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OBJECTIVE To investigate the effect of mono-2-ethylhexyl phthalate(MEHP) on proliferation of primary neural stem cells(NSCs)of rats and NE-4C cells of mice and on the migration of NE-4C cells and the mechanism. METHODS NE-4C or NSCs were treated with MEHP 1,10,100 and 1000 μmol · L-1 for 72 h,respectively. The cytotoxicity was estimated with the cell counting kit-8 (CCK-8). Cell proliferation was analyzed by EdU assay. The mRNA expression levels of the glucocorticoid receptor(GR),signal transducer and activator of transcription 3(Stat3)and sex determining region Y (SRY)-box 2(Sox2) were detected by qRT-PCR. The protein expression levels of total GR,GRβ, Sox2,Stat3 and p-Stat3 were measured by Western blotting. RESULTS Cell viability of NE-4C cells and NSCs at MEHP 1000μmol·L-1 was significantly decreased,which was 70.3%and 40.0%of the control group, respectively. EdU assay showed that MEHP 100 μmol · L-1 decreased NE-4C cells and NSCs by 74.8%and 12.0%(P<0.05)compared with control. The effect of MEHP on the cell migration of NE-4C was evidenced by the fact that the migration was obviously reduced to (63.4±2.0)%(P<0.05)after treatment with MEHP 100μmol · L-1 for 72 h. The mRNA expression levels associated with proliferation and migration in NE-4C of GR,Stat3 and Sox2 in MEHP 100 μmol · L-1 group were down-regulated to 49.8%,26.0% and 14.0%of control(P<0.05). At MEHP 100μmol · L-1,mRNA of GR, Stat3 and Sox2 in NSCs declined to 10.0%,14.0% and 15.3% of normal control. Western blotting results revealed that protein expressions of GR,GRβ,Sox2 and p-Stat3 were remarkably inhibited by MEHP 100 μmol · L-1 in that the relative expression of NE-4C was 0.92 ± 0.17,0.87 ± 0.35,0.81 ± 0.22 and 0.62 ± 0.24(P<0.05). The corresponding protein expression in NSCs was 0.82 ± 0.20,0.56 ± 0.12,0.84 ± 0.36 and 0.53 ± 0.20(P<0.05)when the cells were treated with MEHP 100μmol · L-1 for 72 h. CONCLUSION MEHP can inhibit the proliferation and migration of NE-4C cells and NSCs possibly by decreasing Stat3 and Sox2 that are mediated by GRβ.
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Objective To evaluate the effects of propofol on apoptosis and invasiveness of human lung cancer cell line A549 cells.Methods Human lung cancer cell line A549 were seeded onto 96-well plates (100 μl/well) and 6-well plates (2 000 μl/well) at a density of 2× 105 cells/ml,and cultured for24 h at 37 ℃ in 5％ CO2.The cells were randomly divided into 2 groups (n =60 each) using a random number table:dimethyl sulfoxide (DMSO) group and propofol group (group P).In group P,propofol with the final concentration of 100 μmoYL was added.In group DMSO,0.5％ DMSO with the final concentration of 0.5％ was added.At 24 h of incubation with drugs,caspase-3 expression was detected by high content screening (HCS); the expression of matrix metalloproteinase (MMP-2) was detected by Western blot analysis.At 0.5,1 and 5 h of incubation,ERK1/2 expression was also measured using Western blot analysis.Results Compared with group DMSO,the expression of caspase-3 was up-regulated,the expression of MMP-2 was down-regulated,ERK1/2 expression was up-regulated at 0.5 of incubation and down-regulated at 1 h of incubation,and no significant change was found in ERK1/2 expression at 5 h of incubation in group P.Conclusion Propofol can promote apoptosis in A549 cells and inhibit invasiveness of human lung cancer cell line A549 cells.
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Objective To evaluate the effects of different concentrations of parecoxib sodium on the rat sperm motility,capacitation and acrosome reaction in vitro.Methods The sperm samples from Sprague-Dawley rat epididymis were collected by Klinefelter diffusion method and randomly divided into 4 groups (n =18 each) using a random number table:control group (group C),and parecoxib sodium 100,500,1 000 μmol/L groups (P1-3 groups).Parecoxib sodium with the final concentrations of 100,500 and 1 000 μmol/L was added to the culture medium.The samples were then incubated for 5 h in an airtight container filled with 5 ％ CO2 at 37 ℃.Then sperm motility was examined in vitro at 37 ℃ and analyzed by the computer-assisted sperm analysis,including the sperm motility ((a + b)％),average path velocity (VAP),straight line velocity (VSL),curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH).The capacitation effect was assessed by using the chlortetracycline staining and phase-contract microscopy.The acrosome reaction was evaluated by coomassie brilliant blue staining.Results The VAP,VSL,VCL and capacitation ability of the sperm were gradually decreased in C and P1-3 groups (P ＜ 0.05).Compared with group C,(a + b)％ in P2,3 groups and ALH in P2 group were significantly decreased (P ＜ 0.05).There was no significant difference in the acrosome reaction between groups (P ＞ 0.05).Conclusion Parecoxib sodium has significant inhibitory effects on the rat sperm motility and capacitation in a dose-dependent manner,while has no effect on the acrosome reaction in vitro.
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AIM:To evaluate the effect of curcumin on impaired learning-memory ability and the expression of high mobility group box protein 1 ( HMGB1 ) and c-Jun N-terminal kinase ( JNK ) in a rat model of Alzheimer disease (AD).METHODS:Male Sprague-Dawley rats, weighing 250~270 g, were randomly divided into 4 groups (n=9):blank control group (group A), model group (group B), curcumin treatment group (group C, curcumin injected intraper-itoneally at 100 mg· kg-1· d-1 for 6 consecutive days) and solvent control group (group D).The rats of AD model were induced by injection of ibotenic acid into the nucleus basalis of Meynert ( NBM) bilaterally.All rats were trained in Morris maze to assess the ability of learning and memory .The expression of HMGB1 and JNK in the hippocampus was detected by the methods of immunohistochemistry and Western blotting .RESULTS:Compared with group A , the average escape laten-cy (AEL) in groups B and D were obviously longer (P0.05).No significant difference in the expression of HMGB1 in the hippocampus among the 4 groups was found (P>0.05).However, compared with groups B and D , the expression of JNK in group C was decreased obvi-ously (P<0.05).CONCLUSION: Curcumin significantly improves the learning and memory ability of AD rats .The probable mechanisms may be related to inhibiting the release of HMGB 1 from the nucleus of hippocampal neurons and de-creasing the expression of JNK in the hippocampus .
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Objective To study the protective role of pre-resolving mediator lipoxin A4(LXA4) in the NA+ -K+-ATPase in alveolar type Ⅱ (AT Ⅱ ) epithelial cells of rats exposed to lipopolysaccharide (LPS). Method The AT Ⅱ cells were isolated and purified, and divided randomly into control group (PBS), vehiculum (alcohol 0.7 μL/mL) group, LPS (1 μg/mL) group, LXA4(1/10 mol/mL) group and LPS (1 μg/mL LPS) + LXA4(1/10 mol/mL) group. After exposure to LPS and/or LXA4 for4 hours, NA+-K+ -ATPase and β1-subunits mRNA in AT Ⅱ epithelial cells were detected by using RT-PCR, and ATP, ADP, AMP, total adenine nucleotides (TAN) and energy charge (EC) were measured by using high performance liquid chromatography (HPLC), and then the activities of Na+-K+-ATPase were calculated accordingly. Results The NA+-K+-ATPase α-subunit and β-subunit mRNA were significantly decreased in LPS group ( P ＜ 0.05 vs. control group). However, the expressions of NA+ -K+-ATPase mRNA were significantly enhanced by application of LXA4 to AT Ⅱ epithelial cells exposed to LPS (P ＜0.05 vs. LPS group). The activities of NA+ -K+ -ATPase were enhanced in LPS group (P ＜0.05 vs. control group). Compared with control group and LPS group, the activities of NA+-K+-ATpase in LPS + LXA4 group were significantly increased (P ＜0.01 vs. control group; P ＜0.05 vs. LPS group). The EC of AT Ⅱ epithelial cells were higher in LPS group ( P ＜ 0.01 vs. control group). There were no significant differences in EC between control group and LPS + LXA4group(P ＞0.05). Conclusions The pro-resolving mediator LXA4 can enhance the expressions of NA + -K + -ATPase α-subunit and β-subunit mRNA, and the activities of NA + -K + -ATPase in AT Ⅱ epithelial cells or rats exposed to LPS, and ca also balance the metabolism of AT Ⅱ epithelial cells. These findings suggest that LXA4 plays an important role in lung edema clearance in lung injury induced by endotoxin, and the role is likely associated with the enhancement of the expressions of Na+ -K+ -AT-Pase α-subunit and β-subunit, and the activities of Na+ -K* -ATPase, maintaining the balance of metabolism of AT Ⅱ epithelial cells.
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Objective To study the effects of Lipoxins A4(LXA4)on the expressions of aquaporin(AQP)1,3,5 in type Ⅱ pneumonocytes(ATⅡ)of rat treated with lipopolysaccharide(LPS).Method One pathogenfree male Spree Dawley(SD)rat every time.weighing 200～250 g,were used for the study.The typeⅡpenumonocytes of rats were isolated and purified,and the changes of cellular ultrastructure were observed by electron microscope in order to get the purity quotien＞90%.The type Ⅱ pneumonocytes were divided randomly into five groups,namely,vebicukun group(alcohol 0.7μL/mL),control group,LXA4 group(1×10-7mol/mL),endotoxin group(LPS 1μg/mL)and LXA4+LPS group(LXA4 1×10-7mol/mL,LPS 1μg/mL).AQP-1,3,5 mRNA of in the typeⅡpenumonocytes were assayed by using reversal transcription poly chain reaction(RT-PCR),and the expressions of AQP-1,3,5 protein were detected by using.immunohistochemistry(IHC).One each specimen,these tests were repeated for six times.ANOVA was used for statistical analysis.Results RT-PCR and IHC showed that when AT Ⅱ treated with 1 μg/mL LPS for 4 hours,the AQP-1,3,5 mRNA and the expressions of AQP-1,3,5 protein were significantly decreased in LPS group compared with control group(P＜0.01).However,the AQP-1,3,5 mRNA and the expressions of AQP-1,3,5 protein after application of LXA4 significandy increased in LPS+LXA4 group in comparison with LPS group(LPS+LXA4,AQP1:0.647±0.132,AQP3:0.900±0.856,AQP5:0.879±0.058;LPS,AQP1:0.297±0.133,AQP3:0.512±0.113,AQP5:0.647±0.110;P＜0.01).The AQP-1,3,5 mRNA and the expressions of AQP-1,3,5 protein were aignificandy increased in LXA4 group in comparison with control group(LXA4,AQP1:0.539±0.142,AQP3:0.818 4-0.176,AQP5:0.841±0.066;Blank Control,AQP1:0.518±0.139;AQP3:0.138±0.136,AQP5:0.766±0.066;P＜0.01).Conclusions AQP-1,3,5 exist in typeⅡpenumonoeyte of rata,and the LXA4 can up-regulate the mRNA and protein expressions of AQP-1,3,5 in Type Ⅱ penumonocytes of rats treated with LPS.
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Objective To investigate the effects of lipoxin A4 on store-operated calcium channel (SOC) and production of reactive oxygen species in macrophages induced by hpopolysaccharide (LPS).Method Macrophages were randomly assigned Io one of the following six groups:control group,LPS group,Thapsigargin group,lipoxin A4+LPS group,lipoxin A4+Thapsigargin group,2-Aminoethoxydiphenylborate+Thapsigargin group.The intracellular[Ca2+]iwas analyzed by eonfoeal laser microscopy.The production of reactive oxygen specips(ROS) was assayed by flow cytometry.Results LPS increased intracellular[Ca2+]i and reactive oxygen species in a dose-dependent manner.Lipoxin A4 suppressed approximately 75% of the Ca2+ ertry signal induced by thapsigargin and suppressed approximately 93% of the Ca2+ entry signal induced by LPS.The increase in intracellular[Ca2+]i was associated with increased ROS production which was abolished in the presence of lipoxin A4.Conclusions These findings indicate that the LPS-indueed intracellular[Ca2*]i increase depends on the Ca2+entry through SOC channel,and lipoxin A4 inhibits Ca2+ influx and ROS production through SOC channel in ratine maerophages induced by LPS.