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Background Neurotransmitter secretion disorder induced by chronic manganese poisoning has always been one of the important causes of body injury, but the mechanism of neurotransmitter secretion disorder caused by manganese is not clear at present. Objective To investigate the effects of presynaptic membrane intracellular protein 13-1 (Munc13-1) and synapse fusion protein binding protein 18-1 (Munc18-1) on dopamine secretion dysfunction induced by manganese chloride (MnCl2) in human neuroblastoma (SH-SY5Y) cells. Methods A SH-SY5Y cell model induced by MnCl2 was established. Cell viability was measured by MTT assay. Four experimental groups were set up: control group and low-, medium-, and high-dose manganese groups (0, 100, 200, and 400 μmol·L−1 MnCl2). They were treated with corresponding doses of MnCl2 for 24 h. The secretion of dopamine was measured by enzyme-linked immunosorbent assay. The mRNA expression of Syntaxin-1 was detected by real-time quantitaive PCR. Total cell proteins were extracted, and the protein expression levels of Munc13-1, Munc18-1, and Syntaxin-1 were detected by Western blotting. The correlations of MnCl2 exposure and dopamine secretion with the protein expressions of Munc13-1 and Munc18-1 were also analyzed by Pearson correlation. Results Compared with the control group, the cell viability rate decreased gradually with the increase of manganese exposure concentration, and the difference between the medium- and the high-dose manganese groups was statistically significant (P<0.05). The concentration of dopamine in cell culture medium of all manganese exposure groups decreased with the increase of manganese concentration, and compared with the control group and the low-dose manganese group, the medium- and the high-dose manganese groups were statistically significant (P<0.05). The expression of Syntaxin-1 at mRNA or protein level did not change significantly among groups (P>0.05). Compared with the control group, the protein expression of Munc13-1 decreased and that of Munc18-1 increased with the increase of manganese concentration (P<0.05). Compared with the low-dose manganese group, the changes of Munc13-1 protein in the high-dose manganese group and Munc18-1 protein in the medium- and high-dose manganese groups had statistical significance (P<0.05). Compared with the medium-dose manganese group, the protein changes of Munc18-1 in the high-dose manganese group were statistically significant (P<0.05). The correlation analysis showed that MnCl2 dose was negatively correlated with Munc13-1 protein expression (r=−0.898, P<0.05), and positively correlated with Munc18-1 protein expression (r=0.678, P<0.05). Dopamine secretion was positively correlated with Munc13-1 protein expression (r=0.932, P<0.05), and negatively correlated with Munc18-1 protein expression (r=−0.817, P<0.05). Conclusion The inhibition of dopamine secretion in SH-SY5Y cells induced by manganese exposure is related to up-regulation of Munc18-1 and down-regulation of Munc13-1 expression levels, which may be one of the reasons for nerve injury caused by manganese.
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Background A large amount of iron deposition in the brain can cause neuronal damage by inducing oxidative stress, neuroinflammation, and abnormal mitochondrial function. In addition, iron deposition is also reported to be closely related to the pathogenesis of Alzheimer's disease (AD). The neurofibrillary tangles aggregated by tau hyperphosphorylation are one of the important pathological features of AD. Objective To investigate potential effect of exogenous trivalent iron ions on neuronal activity in human neuroblastoma (SH-SY5Y) cells and tau hyperphosphorylation and aggregation. Methods SH-SY5Y cells were treated with ferric chloride (FeCl3) at four concentrations (10, 100, 200, and 400 mg·L−1). Cell survival rate was then detected by CCK8 assay. Intracellular iron content was determined prussian blue (Perl's) by iron staining after 24 h exposure to FeCl3 at 10 or 200 mg·L−1. Transfection of tau-P301L plasmid was conducted to construct an AD-like cell model for tau overexpression. The differences in the expression of the phosphorylated tau (p-tau) protein in SH-SY5Y cells and SH-SY5Y cells with tau overexpression were detected by Western blotting after 24 h exposure to FeCl3 at 10 and 200 mg·L−1. After dilution with phosphate buffered saline (PBS), FeCl3, human tauR3, and FeCl3 + tauR3 were incubated at 37℃, and the fluorescence intensity reflecting tau aggregation level was measured by thioflavin T(ThT) method at 12, 24, 36, 48, 60, 72, 84, and 96 h, respectively. Meanwhile, after 96 h coincubation of FeCl3 and tauR3, the fibers formed by tau aggregation were observed under a transmission electron microscope (TEM). Results After 24 h of FeCl3 exposure, the cell survival rate of SH-SY5Y cells among all groups was statistically different (F=8.63, P<0.01). The cell survival rates in the 200 and 400 mg·L−1 groups were 80.1% and 68.7% of the control group, respectively (P<0.05). Compared with the control group, the nuclei of the 200 mg·L−1 FeCl3 group were mainly yellowish-brown after iron staining and the positive cell rate was up-regulated by 12.9% (P<0.01). After 24 h of FeCl3 exposure , the p-tau (Ser396) protein expression was statistically different among all groups (F=11.6, P<0.01). Compared with the control group, the p-tau protein expression level of SH-SY5Y cells in the 200 mg·L−1 group was up-regulated by 72.7% (P<0.01). After FeCl3-treated SH-SY5Y cells with tau overexpression for 24 h, the p-tau (Ser396) protein expression was statistically different among all groups (F=27.8, P<0.01). Compared with the tau group, the p-tau (Ser396) protein expression level of SH-SY5Y cells in the tau + 200 mg·L−1 group was up-regulated by 44.6% (P<0.05). Compared with the tauR3 group, the fluorescence intensities in the 84 and 96 h tauR3 + FeCl3 groups were up-regulated by 49.9% and 53.7% (P<0.01) respectively. After 96 h of coincubation, compared with the tauR3 group, FeCl3 + tauR3 aggravated tau aggregation and formed fiber deposition under TEM. Conclusion Exogenous trivalent iron ions may inhibit SH-SY5Y cell viability, promote the phosphorylation of tau in SH-SY5Y cells transfected with tau-P301L plasmid, and aggravate tauR3 aggregation and fiber production.
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【Objective】 To explore the effects of Ligusticum Chuanxiong extract on MPP+-induced SH-SY5Y cell damage and Parkinson’s syndrome. 【Methods】 1-methyl-4phenylpyridine ion (MPP+) interfered with SH-SY5Y to establish a cell model of elderly Parkinson’s syndrome (SH-SY5Y-MPP+). After intervention with Ligusticum Chuanxiong extract, cell proliferation and apoptosis as well as miR-23a-3p and SNCA expressions were detected. In addition, the changes of SH-SY5Y-MPP+ after regulating the expression of miR-23a-3p and SNCA were observed, and the relationship between miR-23a-3p and SNCA was verified by dual luciferase reporter. 【Results】 The cell proliferation capacity of SH-SY5y-MPP+ was significantly lower than that of SH-SY5Y, while the apoptosis rate was higher than that of SH-SY5Y (P<0.05). Under the intervention of Ligusticum Chuanxiong extract, the proliferation ability of SH-SY5Y-MPP+, and Bcl-2 and SNCA protein increased, the apoptosis rate, miR-23a-3p, and Bax proteins decreased (P<0.05). Both silencing miR-23a-3p and increasing SNCA could promote the proliferation of SH-SY5Y-MPP+ and inhibit apoptosis, while increasing miR-23a-3p and silencing SNCA were the opposite (P<0.05). The online target gene prediction website found that miR-23a-3p and SNCA had complementary sites that could bind, and the dual luciferase reporter enzyme showed that the firefly activity of SNCA-wt was significantly inhibited after the miR-23a-3p mimic sequence was transfected (P<0.05). After increasing miR-23a-3p, the expression of SNCA protein in SH-SY5Y-MPP+ decreased, while silencing miR-23a-3p was the opposite (P<0.05). Rescue experiments showed that the intervention effect of Ligusticum Chuanxiong extract on SH-SY5Y-MPP+ was completely reversed by increasing miR-23a-3p or silent SNCA (P>0.05); the effect of increasing miR-23a-3p on SH-SY5Y-MPP+ increased SNCA reversion (P>0.05). 【Conclusion】 Ligusticum Chuanxiong extract can affect the biological behavior changes of SH-SY5Y induced by MPP+ by regulating the miR-23a-3p/SNCA axis, which may be a new direction for the treatment of elderly Parkinson’s syndrome in the future.
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[Abstract] Objective To study whether the regulation of mammalian target of rapamycin complex 2(mTORC2) / Akt signaling pathway has a protective effect on SH-SY5Y cell line damaged by 6-hydroxydopamine (6-OHDA), and to clarify its molecular mechanism. Methods SH-SY5Y cells treated with retinoic acid (RA) were given 6-OHDA, mTORC2 signaling pathway inhibitor PP242 and agonist A-443654 respectively. The changes of cell number in each group were investigated by immunofluorescent staining; The total protein was extracted and the expression level and interaction of key proteins in mTORC2 signaling pathway were determined by Western blotting and co-immunoprecipitation (CoIP); The apoptosis rate of cells in each group was detected by flow cytometry. At the same time, the co-culture Parkinson’ s disease (PD) model was made using SH-SY5Y cell line and Bv-2 cell line; MTT colorimetric method was used to detect the cell viability of each group; ELISA was used to detect the content of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in cell culture supernatant. Results The number of tyrosine hydroxylase(TH) / proliferating cell nuclear antigen (PCNA) / hochest-, TH / 5-bronmo-2’ -deoxyuridine(BrdU) -labeled positive cells in 6-OHDA-lesioned PD cell model group was significantly lower than that in the normal group; The apoptosis rate was higher; The expression of Rictor, p-Akt and regulated in DNA damage and development 1(REDD1) was increased; There was an interaction between Rictor and p-Akt or REDD1; The cell viability was significantly reduced in the co-culture model; the content of TNF-α and IL-β increased in the cell culture supernatant. With further up-regulation of the abovementioned protein expressions, the cell survival, apoptosis and pro-inflammatory cytokine levels in A-443654 group were significantly ameliorated, while PP242 group showed the opposite changes. Conclusion A-443654 activates mTORC2 signaling pathway by p-Akt, which increases the expression of Rictor and REDD1 protein. These changes contribute to the amelioration in cell survival rate, apoptosis rate, and the proliferation and differentiation and decreasion of apoptosis rate of SH-SY5Y cells. These result improved 6-OHDA-induced cell damage and inhibited the release of pro-inflammatory cytokines.
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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.
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ObjectiveTo study the inhibitory effect of Banxia Houputang （BHT） on lipopolysaccharide （LPS）-induced inflammation of microglia （BV2） cells and the neuroprotective effect on human neuroblastoma （SH-SY5Y） cells. MethodAfter the neuroinflammatory model was constructed by LPS inducing BV2 cells， model group （LPS 100 µg·L-1）， administration groups （LPS+1 g·L-1 BHT， LPS+2 g·L-1 BHT， LPS+5 g·L-1 BHT， LPS+10 g·L-1 BHT）， and blank group were given DEME medium at the same volume. In addition， neuronal apoptosis model was established by co-culture of LPS-induced BV2 cell inflammation medium and SH-SY5Y cells （LPS-DMEM） and was administrated according to the above grouping. Cell viability was detected by Cell Counting Kit-8 （CCK-8） assay. The content of nitric oxide （NO） and that of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， and interleukin-6 （IL-6） were determined by Griess aasay and enzyme-linked immunosorbent assay （ELISA）， respectively. The mRNA levels of TNF-α， IL-1β， interleukin-4 （IL-4）， nitric oxide synthase （iNOS）， and interleukin-10 （IL-10） were measured by real-time polymerase chain reaction （Real-rime PCR）. Western blot was used to detect the expression levels of signal transducer and activator of transcription 3 （STAT3）， Janus kinase 2 （JAK2） and nuclear factor kappa-B （NF-κB p65）， protein kinase B （Akt）， inhibitor of nuclear factor κB α （IκBα）， B-cell lymphoma-2 （Bcl-2）， and Bcl-2 associated X protein （Bax）. ResultCompared with blank group， LPS increased the NO release， levels of TNF-α， IL-1β， IL-6， and iNOS and protein expression of Akt， NF-κB p65， IκBα， JAK2 and STAT3， decreased the content of IL-4 and IL-10 in BV2 cells， and induced apoptosis of co-cultured SH-SY5Y cells （P<0.01）. Compared with model group， BHT reduced the content of NO， TNF-α， IL-1β， and iNOS （P<0.01） and protein expression of Akt， NF-κB p65， IκBα， JAK2 and STAT3 （P<0.01）， elevated the content of IL-4 and IL-10 （P<0.01）， and inhibited the apoptosis of SH-SY5Y cells induced by LPS-DMEM （P<0.01）. ConclusionThis experiment reveals that BHT inhibited LPS-induced inflammation in BV2 cells by regulating Akt/NF-κB/JAK2/STAT3 signaling pathway and showed neuroprotective effects on SH-SY5Y cells.
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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.
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: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.
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Humanos , Apoptose , Benzenoacetamidas , Sobrevivência Celular , Glucose , Oxigênio/metabolismo , Piperidonas , Espécies Reativas de Oxigênio/metabolismo , Reperfusão , Canais de Cátion TRPMRESUMO
Puerarin has the anti-Alzheimer's disease (AD) activity,which can reverse nerve injury induced by Aβand inhibit neuronal apoptosis.However,its potential pharmacodynamic mechanism still needs to be further researched.The occurrence and development of AD is due to the change of multiple metabolic links in the body,which leads to the destruction of balance.Puerarin may act on multiple targets and multiple metabolic processes to achieve therapeutic purposes.Quantitative proteomic analysis provides a new choice to understand the mechanism as completely as possible.This research adopted SH-SY5Y cells induced by Aβ_(1-42)to establish AD cell model,and Aβimmunofluorescence detection showed that Aβdecreased significantly after puerarin intervention.The mechanism of puerarin reversing SH-SY5Y cell injured by Aβ_(1-42)was further explored by using label-free non-labeled quantitative technology and Western blot detection based on bioinformatics analysis result.The results showed that most of the differential proteins were related to biological processes such as cellular component organization or biogenesis,cellular component organization and cellular component biogenesis,and they mainly participated in the top ten pathways of P value such as pathogenic Escherichia coli infection,m TOR signaling pathway,regulation of autophagy,regulation of actin cytoskeleton,spliceosome,hepatocellular carcinoma,tight junction,non-small cell lung cancer,apoptosis and gap junction.Annexin V/PI flow cytometry and TUNEL were used to detect apoptosis,and the results showed that Aβdecreased significantly and the rate of apoptosis decreased significantly after puerarin intervention.Western blot analysis found that the protein expression level of autophagy related protein LC3Ⅱwas up-regulated after Aβinduction,and the degree of this up-regulation was further enhanced in puerarin intervention group.The trend of the ratio of LC3Ⅱ/LC3Ⅰamong groups was the same as the protein expression level of LC3Ⅱ，the protein expression level of p62 in the control group,AD model group and puerarin intervention group decreased successively.Protein interaction network analysis showed that CAP1 was correlated with TUBA1B,HSP90AB2P,DNM1L,TUBA1A and ERK1/2,and the correlation between CAP1 and ERK1/2 was the highest among them.Western blot showed that the expressions of p-ERK1/2,Bax and CAP1 were significantly down-regulated and the protein expression level of Bcl-2 was significantly up-regulated after puerarin intervention.Therefore,puerarin might improve the SH-SY5Y cells injured by Aβ_(1-42)through the interaction of multiple biological processes and pathways in cells multiple locations,and CAP1 might play an important role among them.
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Humanos , Peptídeos beta-Amiloides , Apoptose , Carcinoma Pulmonar de Células não Pequenas , Linhagem Celular Tumoral , Isoflavonas/farmacologia , Neoplasias Pulmonares , ProteômicaRESUMO
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.
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The effects of new derivatives of caffeine-8-thioglycolic acid (100 µM) on isolated rat brain synaptosomes, human neuroblastoma cell line SH-SY5Y and human recombinant MAOB enzyme (hMAOB) (1 µM) were evaluated. Most of the compounds, administered alone, didn't show statistically significant neurotoxic effects on SH-SY5Y, when compared to the control (non-treated cells). Of all studied structures JTA-2Ox, JTA-11, JTA-12 and JTA-13 decreased cell viability. In combination with 6-hydroxydopamine (6-OHDA) (100 µM), only JTA-1 and JTA-2 revealed neuroprotective effects, stronger than those of caffeine. All compounds administered alone revealed, neurotoxic effects on synaptosomes, as compared to non-treated synaptosomes. JTA-1, JTA-2 and JTA-3 showed lowest neurotoxic effects and were investigated in a model of 6-OHDA-induced oxidative stress. In this model of neurotoxicity, only JTA-1 and JTA-2 showed statistically significant neuroprotective effect, by preserving the synaptosomal viability and the level of reduced glutathione. Inhibition of hMAOB, was revealed by JTA-1 and JTA-2. They inhibited the enzyme by 23% and 25% respectively, thus approaching the selegiline activity, which was 42%. The possible mechanisms of neuroprotection of JTA-1 and JTA-2 might be a result from the inhibition of hMAOB, which catalyze the production of neurotoxic p-quinone from 6-OHDA.
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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.
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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.
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Acetatos , Compostos Fitoquímicos/análise , Extratos Vegetais/química , Prunus/química , SolventesRESUMO
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.
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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.
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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.
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Background: Proton pump inhibitors (PPIs) largely used a drug to treat gastroesophageal disease such as gastric ulcers. Moreover, in recent years, several studies suggest that PPIs have an important anti-cancer effect in monotherapy and or combination with chemotherapy. The aim of this study was to investigate whether esomeprazole and pantoprazole exhibit anti-cancer effect alone or could enhance chemosensitivity on the human neuroblastoma cell line SH-SY5Y to cisplatin.Methods: The human neuroblastoma SH-SY5Y cells were cultured and treated with different concentrations of esomeprazole, pantoprazole, and cisplatin alone. Also, these cells exposed to cisplatin+ esomeprazole and cisplatin + pantoprazole combinations, respectively and incubated 24 h. The antiproliferative activities of the (PPIs) alone or in a combination of cisplatin was evaluated using the XTT colorimetric assay.Results: According to experimental data, neither PPIs showed no cytotoxicity on the human neuroblastoma cell line SH-SY5Y at all concentrations. However, when combined with cisplatin separately, they were found to have significant antiproliferative effects on the human neuroblastoma SH-SY5Y cell lines when compared to cell lines treated with cisplatin alone (p<0.05).Conclusions: Taken together, the inhibition of V-ATPase via esomeprazole and pantoprazole might enhance the chemosensitivity of cisplatin on the human neuroblastoma cell line SH-SY5Y. However, further studies are needed to be able to utilize PPIs in human neuroblastoma cells.
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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.
Assuntos
Humanos , Anemarrhena , Química , Benzofenonas , Farmacologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Fármacos Neuroprotetores , Farmacologia , Compostos Fitoquímicos , Farmacologia , Raízes de Plantas , QuímicaRESUMO
Objective: To establish a model for the injury of human neuroblastoma cell (SH-SY5Y) induced by sodium glutamate, and to observe the protective effect of syringaresinol on cell damage from Viscum liquidambaricolum hayataon, and to explore its mechanism. Method: Construction of SH-SY5Y cell injury model using sodium glutamate.The experiment was divided into normal cell group, injury model group (sodium glutamate 50 mmol·L-1, sodium glutamate 50 mmol·L-1 + DMSO),syringaresinol experimental group (6.25, 12.5, 25 μmol·L-1), by cell counting, cell morphology observation, Annexin V-FITC/PI apoptosis detection, ROS reactive oxygen species detection, mitochondrial membrane potential, and Western blot, evaluation of syringaresinol on glutamate-induced neuronal excitability injury neuroprotective activity. Result: Compared with normal group, the cell survival rate of the model group was significantly decreased (PPPPP-1) showed a concentration-dependent increase in cells. Survival rate (PPPPPConclusion: Syringaresinol has significant protective activity against excitatory damage induced by sodium glutamate in SH-SY5Y neurons, the mechanism may be through anti-oxidative stress, repairing mitochondrial function and DNA damage to significantly reduce sodium glutamate-induced neuronal apoptosis.