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Objective To observe periventricular white matter changes and matrix metalloproteinase-3 (MMP-3) expression after two-vessel occlusion in rats.Methods A total of 40 Wistar rats were randomly divided into four groups:sham operation group,model group 1 (20 days after operation),model group 2 (40 days after operation),model group 3 (60 days after operation)(n =10 per group).Model of chronic cerebral hypoperfusion was induced by permanent ligation of the bilateral common carotid arteries in the rat.The change of periventricular white matter was observed by electron microscope.The myelin basic protein (MBP) levels was determined by Western blotting,and the expression of MMP-3 was analysed by immunohistochemical stain and Western blotting.Results Permanent ligation of the bilateral common carotid arteries resulted in demyelination in the corpus callosum and internal capsule in rats.The expressions of MMP-3 in model groups were markedly elevated compared with sham group (model group 1,39.17± 4.167; model group 2,43.33±3.502; model group 3,54.16±2.787; sham group,26.67±3.830; all P＜0.01).The content of MBP in model groups was gradually decreased compared with sham group (model group 1,54.50±4.087; model group 2,47.83±4.875; model group 3,36.50±4.231; sham group,65.01±7.688; all P＜0.01).Additionally,the upregulation of MMP-3 had significant correlation with the loss of MBP in the periventricular white matter (r =-0.883,P＜0.01).Conclusions Chronic cerebral hypoperfusion induces progressive periventricular white matter demyelination,and the upregulation of MMP-3 may involve in the pathological process.
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Objective To observe the effects of histone deacetylases inhibitor (HDACi) on cognitive performance and cerebral tau phosphorylation in transgenic mice coexpressed five familial Alzheimer' s disease mutations (5XFAD).Method The total 12 5XFAD-CC and 12 wild type (WT) mice were administrated with suberoylanilide hydroxamic acid ( SAHA,n =7) and vehicle ( n =5 ),respectively.The cognitive performance was assessed by Y-maze and Morris water maze.The protein levels of acetylated α-tubulin,total tau and phosphorylated tau and phosphorylated glycogen synthase kinase-3β (GSK3β) were determined by Western blotting.Results SAHA ameliorated learning and memory deficits in 5XFAD-CC mice (39.10％ ±2.25％,t =2.688,P =0.0312 for total numbers of entrance in novel arm; 26.81％ ±0.78％,t =3.271,P =0.017 for time spending in novel arm; F =5.936,P =0.045 for hidden platform;31.70％ ±4.21％,t =2.317,P =0.049 for probe trial).Administration of SAHA significantly increased acetylated α-tubulin in hippocampus of WT and 5XFAD-CC mice (26.42％ and 29.64％,respectively).Additionally,SAHA attenuated tau-pSer396,tau-pSer404 and tau-pThrThr231 in hippocampus of 5XFAD-CC mice (24.22％,48.98％ and 26.95％,respectively). Moreover,hippocampal phosphorylated GSK3β was markedly reduced in SAHA-treated 5XFAD-CC mice (31.29％). Conclusion SAHA may improve cognitive performance in 5XFAD-CC mice, which is associated with its significant effects on the phosphorylation of tau and GSK3β.
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Objective To investigate the relationship between the neuroprotective effect of epigallocatechin gallate ( EGCG ) for PC12 cells induced by 1-methyl-4-phenyl-pyridinium ( MPP+ ) and activating nuclear factor-related factor 2 ( NRF2 ).Methods Well differentiated PC12 cells treated with MPP+ were used as the in vitro cell models,and PC12 cells were pretreated with different concentrations of EGCG.MTT assay was used to investigate the cell viability.Western blot was used to observe the expression of NRF2 in cells and distribution in the nucleus and the cytoplasm.Real-time PCR was used to observe the antioxidant enzymes,HO-1 and NQO1 mRNA expression.Results Pretreatment of PC12 cells with different concentrations of EG CG for an hour could restore cell viability.Western blot showed that expression of NRF2 in cells treated with MPP+ for 24 hours was increased 148％ +5％ compared with the control group (t =6.102,P ＜0.01 ).The level of NRF2 in EGCG pretreated group was 188％ + 6％ compared with the control group(t =11.172,P ＜0.01 ).Moreover the NRF2 protein level in the nuclear was also increased.Western blot showed that the NRF2 protein level in the nuclear was 258％ +2％ compared with the control group (t =21.995,P ＜ 0.01 ).Further research found U 120,an inhibitor of ERK,could inhibit the effect of EGCG.The levels of NRF2 in both samples were 148％ ± 15％ and 158％ ± 1％ compared with their respective control groups(t =6.118,8.058,both P ＜0.01 ).In accordance with the NRF2 data,real-time PCR indicated that the levels of HO-1 and NQO1 mRNA expression increased obviously in the group pretreated with EGCG.Likewise,U120 could also inhibit HO-1 and NQO1 mRNA expression induced by EGCG.Conclusions EGCG can repair oxidative damage to PC12 cells induced by MPP+.The protective effect may be related through the ways to activate ERK-NRF2 and induce downstream of antioxidant enzyme expression,such as HO-1 and NQO1.
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This study is to observe the effect of ilexonin A (IA) on the expression of basic fibroblast growth factor (bFGF) and growth associated protein-43 (GAP-43), and neurogenesis after cerebral ischemia-reperfusion in rats and explore its possible mechanism of protecting neuronal injury. Models of middle cerebral artery occlusion (MCAO) were established in SD rats. Before and after two hours ischemia-reperfusion, IA (20 and 40 mg x kg(-1)) was injected immediately and on 3, 7, 14, and 28 d once a day. The neurological severity was evaluated by neurological severity scores (NSS); neuronal injury in the boundary zone of the infarction area was evaluated by TUNEL and Niss1 staining. The expressions of bFGF and GAP-43 and neurogenesis were evaluated by Western blotting and 5-bromodeoxyuridine (Brdu) fluorescence staining, respectively. After treatment with IA, the NSS of treatment groups were lower than that of the models (3 and 7 d). The number of TUNEL positive neurons decreased and Nissl positive neurons increased at the same time (3 d). The expressions of bFGF and GAP-43 increased significantly in the boundary zone of the infarction area when compared to model group. Moreover, IA markedly enhanced the neurogenesis in the brain after ischemia-reperfusion, which revealed an increase of Brdu/NeuN positive cells in the boundary zone of the infarction area. The possible mechanism of protecting neuronal injury of IA may be related to inhibition on neuronal apoptosis, upregulation of bFGF and GAP-43, and neurogenesis in boundary zone of infarction after cerebral ischemia-reperfusion.
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This study is to explore whether the Wnt/beta-catenin signaling pathway is involved in the process of tripchlorolide (T4) protecting against oligomeric Abeta(1-42)-induced neuronal apoptosis. Primary cultured cortical neurons were used for the experiments on day 6 or 7. The oligomeric Abeta(1-42) (5 micromol x L(-1) for 24 h) was applied to induce neuronal apoptosis. Prior to treatment with Abeta(1-42) for 24 h, the cultured neurons were pre-incubated with T4 (2.5, 10, and 40 nmol x L(-1)), Wnt3a (Wnt signaling agonists) and Dkk1 (inhibitors) for indicated time. Then the cell viability, neuronal apoptosis, and protein levels of Wnt, glycogen synthase kinase 3beta (GSK3beta), beta-catenin and phospho-beta-catenin were measured by MTT assay, TUNEL staining and Western blotting, respectively. The result demonstrated that oligomeric Abeta(1-42) induced apoptotic neuronal cell death in a time- and dose-dependent manner. Pretreatment with T4 significantly increased the neuronal cell survival and attenuated neuronal apoptosis. Moreover, oligomeric Abeta(1-42)-induced phosphorylation of beta-catenin and GSK3beta was markedly inhibited by T4. Additionally, T4 stabilized cytoplasmic beta-catenin. These results indicate that tripchlorolide protects against the neurotoxicity of Abeta by regulating Wnt/beta-catenin signaling pathway. This may provide insight into the clinical application of tripchlorolide to Alzheimer's disease.
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Objective To explore the possible molecular mechanism of Ginsenoside Rg1 preventing against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced substantia nigra neurons apoptosis in Parkinson disease(PD) mouse model. Methods C57BL mice were administrated(sc) with MPTP to produce PD mouse model.Different doses of Rg1(5.0,10.0,20.0*!mg/kg) were given(ip) prior 3*!d to MPTP in the pretreatment groups.Nissl staining,tyrosinehydroxythase(TH) immunostatining,cleaved caspase-3 immunostatining and TUNEL staining were used to observe the changes of nigra neurons,meanwhile,Western blot was used to detect the phosphorylated protein of JNK and c-Jun in substantia nigra. Results Pretreatment with Rg1 could prevent the loss of Nissl staining neurons and TH-positive neurons,inhibit JNK and c-Jun phosphorylation in SN,decrease the percent of cleaved caspase-3 and TUNEL-positive cell.Conclusion Rg1 can attenuate MPTP-induced apoptosis in substantia nigra neurons through blocking JNK signaling cascade.
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AIM: To explore the possible anti apoptotic mechanism of ginsenoside Rg1 on MPP + induced cellular apoptosis. METHODS: The apoptosis of SHSY5Y induced by MPP + was observed by AO EB staining. Flow cytometry was used to quantitate mitochondrial transmembrane potential (△?m) and reactive oxygen species (ROS) . Western Blot was used to detect the expression of bcl 2 and bax proteins in SHSY5Y cells. RESULTS: MPP + induced apoptosis in SHSY5Y cells was obviously inhibited by pretreatment with 10 ?mol?L -1 Rg1. Although there was no difference of mitochondrial transmembrane potential between Rg1 pretreated groups and MPP + groups, the level of ROS in Rg1 pretreated groups decreased, the expression of bcl 2 protein increased and expression of bax protein decreased. CONCLUSION: Rg1 protects against MPP + induced apoptosis in SHSY5Y cells and the effect may be attributed to its remove of ROS and its regulation of expression of bcl 2 and bax proteins.
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AIM: To investigate the effects of nitric oxide(NO) and caspase-3 on dopamine-induced apoptosis in PC12 cells. METHODS: Flow cytometric assay was used to quantify the apoptotic cells. The morphological of apoptotic cells was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL). Nitrite was quantified by Griess reaction. Inducible nitric oxide synthase(iNOS) mRNA was identified by semiquantitative reverse transcription polymerase chain reaction(RT-PCR). Caspase-3 activity was determined by fluorescent spectrofluorometer. RESULTS: Dopamine induced PC12 cells apoptosis in a concentration-dependent manner (0.15-0.60 mmol/L), with positive TUNEL staining. During the development of apoptosis, the expression of iNOS mRNA and the levels of NO increased markedly, so did caspase-3 activity(P
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AIM: To study the possible molecular mechanism of beta-amyloid peptide_ 1-40 -induced apoptosis in rat cortical neurons. METHODS: 40 mg/L beta-amyloid peptide_ 1-40 (A?_ 1-40 ) was used to induce apoptosis in cultured rat cortical neurons. The level of CDK4, phosphorylated pRB were detected by flow cytometry and immunoblotting; RT-PCR was used to examine the mRNA expression of E2F1 while fluorescent spectrofluorometer was used to measure caspase-3 activity. All of the above study was designed to observe whether the level of CDK4, phosphorylated pRB and E2F1 mRNA expression could be affected by A?_ 1-40 . RESULTS: (1)The level of CDK4, phosphorylated pRB increased markedly 2-4 hours after treatment with A?_ 1-40 , and caspase-3 activity elevated remarkably 12-24 hours after treatment with A?_ 1-40 ; (2) E2F1 mRNA expression was upregulated 3 hours after incubation with A?_ 1-40 . CONCLUSION: A?_ 1-40 may induce apoptosis in rat cortical neurons in a manner dependent on CDK4-pRB-E2F1 pathway.
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AIM: To explore the possible signal transduction pathway of 1-methyl-4-phenylpyridinium(MPP +)-induced apoptosis. METHODS: The apoptosis of SHSY5Y cells induced by MPP + was observed by acridine orange-ethidium bromide(AO-EB) staining. Western blot was used to detect the activity of JNK in SHSY5Y cells, and the antisense oligo- neucleutide of JNK were used as inhibitor of JNK. RESULTS: MPP + induced apoptosis in SHSY5Y cells. During the apoptotic process, JNK activity increased. MPP +-induced apoptosis in SHSY5Y cells was obviously inhibited by pretreatment with NAC or by transfection with antisense oligonucleotide of JNK into SHSY5Y cells. Simultaneously, decrease in JNK activity and percentage of positive cleaved caspase-3 cells in these groups were also observed. CONCLUSION: The possible signal transduction pathway of MPP +-induced apoptosis in SHSY5Y cells might be attributed to the production of ROS ,activation of JNK and then activation of caspase 3.
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Objective To investigate the effects of caspase\|3 on ? ?? 1 40 induced apoptosis in rat cortical neurons. Methods Apoptosis was induced by 40?mg\5L -1 ?\|AP 1 40 .The apoptotic neurons were observed by TUNEL staining and DNA gel electrophoresis.The activity and mRNA expression of caspase\|3 was measured by fluorescent spectrofluorometer and RT\|PCR.The cleaved caspase\|3 was detected with immunocytochemistry. Results After treatment with ?\|AP 1\|40 ,the rat cortical neurons showed DNA fragmentation.The expression of caspase\|3 mRNA ratio to ? actin was 0^78,0^85 and 0^39 respectively after treatment for 12,24 and 48?h.Caspase\|3 activity was 133 24?7 47,192 16?11 03,88 87?4 24 MFI\5?g protein -1 \5h -1 .The cleaved caspase\|3 was observed within cytoplasm.Specific inhibitor of caspase\|3 Ac\|DEVD\|CHO inhibited caspase\|3 activation and blocked cortical neurons apoptosis markedly.Conclusion\ Caspase\|3 might be an executor during ? AP 1 40 induced apoptosis in rat cortical neurons.[