亚砷酸钠对SH-SY5Y细胞生长和3MST表达的影响

神经母细胞瘤是儿童最常见的恶性肿瘤之一。因其恶性程度高，手术加化疗的方案对于侵润性的神经母细胞瘤效果不佳。砷，俗名砒霜，很早被用来治疗急性粒性白血病。现在发现砷对神经母细胞瘤、胶质瘤，以及来源于肝脏、前列腺、肾脏、子宫颈和胆囊等固体肿瘤也有效［1］。然而，其作用机制尚未完全明了。有报道，肿瘤细胞H2 S合成酶表达上调［2］。那么，砷是否可通过下调H2 S合成酶的表达发挥抗癌作用？本实验采用SH－SY5Y细胞和亚砷酸钠（ NaAsO2）对此问题进行了探讨。

Is X-linked methyl-CpG binding protein 2 a new target for the treatment of Parkinson's disease?

X-linked methyl-CpG binding protein 2 mutations can induce symptoms similar to those of Parkinson’s disease and dopamine metabolism disorders, but the specific role of X-linked methyl-CpG binding protein 2 in the pathogenesis of Parkinson’s disease remains unknown. In the present study, we used 6-hydroxydopamine-induced human neuroblastoma cell （SH-SY5Y cells） injury as a cell model of Parkinson’s disease. The 6-hydroxydopamine （50 μmol/L） treatment decreased protein levels for both X-linked methyl-CpG binding protein 2 and tyrosine hydroxylase in these cells, and led to cell death. However, overexpression of X-linked methyl-CpG binding protein 2 was able to ameliorate the effects of 6-hydroxydopamine, it reduced 6-hydroxydopamine-induced apoptosis, and increased the levels of tyrosine hydroxylase in SH-SY5Y cells. These findings suggesting that X-linked methyl-CpG binding protein 2 may be a potential therapeutic target for the treatment of Parkinson’s disease.

Discovery of a ULK1 activator that induces autophagy in vitro and in vivo Parkinson＇ s disease models

Aim It has been widely accepted that autophagy plays a key role in some human diseases such as Par- kinson＇ s disease （PD）. UNC-51-1ike kinasesl （ ULK1 ） has been widely reported to initiate autophagy via its com- plex ULKl-mAtg13-FIP200 at the first stage; however, targeting ULK1 as a therapeutic strategy in PD still remains in its infancy. This study aimed at developing a novel ULK1 activator as candidate drugs for PD therapy and valida- ting the possible mechanism and efficacy in vitro and in vivo. Methods Sequence alignment, phylogenetic analy- sis, homology modeling, molecular dockingand structure modificationwere applied forscreening of candidate com- pounds. Surface plasmon resonance （SPR） analysis and molecular dynamics （MD） simulations were carried outto prove the binding betweenULKland BL-UA07. Observations of cell morphology were executed through several methods including MDC staining and GFP-LC3 transfection. Flow cytometric analysis of MDC was used for quantifi- cation of autophagy ratio. Western blot and RNAi transfection were used to explore the detailed mechanisms of BL- UA07-induced autophagy. Furthermore, an in vivo PD mouse model was established for validating the PD treatment efficacy of BL-UA07. Results After a series of screening and structure modification, a novel compound BL-UA07 targeting ULK1 was obtained, which couldeffectivelybind with its target. Then, our results showed that BL-UA07 could induce autophagy via ULK1 complex and decrease damage induced by MPP ~ in SH-SY5Y cells. In addition, in vivomouse model was established to evaluate the protective effect of BL-UA07. The results demonstrated that BL- UA07 has a therapeutic effect on the in vivomouse model without apparent toxicity, which is dependent on the cyto- protective autophagy mediated by ULK1. Conclusion In this study, a novel specific ULK1 activator （BL-UA07） was computationally designed, chemically synthesized and biologically validatedthat could induce cytoprotective au- tophagy in neuroblastoma SH-SYSY cells and in vivo mouse models. Together, these results may uncover this small-molecule compound BL-UA07 as a novel ULK1 activator in autophagy and thus would provide a new clue for exploring more candidate drug targeting ULK1 for future PD therapy.

A Nucleotide-based Drug Protects Against Glutamate-and MPP^(+)-Induced Neurotoxicity

Nucleo CMP Forte? is a nucleotide-based drug consisting of cytidinemonophosphate, uridinemonophosphate, uridin-ediphosphate and uridinetriphosphate. It has been prescribed for peripheral nervous system disorders, such as lum-bosciatalgia, diabetic or alcoholic polyneuropathy, or trigeminal neuralgia. Its effects on brain pathologies has re-ceived little attention. We examined its neuroprotective effects on cell toxicity induced by glutamate excitotoxicity or by 1-methyl-4-phenyl-pyridinium (MPP+), an in vitro cell model of Parkinson’s disease. We used the human dopaminergic cell line SH-SY5Y and a primary culture of rat cortical cells pre-treated with the drug for 24 hours and then exposed to MPP+ or glutamate at a range of concentrations. Cell viability was measured at different times. Nucleo CMP Forte? pre-treatment significantly increased the rate of cell division in SH-SY5Y cells, as well as the synthesis of triglycerides and phospholipids. More interestingly, drug pre-treatment significantly reduced MPP+- and glutamate-induced cell death in SH-SY5Y cells and in rat cortical cells. These results indicate that the nucleotides included in Nucleo CMP Forte? are promising therapeutic molecules for the prevention of neuronal death in brain caused by focal ischemia, Parkinson’s disease or other neurodegenerative pathologies.

SH-SY5Y human neuroblastoma cell line： in vitro cell model of dopaminergic neurons in Parkinson＇s disease

Objective To evaluate the human neuroblastoma SH-SY5Y cell line as an in vitro model of dopaminergic （DAergic） neurons for Parkinson＇s disease （PD） research and to determine the effect of differentiation on this cell model. Date sources The data of this review were selected from the original reports and reviews related to SH-SY5Y cells published in Chinese and foreign journals （Pubmed 1973 to 2009）. Study selection After searching the literature, 60 articles were selected to address this review. Results The SH-SY5Y cell line has become a popular cell model for PD research because this cell line posses many characteristics of DAergic neurons. For example, these cells express tyrosine hydroxylase and dopamine-13-hydroxylase, as well as the dopamine transporter. Moreover, this cell line can be differentiated into a functionally mature neuronal phenotype in the presence of various agents. Upon differentiation, SH-SY5Y cells stop proliferating and a constant cell number is subsequently maintained. However, different differentiating agents induce different neuronal phenotypes and biochemical changes. For example, retinoic acid induces differentiation toward a cholinergic neuronal phenotype and increases the susceptibility of SH-SY5Y cells to neurotoxins and neuroprotective agents, whereas treatment with retinoic acid followed by phorbol ester 12-O-tetradecanoylphorbol-13-acetate results in a DAergic neuronal phenotype and decreases the susceptibility of cells to neurotoxins and neuroprotective agents. Some differentiating agents also alter kinetics of 1-methyl-4-phenyl-pyridinium （MPP~） uptake, making SH-SY5Y cells more similar to primary mesencephalic neurons. Conclusions Differentiated and undifferentiated SH-SY5Y cells have been widely used as a cell model of DAergic neurons for PD research. Some differentiating agents afford SH-SY5Y cells with more potential for studying neurotoxiclty and neuroprotection and are thus more relevant to experimental PD research.

Mesencephalic astrocyte-derived neurotrophic factor reduces cell apoptosis via upregulating HSP70 in SHSY-5Y cells

Background:Mesencephalic astrocyte-derived neurotrophic factor(MANF)is a new candidate growth factor for dopaminergic neurons against endoplasmic reticulum stress(ER stress).HSP70 family,a chaperon like heat shock protein family,was proved to be involved in the MANF induced survival pathway in 6-OHDA treated SHSY-5Y cells.However,the ER stress relative transcriptome,in MANF signaling cascades is still investigated.The involvement of HSP70,a 70kd member of HSP70 family,need further to be verified.Methods:The cell apoptosis was assayed by MTT,TUNEL staining and western blot of cleaved Caspase-3.The differentially expressed genes in SHSY-5Y cells under different conditions(control,6-OHDA,6-OHDA+MANF)were investigated by RNA-seq.Expression of HSP70 was further confirmed by real-time PCR.RNAi knockdown for HSP70 was performed to investigate the role of HSP70 in the MANF signaling pathway.Results:MANF inhibits 6-OHDA-induced apoptosis in SHSY-5Y cells.Six ER stress relative genes(HSP70,GRP78,xbp-1,ATF-4,ATF-6,MAPK)were found enriched in 6-OHDA+MANF treatment group.HSP70 was the most significantly up-regulated gene under 6-OHDA+MANF treatment in SHSY-5Y cells.RNAi knockdown for HSP70 inhibits the protective effects of MANF against 6-OHDA toxicity in SHSY-5Y cells.Conclusion:MANF exerts a protective role against 6-OHDA induced apoptosis in SHSY-5Y cells via up-regulating some ER stress genes,including HSP70 family members.The HSP70 expression level plays a key role in MANFmediated survival pathway.

TiO_2对水热法制备钛酸锶钡的影响

以乙酸锶、乙酸钡、钛酸丁酯为原料,通过溶胶-凝胶法制备出钛酸锶钡(BST)前驱体。在前驱体中分别加入10%金红石TiO_2、锐钛矿TiO_2和用水热法制备的一维TiO_2,再进行水热合成,得到3种不同的水热产物。用傅里叶变换红外光谱仪(FTIR)、X线衍射仪(XRD)、扫描电子显微镜镜(SEM)对前驱体和3种水热产物进行物相和形貌的表征,并对水热产物的生长机理和形成原因进行探讨。结果表明,在前驱体中添加锐钛矿TiO_2能够制备出柱状晶结构的BST;添加一维TiO_2的前驱体生成了一维的BST晶体。

绍兴中小企业人才流动现状与对策研究

近年来,绍兴的中小企业在促进绍兴经济增长、增加就业机会、活跃绍兴经济等方面发挥着越来越重要的作用,与此同时绍兴中小企业的人才流动问题也倍受关注.文章从绍兴中小企业人才流动的现状出发,分析其影响流动的要素,运用人才流动决策模式,提出促进人才合理流动的相应对策.