Types and control methods of black dot⁃like contaminants in cell cultures / 安徽医科大学学报

Objective @#To explore the type and control measure of black dot⁃like contaminants in cell cultures.@*Methods@#The growth state of bacteria was investigated under an inverted microscope ; Their morphological characteristics were analyzed by Gram and auramine O staining as well as electron microscopy; 16S rDNA gene sequencing was used to analyze bacterial species ; Drug sensitivity test was used to screen antibiotics against the bacteria;Cryopreserved SH⁃SY5Y cells were resuscitated by cell culture supernatant of RAW264 cells.@*Results@#Inverted microscopic real⁃time observations showed that black dot⁃like substances had two growth states : static and moving.They were negative for Gram staining while positive for auramine O staining. Electron microscopy revealed that they were short rod⁃shaped bacteria with a polar flagellum during moving phase. 16S rDNA gene sequencing showed that these bacteria were phenylobacterium zucineum HLK1. Ceftriaxone , carboxycillin and imipenem were screened by drug sensitivity test to have inhibitory effects on the bacteria , but cell culture experiments showed that they could not remove the bacteria from SH⁃SY5Y cells. Contaminated cells could not be cryopreserved for a long time , but resuscitation with RAW264. 7 cell culture supernatant significantly improved the survival rate of cells.@*Conclusion@#The black dot⁃like contaminants in cell cultures are a special type of oligotrophic bacterium with strong viability that can invade the cells and cannot be cleared with antibiotic treatment. RAW264. 7 cell culture supernatant seems contain some substances against bacteria , and resuscitating frozen cells with RAW264. 7 cell culture supernatant may significantly improve the survival rate of cells.

Effects of repetitive magnetic stimulation on the growth and differentiation of SH-SY5Y cells / 中华物理医学与康复杂志

Objective:To explore the effect of repeated magnetic stimulation (rMS) on the growth and differentiation of SH-SY5Y human neuroblastoma cells.Methods:SH-SY5Y cells were subjected to rMS at 15%, 30% and 60% of the maximum output intensity at frequencies of 0.5Hz, 1Hz, 5Hz, 10Hz and 20Hz. They received either 800 or 1600 pulses per day for 4 days. Cell viability was detected using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. Apoptosis was induced using 1-methyl-4-phenylpyridine ion (MPP + ) and all-trans retinoic acid was used to induce differentiation. The expression of neuron-specific nuclear proteins and the degree of cell differentiation were observed by immunohistochemistry. Results:0.5Hz rMS inhibited proliferation and 10Hz rMS promoted it. With 5Hz rMS significantly greater cell proliferation was observed at 15% and 30% of the maximum output intensity. The stimulatory effect of 1600 pulses per day was significantly greater than that of 800 pulses, especially at 10Hz. Apoptosis was inhibited at both 0.5Hz and 10Hz with 30% of the maximum output intensity. Meanwhile, both 0.5Hz and 10Hz rMS promoted differentiation of the SH-SY5Y cells into neurons.Conclusions:rMS at low frequency inhibits the proliferation of SH-SY5Y cells, but at higher frequency it promotes it. The effect strengthens with more pulses administered. rMS has a protective effect on MPP + -induced SH-SY5Y apoptosis, and it can promote the cells′ differentiation into neurons.

Protective effect of transient receptor potential melastatin 2 inhibitor A10 on oxygen glucose deprivation/reperfusion model / 浙江大学学报·医学版

:To investigate the effect of transient receptor potential melastatin 2 （TRPM2） inhibitor A10 on oxygen glucose deprivation/reperfusion （OGD/R） injury in SH-SY5Y cells．:Human neuroblastoma SH-SY5Y cells were subject to OGD/R injury，and then were divided into blank control group，model control group and A10 group randomly. The cell survival rate was detected by cell counting kit 8 （CCK-8）; the level of cellular reactive oxygen species （ROS） was detected by reactive oxygen detection kit; the mitochondrial membrane potential was detected by tetramethylrhodamine （TMRM） method; the number of apoptotic cells was detected by TUNEL apoptosis assay kit; the protein expression level of cleaved caspase 3 was detected by Western blot．:Compared with 3，20，30，50， has lower cytotoxicity and better inhibition effect on channel activity. Compared with the model control group，ROS level was reduced，the mitochondrial membrane potential was improved，the number of apoptosis cells was reduced ，and the expression of cleaved caspase 3 was significantly reduced in the A10 group（all <0.05）. : A10 can alleviate cell damage after OGD/R by inhibiting TRPM2 channel function，reducing extracellular calcium influx，reducing cell ROS levels，stabilizing mitochondrial membrane potential levels，and reducing apoptosis.

Effects of Panaxadiol on Tau Protein Phosphorylation and Fyn/GluN 2B Signaling Pathway in APP-SH-SY 5Y Cells / 中国药房

OBJECTIVE：To study the effects and mechanism of panaxadiol （PD） on Tau protein phosphorylation in the SH-SY5Y cells transfected with APP gene（APP-SH-SY5Y）. METHODS ：The target of PD and non-receptor tyrosine kinases Fyn was verified by molecular docking. SH-SY 5Y cells were cultured in vitro ，and the APP-SH-SY 5Y cell models and green fluorescent （GFP）-SH-SY5Y cell model （control cell ）was constructed. The expression of Aβ1-42 was detected so as to verify the success of APP-SH-SY5Y cell model. Taking GFP-SH-SY 5Y cells as control ，the effects of 5，10，20，30，40 μmol/L PD and 125，250， 500，1 000，2 000 nmol/L PP 2（Fyn inhibitor ，positive control ）on the survival rate of APP-SH-SY 5Y cells were detected by CCK-8 assay after treated for 24 h，so as to confirm the optimal concentration. The concentration of Ca 2 + ，the ratio fophosphorylated Tau protein （p-Tau）/Tau，phosphorylatedn Src（p-Src）/Fyn and phosphorylated glutamate receptor 2B（p-GluN2B）/ GluN2B were detected in APP-SH-SY 5Y cells after trated with the optimal concentration of PD and PP 2 for 24 h. RESULTS ：The results of molecular simulation docking showed that PD could target Fyn protein. Compared with GFP-SH-SY 5Y cells ，the protein expression of Aβ1-42 in APP-SH-SY 5Y cell were increased significantly （P＜0.01）. The optimal concentration of PD and PP 2 were 20 μmol/L and 500 nmol/L. The 20 μmol/L PD and 500 nmol/L PP 2 could increase the survival rate of the cells and reduced the concentration of Ca 2+，the ratio of p-Tau/Tau ，p-Src/Fyn，and p-GluN 2B/GluN2B. CONCLUSIONS：PD can reduce the the phosphorylation of Tau protein through inhibiting Fyn/GluN 2B signaling pathway.

LINC00612 targeted Bcl-2 to inhibit the apoptosis of neurons incubated with Aβ1-42 / 中风与神经疾病杂志

@#Objective To investigate the effect of LINC00612 on apoptosis of SH-SY5Y cells incubated with Aβ1-42.Methods Neuronal injury model of Alzheimer’s disease were established by SH-SY5Y cells incubated with Aβ1-42.The expression of LINC00612 in SH-SY5Y cells incubated with Aβ1-42 was detected by qRTPCR. The LINC00612 overexpressed plasmid was transfected into SH-SY5Y cells incubated with Aβ1-42，and the expression of Bcl-2 was detected by Western blot. Apoptosis was detected by Annexin V-FITC /PI staining. RNA-pulldown and RIP experiments were performed to detect the binding effect of LINC00612 and Bcl-2.Results LINC00612 was low expressed in SH-SY5Y cells incubated with Aβ1-42.Overexpression of LINC00612 significantly increased Bcl-2 expression and decreased apoptosis. RNA pulldown and RIP results showed that LINC00612 could bind to Bcl-2.Conclusion Overexpression of LINC00612 can up-regulate Bcl-2 expression and inhibit the apoptosis of SH-SY5Y cells incubated with Aβ1-42.

Effects of arsenite and MPST over-expressin on GRP78/CHOP endoplasmic reticulum stress pathway in SH-SY5Y cells / 中国药理学通报

Aim To investigate whether endoplasmic reticulum stress is involved in the neurotoxicity of sodi¬um arsenite and clarify whether over-expression of 3-mercaptopyruvate sulfurtransferase (MPST) regulates endoplasmic reticulum stress induced by arsenic. Methods The SH-SY5Y cell line stably expressing the exogenous MPST gene was obtained by constructing the lentiviral vector of MPST gene. The SH-SY5Y cells were randomly divided into six groups, the SR-MPST over-expression group stably expressing the exogenous MPST gene, SH-PEB control group transfected with empty vector, the arsenite treatment group ( NaAs02 group ), TUDC A treatment group ( blocker of endoplasmic reticulum stress ) and TUDC A + NaAs02 group. Western blot was used to examine the protein expression of GRP78 and CHOP after different treatment. Results Although MPST overexpression had no significant effects on the expression of GRP78 and CHOP proteins, NaAs02 could significantly increased the protein levels of GRP78 and CHOP ( P < 0. 01 ) and the up-regulation of GRP78 and CHOP proteins caused by NaAs02 could be blocked by the treatment of TUDC A. In addition, the inhibition by MPST overexpression on the arsenic-induced increase of GRP78 and CHOP proteins (P <0. 01 ) could also be reversed by the TUDC A treatment significantly. Conclusions The GRP78/ CHOP endoplasmic reticulum stress pathway is involved in the neurotoxic damage induced by arsenic; MPST overexpression may decrease arsenic-induced endoplasmic reticulum stress.

Effect of oxidative stress in SH-SY5Y cell injury induced by deguelin / 中国药理学通报

Aim To study the correlation between oxidative stress and neurotoxicity induced by deguelin, providing the mechanism basis for the structural modification and drug combination about deguelin. Methods SH-SY5Y cells were exposed to deguelin ranging from 1. 56-100 jimol • L"1 for 24 to 72 h, whose survival rate was determined by CCK-8 assay. The content of LDH, MDA levels, GPx and antioidant enzyme activities of SOD were determined using the assay kits ac-cording the manufacturer's protocol. And the content of ROS was determined by flow cytometry. The expressions of MEK, EGF, ERK and CREB were determined by immunoblotting. Results Deguelin obviously inhibited the growth of SH-SY5Y cells in a concentra-tion-and time-dependent manner ( P < 0. 05 ). After the cells were injured by deguelin, the content of ROS, LDH and MDA in SH-SY5Y cells increased obviously (P <0. 05), while the vitality of GPx and SOD decreased obviously ( P < 0. 05) in a dose-dependent manner. Furthermore, the protein expression levels of MEK, EGF, RAS and CREB in MAPK/ERK signaling pathway decreased obviously when treated with deguelin in a dose-dependent manner. (P < 0. 05). Conclusions The trauma of SH-SY5Y cells induced by deguelin is closely related to oxidative stress, in which MAPK/ERK signaling pathway may play a mediating role.

Inhibitory effect of foodborne procyanidins on growth of SH-SY5Y cells and its mechanism / 吉林大学学报(医学版)

Objective: To investigate the inhibitory effect of foodborne procyanidins on the growth of human neuroblastoma SH-SY5Y cells, and to elucidate its mechanism. Methods: The SH-SY5Y cells were cultured and divided into control group, 10 mg · L-1 foodborne procyanidins group, 20 mg · L-1 foodborne procyanidins group and 40 mg · L-1 foodborne procyanidins group; the medium containing different concentrations (0, 10, 20 and 40 mg · L-1) of foodborne procyanidins was added into each group. The proliferation rates of SH-SY5Y cells were measured by MTT method at 24, 48 and 72 h after the drug treatment. Flow cytometry was used to detect the cell cycle of SH-SY5Y cells at 72 h after the drug treatment, and the apoptotic rate of SH-SY5Y cells was detected by Annexin V apoptotic assay kit at 72 h after the drug treatment. Results: Compared with control group, the proliferation rates of SH-SY5Y cells at 24, 48 and 72 h in 10, 20 and 40 mg · L-1 foodborne procyanidins groups were decreased; there were significant differences at 48 and 72 hours in 20 mg · L-1 foodborne procyanidins group (P<0.05 or P<0.01); there were also significant differences at 24, 48 and 72 h in 40 mg · L-1 foodborne procyanidins group (P < 0.01). Compared with control group, the percentage of cells in G0/G1 phase in 40 mg · L-1 foodborne procyanidins group were increased (P<0.01) and the percentage of cells in G2/M phase were decreased (P<0.01). Compared with control group, the apoptotic rates of cells in 10, 20 and 40 mg · L-1 foodborne procyanidins groups were increased significantly (P<0.01). Conclusion: Foodborne procyanidins can inhibit the growth of human neuroblastoma SH-SY5Y cells, and its mechanism is mainly to block the cell cycle and induce the apoptosis.

Chemical constituents from ethyl acetate extraction of Prunus mume / 中国中药杂志

Eight compounds were isolated from the ethyl acetate extraction of Prunus mume by column chromatography. On the basis of physicochemical properties and spectrum analysis, these compounds were identified as isoquercitrin-6″-O-benzoate(1), pinoresinol(2), naringin(3), ethyl-β-D-glucopyranoside(4), astragalin(5), quercetin(6), hypericin(7), and rutin(8). Among them, compound 1 was a new natural product, and compounds 2-5 were isolated from this plant for the first time. In vitro study, compounds 1, 3, 5-8 could significantly increase the cell survival ratio.

Evaluating the Effect of Iron (II) Chloride Induced Oxidative Stress in SH-SY5Y Cells and Its Role in Upregulation of α-Synuclein Expression

The α-synuclein (SNCA) gene is a pathogenic gene identified in rare familial Parkinson Disease (PD). Recent studies highlight the role of DNA methylation in the pathogenesis of familial and sporadic PD. Hypomethylation in SNCAgene has been associated with increased SNCA gene expression and was observed in post mortem brains of patients with sporadic PD. This study was aimed at evaluating the effect of iron (II) chloride on SH-SY5Y cell models as pertain to cell death caused by oxidative stress, upregulation of SNCA gene expression and reduced SNCA gene methylation. Result obtained from LDH assay showed significant (p<0.05) evidence of cell death in treated cells as compared to the control sample. Analysis for SNCAgene quantification using RT-PCR showed significant increases in fold change. Cells treated with 1000μM of FeCl₂showed the highest fold change of 6.0 while cells treated with 250μM had the lowest fold change of 1.8. In DNA methylation assay using pyrosequencing, cells treated with varying concentrations of FeCl₂showed significant (p<0.05) decrease in DNA methylation. At 250μM, 500μM and 750μM concentrations of FeCl₂, an average mean methylation levels of 1.84%, 1.40% and 1.23% was obtained respectively while cells treated with 1000μM had the lowest average mean methylation level of 1.0%. Thus, the decrease in methylation is linked to the upregulation of the SNCAgene which has been reported to be among the causative factors in the pathogenesis of Parkinson’s disease.

Protein profiling of SH-SY5Y neuroblastoma cells: The effect of rhein

Bone marrow mesenchymal stem cells (BMSCs) play an important role in the process of bone repair. The present studyinvestigated the effect of 5-azacytidine (AZA) and trichostatin A (TSA) on BMSC behaviors in vitro. The role of WNTfamily member 5A (WNT5A)/WNT family member 5A (WNT7A)/b-catenin signaling was also investigated. BMSCs wereisolated from a steroid-induced avascular necrosis of the femoral head (SANFH) rabbit model. The third-generation ofBMSCs was used after identification. The results revealed obvious degeneration and necrosis in the SANFH rabbit model.AZA, TSA and TSA ? AZA increased BMSC proliferation in a time-dependent fashion. AZA, TSA and TSA ? AZAinduced the cell cycle release from the G0/G1 phase and inhibited apoptosis in BMSCs. AZA, TSA and TSA ? AZAtreatment significantly decreased caspase-3 and caspase-9 activities. The treatment obviously increased the activity andrelative mRNA expression of alkaline phosphatase. The treatment also significantly up-regulated the proteins associatedwith osteogenic differentiation, including osteocalcin and runt-related transcription factor 2 (RUNX2), and Wnt/b-cateninsignal transduction pathway-related proteins b-catenin, WNT5A and WNT7A. The relative levels of Dickkopf-relatedprotein 1 (an inhibitor of the canonical Wnt pathway) decreased remarkably. Notably, TSA ? AZA treatment exhibited astronger adjustment ability than either single treatment. Collectively, the present studies suggest that AZA, TSA and TSA ?AZA promote cell proliferation and osteogenic differentiation in BMSCs, and these effects are potentially achieved via upregulation of WNT5A/WNT7A/b-catenin signaling.

Effects of METH on mitochondrial membrane potrntial, ultrastaucture and expression of Mfnl and Fisl protein in SH-SY5Y cells cultured in vitro / 中国药理学通报

Aim To evaluate the influence of METH on MMP, mitochondrial ultrastructure, and the expression levels of mitochondrial proteins, Mfnland Fisl, in human neuroblastoma SH-SY5Y cells in vitro. Methods A stable and feasible culture method of SH-SY5Y cells in vitro was established with different concentrations of METH(0. 0, 1. 0, 1. 5 and 2. 0 mmol L-1), and for various periods of exposure for 3, 6, 12, 24 h, the MMP of SH-SY5Y cells was stained by MMP assay kit (JC-1) , the mitochondrial ultrastructure of SH-SY5Y cells exposed to METH was observed by transmission electron microscope, and the expression levels of Mfnl and Fisl proteins were detected by Western blot. Results Compared with control group for various periods of exposure(3,6,12,24 h), the red/green fluorescence ratios of MMP and the expression levels of Mfn1 protein decreased significantly in METH groups (P<0. 05) , while the expression levels of Fisl pro-tein increased significantly (P <0.05). SH-SY5Y cells were treated with METH for 24 h prior to observation under transmission electron microscope ( TEM ). The mitochondria of SH-SY5Y cells in unprocessed group showed the oval, rodlike and double-layer membrane structure, along with clear normal mitochondrial cristae. However, the oval and rodlike structure of mitochondria in SH-SY5Y cells of METH treatment groups had been split into small ball structures. Moreover , mitochondrial autophagosome and autophagic iyso-some could also be found. Conclusions METH could induce a decrease in MMP, mitochondrial ultrastruc-tural changes, and changes in the expression levels of Mfnl and Fisl in SH-SY5Y cells, which might be associated with nerve cell damage caused by METH.

Effect of sodium arsenite on cell cycle of SH-SY5Y cells and intervention of MPST over-expression / 中国药理学通报

Aim To investigate the effects of sodium arsenite (NaAsO2) on the cell cycle and growth, and the intervention of MPST over-expression in the neural cells. Methods NaAsO2 was used to treat SH-SY5Y neuroblastoma cells for 48 h from the blank control (BC), empty vector control ( transfected with empty vector, NC ) and over-expression group ( lentiviral transfection with MPST,OP). The methods of CCK-8, crystal violet staining,flow cytometry and Western blot were used to examine cell viability,adherent rate,cell cycle and protein expression of p53,CDC25A,CyclinA and CDK2. Results The cell viability and adherent rate significantly decreased after treatment with NaAsO2 for 48 h,which was reversed in OP group (P <0. 01). Meanwhile, NaAsO2 also significantly increased the proportion of S phase cells and p53 protein expression, and down-regulated the protein levels of CDC25A,Cyc-lin A and CDK2 in BC and NC groups ( P < 0. 01), whereas the above changes of protein levels were significantly antagonized in OP group compared with NC group (P < 0. 05, P < 0. 01). Conclusions NaAsO2 inhibits the cell growth by inducing S-phase arrest and over-expression of MPST could reverse the noxious effects caused by NaAsO2 in SH-SY5Y cells.

Effect of Tamibarotene on the proliferation and Tyrosine kinase receptor A,N-myc expressions of neuroblastoma SH-SY5Y cells / 中华实用儿科临床杂志

Objective To study the effect of Tamibarotene on the SH-SY5Y cell proliferation inhibition ability and the mRNA and protein expressions of tyrosine kinase receptor a (TrkA) and N-myc (MYCN) in order to provide some experimental bases for the treatment of neuroblastoma.Methods The SH-SY5Y cells were treated with different concentrations of Am80 (0,10,20,40,80,160 μmol/L) for 48 h,then Cell Counting Kit-8 (CCK-8) was used to test the cell proliferation.Reverse transcription PCR(RT-PCR) and Western blot were used to test the mRNA and protein expressions of TrkA and MYCN at 48 hours.Results When the concentration was 10 μmol/L,Am80 had no significant inhibitory effect on SH-SY5Y cells [(3.51 ± 1.68)％,inhibition ratio ＜ 5 ％];but when the concentration was 20 μmol/L,it showed weak inhibition [(9.60 ± 1.97) ％,inhibition ratio ＜ 10％].The inhibition rate of SH-SY5Y cell proliferation[(57.43 ± 4.95)％] was significantly enhanced at Am80 with a concentration of 80 μmol/L.The concentrations of Am80 could effectively inhibit SH-SY5Y cell proliferation in a dose-dependent manner(P ＜0.05).The expression of TrkA increased with the increase of Am80 concentration.Am80 significantly decreased the expression of MYCN in SH-SY5Y cells(10 μmol/L:0.65 ±0.05 vs.20 μmol/L:0.36 ±0.06),and the difference was statistically significant(P ＜ 0.05).Conclusions It is suggested that Am80 can effectively inhibit SH-SY5Y cell proliferation in a concentration-dependent manner.The underlying mechanism involves increasing the expression of TrkA by down-regulation of MYCN.

A new benzophenone isolated from fibrous roots of Anemarrhena asphodeloides / 中国中药杂志

Five compounds were isolated from the fibrous roots of Anemarrhena asphodeloides by silica gel, Sephadex LH-20 and semi-HPLC column chromatography. On the basis of physic-chemical properties and spectroscopic data analysis, these compounds were identified as methyl 2-[2,4-dihydroxy-3-(4-hydroxybenzoyl)-6-methoxyphenyl]acetate(1), 4-[formyl-5-(methoxymethyl)-1H-pyrrol-1-yl]butanoate(2), perlolyrine(3),syringaresinol-4'-O-β-D-glucoside(4) and 4',6-dihydroxy-4-methoxybenzophenone-2-O-(2″),3-C-(1″)-1″-desoxy-α-L-fructofuranoside(5). Among them, 1 was a new benzophenone. Compounds 2-5 were isolated from this plant for the first time. Compound 1 was tested for neuroprotective effects against H_2O_2-induced damage in SH-SY5 Y cells.

Sodium valprovate suppresses autophagy in SH-SY5Y cells activating miR-34c-5p/ATG4B signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effect of sodium valproate (VPA) on activation of miR-34c-5p/ATG4B signaling pathway and autophagy in SH-SY5Y cells.@*METHODS@#Routinely cultured SH-SY5Y cells were treated with VPA at different doses for 24 h, and the changes in the mRNA levels of ATG4B and miR-34c-5p and the protein expression of ATG4B were assessed using qRTPCR and immunoblotting, respectively. The effect of transfection with a plasmid containing ATG4B promoter on the promoter activity of ATG4B in VPA-treated SH-SY5Y cells was assessed using the reporter gene assay. The stability of ATG4B mRNA was analyzed with qPCR in SH-SY5Y cells treated with VPA alone or with VPA combined with the transcription inhibitor actinomycin D. The expression level of miR-34c-5p was detected using qPCR in SH-SY5Y cells treated with VPA alone or with VPA combined with miR-34c-5p mimics or antagonist, and the role of miR-34c-5p in VPA-induced ATG4B down-regulation was evaluated. The changes in the level of autophagy were evaluated by detecting LC3-Ⅱ expression in the cells after treatment with VPA or VPA combined with miR-34c-5p antagonist.@*RESULTS@#VPA dose-dependently down-regulated the expression of ATG4B at both the mRNA and protein levels in SH-SY5Y cells. VPA treatment did not significantly affect the promoter activity of ATG4B, but obviously lowered the mRNA stability of ATG4B in SH-SY5Y cells. VPA treatment up-regulated the expression of miR-34c-5p, and the miR-34c-5p antagonist reversed VPA-induced down-regulation of ATG4B in SH-SY5Y cells. VPA also down-regulated the expression level of LC3-Ⅱ in SH-SY5Y cells.@*CONCLUSIONS@#VPA suppresses autophagy in SH-SY5Y cells possibly via activating miR-34c-5p/ATG4B signaling pathway.

Effects of puerarin on H2O2-induced SH-SY5Y cell apoptosis / 中国药理学通报

Aim To investigate the neuroprotective effects of puerarin on H2O2-induced SH-SY5Y cell ap-optosis and the molecular mechanisms underlying the neuroprotective effects. Methods Neuron injury mod-el was established in vitro through H2O2-induced SH-SY5Y injury. MTT assay was performed to detect the effect of puerarin on H2O2-induced SH-SY5Y survival rates. Hoechst 33342 staining was used to observe the cell apoptosis. JC-1 staining was employed to detect the level of mitochondria membrane poential. Caspase-3 was determined by caspase-3 catalyze the substrate specificity Ac-DEVD-pNA. Caspase-9 was determined by caspase-9 catalyze the substrate specificity Ac-LE-HD-pNA. The effects of puerarin on the protein level of Bcl-2,Bax,p-Akt and Akt were determined by West-ern blot. Results The cell survival rate significantly increased after puerarin pretreatment compared with H2O2model group. Furthermore, puerarin pretreat-ment not only inhibited the decreasing of mitochondrial membrane potential,increasing of caspase-3, caspase-9 enzymatic activity and the expression of Bax,but also promoted the expression of p-Akt and Bcl-2, which was prevented by LY294002, an inhibitor of PI3K/Akt. Conclusion Puerarin can play a neuroprotective role for SH-SY5Y cell apoptosis induced by H2O2, maybe via activating PI3K/Akt signaling pathway.

Effects of gastrodin against SH-SY5Y cell autophagy induced by methamphetamine and underlying mechanisms / 中国药理学通报

Aim To investigate the effects of gastrodin on SH-SY5Y cell autophagy induced by methamphet-amine (METH) and the underlying mechanisms. Methods SY5Y cells were treated by METH with the concentration of 0.5,1.0,1.5,2.0,2.5,3.0 mmol·L-1for 24 h. The morphological changes were ob-served by microscopy,the expression of LC3-Ⅱ,Bec-lin-1,Akt,p-Akt,mTOR and p-mTOR were detected by Western blot. Gastrodin was added to the medium 1 h before METH treatment. Results The SY 5 Y cells were morphologically featured by shrinkage and den-drite disruption after exposed to METH(0~3 mmol· L-1),and autophagic vacuoles occurred in cytoplasm. The expression of LC3-Ⅱ increased over METH dose. Confocal results showed that LC3-Ⅱsignificantly in-creased in METH group as compared with control, while decreased in METH+ Gastrodin group. The ex-pression levels of LC3-Ⅱand Beclin-1 significantly in-creased (P<0.01) in METH group, p-mTOR and p-Akt decreased, and mTOR and Akt showed no signifi-cant difference as compared with control. However, the gastrodin could decrease the expression of LC3-Ⅱand Beclin-1 and increase the expression of mTOR,p-mTOR,Akt and p-Akt as compared with METH-trea-ted groups. Conclusions METH can induce SY5Y cells autophagy. The protective effect of gastrodin a-gainst METH-induced autophag may be related to gast-rodin regulation mTOR and Akt signaling pathway.

Expression and significance of MeCP2 and TH protein in normal SH-SY5Y cells and Parkinson's disease cell model / 国际检验医学杂志

Objective To investigate the expression and significance of TH and MeCP2 protein in normal SH-SY5Y cells and Parkinson's disease cell model.Methods The experiment was divided into 3 groups:blank control group,non transfection;model control group;pEGFP-N1-MeCP2 group,transfection solution added to the recombinant pEGFP-N1-MeCP2 plasmid.In addition to the blank group,the remaining two groups were added 50.0 μmol/L 6-OHDA treatment for 24 hours.CCK-8 assay and flow cytometry were used to detect cell activity and apoptosis in normal SH-SY5Y cells and the SH-SY5Y cell with up-regulation of MeCP2 induced by 6-OHDA.Immunofluorescence and Western blot analysis were used to detect the changes of MeCP2 protein and TH protein expression in each group of SH-SY5Y cells.Results As for the cell survival and apoptosis of SH-SY5Y cells detected by CCK-8 assay and induced 6-OHDA of flow cytometry and SH-SY5Y cells with up-regulation of MeCP2 in model control group,compared to those in pEGFP-N1-MeCP2 group and the thank control group,the difference was statistically significant (P＜0.05).The expression of MeCP2 and TH in SH-SY5Y induced 6-OHDA cells was detected by double immunofluorescence,it was found that in model control group,with 6-OHDA (50.0 μmol/L) inducing for 24 hours,and the expression of MeCP2 and TH decreased significantly (P＜0.05).After Western blot was used to detect the up-regulation of MeCP2 expression,in 6-OHDA induced SH-SY5Y cells of MeCP2 and TH expression,the expression of MeCP2 and TH protein in pEGFP-N1-MeCP2 group and blank control group was statistically different from that in model control group (P＜0.01).Conclusion The transfection of pEGFP-N1-MeCP2 recombinant plasmid could inhibit the apoptosis of SH-SY5Y cells induced by 6-OHDA,improve the expression of TH and increase the cell survival rate.