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Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase‐9 (MMP‐9) activity in the ischemic brain, which exacerbates blood‐brain barrier injury and increases the risk of symptomatic cerebral hemorrhage. The mechanism through which tPA enhances MMP‐9 activity is not well understood. Here we report an important role of caveolin‐1 in mediating tPA‐induced MMP‐9 synthesis. Brain microvascular endothelial cell line bEnd3 cells were incubated with 5 or 20 μg/ml tPA for 24 hrs before analyzing MMP‐9 levels in the conditioned media and cellular extracts by gelatin zymography. tPA at a dose of 20 μg/mL tPA, but not 5 μg/mL, significantly increased MMP‐9 level in cultured media while decreasing it in cellular extracts. Concurrently, tPA treatment induced a 2.3‐fold increase of caveolin‐1 protein levels in endothelial cells. Interestingly, knockdown of Cav‐1 with siRNA inhibited tPA‐induced MMP‐9 mRNA up‐regulation and MMP‐9 increase in the conditioned media, but did not affect MMP‐9 decrease in cellular extracts. These results suggest that caveolin‐1 critically contributes to tPA‐mediated MMP‐9 up‐regulation, but may not facilitate MMP‐9 secretion in endothelial cells.

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This study involved mice that received 4 days of ethanol (EtOH) vapor inhalation and then were assessed for type 1 inositol 1,4,5‐trisphosphate receptor (IP3Rs‐1) expression and the development of EtOH‐induced place preference at various time points in withdrawal. IP3R‐1 protein was found to be significantly increased in the nucleus accumbens (NAcc) of mice immediately after 4‐day EtOH vapor inhalation, while it significantly reduced to the control level during the next 3 days of withdrawal from EtOH inhalation. EtOH (2 g/kg, i.p.)‐induced place preference after 3 days of withdrawal from EtOH vapor inhalation increased dose dependently for 4 days, which was significantly inhibited by 2‐aminophenoxyethane‐borate, an antagonist for IP3Rs. EtOH conditioning significantly increased, compared to alcohol‐naïve control mice, both IP3R‐1 protein and the release of dopamine in the NAcc of mice after 3 days of withdrawal from EtOH vapor inhaled for 4 days, and this increase of IP3R‐1 protein was completely abolished by intracerebroventricular injection of FK506, an inhibitor for calcineurin. These results indicate that the sensitization of EtOH‐induced place preference is due to up‐regulated IP3R‐1 via calcineurin‐mediated pathway after enhanced release of dopamine in the NAcc on EtOH administration during EtOH conditioning.

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Glycogen synthase kinase‐3 beta (GSK‐3β) dysfunction may play an essential role in the pathogenesis of psychiatric, metabolic, neurodegenerative diseases, in which oxidative stress exists concurrently. Some studies have shown that GSK‐3β activity is up‐regulated under oxidative stress. This study evaluated how oxidative stress regulates GSK‐3β activity in human embryonic kidney 293 (HEK293)/Tau cells treated with hydrogen peroxide (H2O2). Here, we show that H2O2 induced an obvious increase of GSK‐3β activity. Surprisingly, H2O2 dramatically increased phosphorylation of GSK‐3β at Ser9, an inactive form of GSK‐3β,while there were no changes of phosphorylation of GSK‐3β at Tyr216. Moreover, H2O2 led to a transient [Ca2+]i elevation, and simultaneously increased the truncation of GSK‐3β into two fragments of 40 kDa and 30 kDa, whereas inhibition of calpain decreased the truncation and recovered the activity of GSK‐3β. Furthermore, tau was hyperphosphorylated at Ser396, Ser404, and Thr231, three most common GSK‐3β targeted sites after 100 μM H2O2 administration in HEK293/Tau cells, whereas inhibition of calpain blocked the tau phosphorylation. In addition, we found that there were no obvious changes of Cyclin‐dependent kinase 5 (CDK5) expression (responsible for tau phosphorylation) and of p35 cleavage, the regulatory subunit of CDK5 in H2O2‐treated HEK293/Tau cells. In conclusion, Ca2+‐dependent calpain activation leads to GSK‐3β truncation, which counteracts the inhibitory effect of Ser9 phosphorylation, up‐regulates GSK‐3β activity, and phosphorylates tau in H2O2‐treated HEK293/Tau cells.  相似文献   

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Vascular dysfunction is emerging as a key pathological hallmark in Alzheimer’s disease (AD). A leaky blood–brain barrier (BBB) has been described in AD patient tissue and in vivo AD mouse models. Brain endothelial cells (BECs) are linked together by tight junctional (TJ) proteins, which are a key determinant in restricting the permeability of the BBB. The amyloid β (Aβ) peptides of 1–40 and 1–42 amino acids are believed to be pivotal in AD pathogenesis. We therefore decided to investigate the effect of Aβ 1–40, the Aβ variant found at the highest concentration in human plasma, on the permeability of an immortalized human BEC line, hCMEC/D3. Aβ 1–40 induced a marked increase in hCMEC/D3 cell permeability to the paracellular tracer 70 kD FITC‐dextran when compared with cells incubated with the scrambled Aβ 1–40 peptide. Increased permeability was associated with a specific decrease, both at the protein and mRNA level, in the TJ protein occludin, whereas claudin‐5 and ZO‐1 were unaffected. JNK and p38MAPK inhibition prevented both Aβ 1–40‐mediated down‐regulation of occludin and the increase in paracellular permeability in hCMEC/D3 cells. Our findings suggest that the JNK and p38MAPK pathways might represent attractive therapeutic targets for preventing BBB dysfunction in AD.  相似文献   

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Accumulating evidence indicates that abnormal deposition of amyloid‐β (Aβ) peptide in the brain is responsible for endothelial cell damage and consequently leads to blood–brain barrier (BBB) leakage. However, the mechanisms underlying BBB disruption are not well described. We employed an monolayer BBB model comprising bEnd.3 cell and found that BBB leakage was induced by treatment with Aβ1–42, and the levels of tight junction (TJ) scaffold proteins (ZO‐1, Claudin‐5, and Occludin) were decreased. Through comparisons of the effects of the different components of Aβ1–42, including monomer (Aβ1–42‐Mono), oligomer (Aβ1–42‐Oligo), and fibril (Aβ1–42‐Fibril), our data confirmed that Aβ1–42‐Oligo is likely to be the most important damage factor that results in TJ damage and BBB leakage in Alzheimer's disease. We found that the incubation of bEnd.3 cells with Aβ1–42 significantly up‐regulated the level of receptor for advanced glycation end‐products (RAGE). Co‐incubation of a polyclonal antibody to RAGE and Aβ1–42‐Oligo in bEnd.3 cells blocked RAGE suppression of Aβ1–42‐Oligo‐induced alterations in TJ scaffold proteins and reversed Aβ1–42‐Oligo‐induced up‐regulation of RAGE, matrix metalloproteinase (MMP)‐2, and MMP‐9. Furthermore, we found that these effects induced by Aβ1–42‐Oligo treatment were effectively suppressed by knockdown of RAGE using small interfering RNA (siRNA) transfection. We also found that GM 6001, a broad‐spectrum MMP inhibitor, partially reversed the Aβ1–42‐Oligo‐induced inhibitor effects in bEnd.3 cells. Thus, these results suggested that RAGE played an important role in Aβ‐induced BBB leakage and alterations of TJ scaffold proteins, through a mechanism that involved up‐regulation of MMP‐2 and MMP‐9.

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Hydrogen peroxide is the most stable of the reactive oxygen species (ROS) and is a regulator of development, immunity and adaptation to stress. It frequently acts by elevating cytosolic free Ca2+ ([Ca2+]cyt) as a second messenger, with activation of plasma membrane Ca2+‐permeable influx channels as a fundamental part of this process. At the genetic level, to date only the Ca2+‐permeable Stelar K+ Outward Rectifier (SKOR) channel has been identified as being responsive to hydrogen peroxide. We show here that the ROS‐regulated Ca2+ transport protein Annexin 1 in Arabidopsis thaliana (AtANN1) is involved in regulating the root epidermal [Ca2+]cyt response to stress levels of extracellular hydrogen peroxide. Peroxide‐stimulated [Ca2+]cyt elevation (determined using aequorin luminometry) was aberrant in roots and root epidermal protoplasts of the Atann1 knockout mutant. Similarly, peroxide‐stimulated net Ca2+ influx and K+ efflux were aberrant in Atann1 root mature epidermis, determined using extracellular vibrating ion‐selective microelectrodes. Peroxide induction of GSTU1 (Glutathione‐S‐Transferase1 Tau 1), which is known to be [Ca2+]cyt‐dependent was impaired in mutant roots, consistent with a lesion in signalling. Expression of AtANN1 in roots was suppressed by peroxide, consistent with the need to restrict further Ca2+ influx. Differential regulation of annexin expression was evident, with AtANN2 down‐regulation but up‐regulation of AtANN3 and AtANN4. Overall the results point to involvement of AtANN1 in shaping the root peroxide‐induced [Ca2+]cyt signature and downstream signalling.  相似文献   

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MiRNAs contribute greatly to epithelial to mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs), which is a crucial step in peritoneal fibrosis (PF). In this study, we tried to profile whether miRNA expression differences exist after human umbilical cord mesenchymal stem cells (hUCMSCs) treatment in PF rats and investigate the possible role of miR‐153‐3p involved in anti‐EMT process. We randomly assigned 34 rats into three groups: control group (Group Control), MGO‐induced PF rats (Group MGO) and hUCMSCs‐treated rats (Group MGO + hUCMSCs). MiRNA microarrays and real‐time PCR analyses were conducted in three groups. α‐SMA, Snail1 and E‐cadherin expression were detected by Western blot. Luciferase reporter assays were used to detect the effects of miR‐153‐3p overexpression on Snai1 in rat peritoneal mesothelial cells (RPMCs). We identified differentially expressed miRNAs related to EMT, in which miR‐153‐3p demonstrated the greatest increase in Group MGO + hUCMSCs. Transient cotransfection of miR‐153‐3p mimics with luciferase expression plasmids resulted in a significant repression of Snai1 3′‐untranslated region luciferase activity in RPMCs. These studies suggest that miR‐153‐3p is a critical molecule in anti‐EMT effects of hUCMSCs in MGO‐induced PF rats. MiR‐153‐3p might exert its beneficial effect through directly targeting Snai1.  相似文献   

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Fusion of monomyoblasts to form multinucleated myotubes is a prerequisite for skeletal myogenesis, and muscle fibroblast–myoblast interaction plays an important role in the process; however, relative studies are limited. In the current study, SLD (sex‐linked dwarf) chicken, a myogenic deficient model caused by GH (growth hormone)–IGF‐I axis deficiency due to dw gene mutation, was introduced to study effects of fibroblasts on myodifferentiation. Using a membrane insert co‐culture system, we identified that, compared with SLD fibroblasts, normal fibroblasts promoted myogenesis of primary SLD myoblasts by improving their differentiation potential in a paracrine fashion, and this effect was involved in both primary and secondary fusions. This process was also coupled with up‐regulation of β1 integrin, and reduced myogenesis, resulting from siRNA interference demonstrated that β1 integrin was required for the response. Further, in terms of genetic discrepancy between normal and SLD fibroblasts, GH–IGF‐I signalling might play a role in this paracrine control.  相似文献   

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Securin and γ‐H2AX have been shown to regulate cell survival and genomic stability. However, it is still unknown how the expression and regulation of these proteins is altered following treatment with baicalein, a natural flavonoid extracted from the Scutellaria baicalensis root. In the present study, we investigate the possible roles of securin and γ‐H2AX in baicalein‐induced cancer cell death. Baicalein reduced cell viability in a variety of human cancer cell lines, including bladder, cervical, colon, and lung cancer cells. Interestingly, baicalein treatment (40–80 µM for 24 h) markedly inhibited securin expression, while the levels of γ‐H2AX were elevated. Abnormal spindle formation and chromosomal segregation were induced by baicalein. Furthermore, wild type HCT116 cancer cells had a higher incidence of cytotoxicity and apoptosis than securin‐null HCT116 cells following treatment with baicalein. In contrast, baicalein increased the levels of γ‐H2AX to a similar extent in both cell types. Transfection with H2AX siRNA further increased baicalein‐induced cell death. Additionally, blockade of the AKT pathway by treatment with wortmannin or AKT shRNA lowered the levels of γ‐H2AX and enhanced cytotoxicity in baicalein‐treated cells. Taken together, our findings suggest that the opposing effects of baicalein on securin and γ‐H2AX levels may be involved in the regulation of cell viability and genomic stability by this compound. J. Cell. Biochem. 111: 274–283, 2010. © 2010 Wiley‐Liss, Inc.  相似文献   

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Resveratrol, a polyphenol found in fruits, has been demonstrated to activate Sir2. Though many studies have demonstrated that resveratrol can activate SIRT1, whether it has effect on other sirtuins (SIRT2–7) are unknown. The present study shows that exposure of H9c2 cells to 50 µM H2O2 for 6 h caused a significant increase in apoptosis, as evaluated by TUNEL and flow cytometry (FCM), but pretreatment of resveratrol (20 µM) eliminated H2O2‐induced apoptosis. Resveratrol also prevented H2O2‐induced caspase‐3 activation. Exposure of cells to resveratrol caused rapid activation of SIRT1,3,4,7. Sirtuin inhibitor, nicotinamide (20 mM) attenuated resveratrol's inhibitory effect on cell apoptosis and caspase‐3 activity. These results suggest that resveratrol protects cardiomyocytes from H2O2‐induced apoptosis by activating SIRT1,3,4,7. J. Cell. Biochem. 107: 741–747, 2009. © 2009 Wiley‐Liss, Inc.  相似文献   

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