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Objective:To explore the role and mechanism of kidney brain protein (KIBRA) down-regulation in cognitive dysfunction caused by chronic cerebral hypoperfusion.Methods:Ninety male SPF grade Sprague Dawley (SD) rats were divided into four groups according to random number table: sham operation group ( n=15), chronic hypoperfusion group (2VO group, n=25), chronic hypoperfusion stereotaxic injection of AAV-KIBRA group (2VO+ AAV-KIBRA group, n=25), chronic hypoperfusion stereotaxic injection of AAV-Vector group (2VO+ AAV-vector group, n=25). Chronic cerebral hypoperfusion model was established by bilateral ligation of common carotid artery, and stereotactic injection of 2 μL AAV-KIBRA or AAV-vector was performed for 30 days.Morris water maze, in vitro electrophysiology, p21-activated kinase 3(PAK3) activity detection, Western blot, immunoprecipitation and Golgi staining were used to detect spatial learning and memory ability, long-term potentiation(LTP), KIBRA level expression, PAK3 activity changes and the distribution of dendritic spines.SPSS 16.0 statistical software was used for statistical data.One-way ANOVA was used to compare the differences between groups.LSD test was used to compare the significance of data differences between the two groups.Welch test was used for uneven variance. Results:After 1 month of chronic cerebral hypoperfusion, the level of KIBRA in the hippocampus of rats was detected by homogenate and Western blot, and it was found that the level of KIBRA in 2VO group was lower than that of sham group(73.49±4.12)% ( P<0.01). AAV-KIBRA injection in hippocampal CA1 region significantly up-regulated the level of KIBRA to (91.91±7.01)% over 2VO group ( P<0.01). Morris water maze test showed that the latency of the 2VO group(3rd-7th day trail data: (48.18±2.82)s, (43.45±2.27)s, (32.27±2.22)s, (26.55±2.37)s, (17.18±2.67)s) were significantly longer than those of the sham group((41.67±2.74)s, (32.58±2.57)s, (22.50±2.94)s, (16.91±2.39)s, (8.75±1.52)s) (all P<0.05), and the latencies of the 2VO+ AAV-KIBRA group 3rd-7th day trail data: (43.83±2.95)s, (35.25±2.15)s, (26.58±2.03)s, (19.92±2.17)s, (17.75±1.35)s) was significantly shorter than that of the 2VO group ((all P<0.01). The Morris water maze test with the platform removed showed that the latency of rats in the 2VO group to reach the platform region was significantly longer than that of the sham group, while the latency of rats in the 2VO+ AAV-KIBRA group to reach the platform region was significantly shorter than that in the 2VO group ( P<0.01). At the same time, the retention time and the crossing times in the platform region of 2VO group were less than those of the sham group ( P<0.01), but the retention time and the crossing times in the platform region of 2VO+ AAV-KIBRA group were significantly higher than those in the 2VO group ( P<0.01). The electrophysiological records of the brain slices showed that the relative excitatory postsynaptic field potential of 2VO group (1.43±7.43) was significantly lower than that of sham group (2.21±6.54) after high frequency stimulation, while the relative excitatory postsynaptic field potential of 2VO+ AAV-KIBRA group (1.90±8.15) was higher than that of 2VO group ( P<0.01). Immunoprecipitation in rat hippocampus revealed that PAK3 could be detected by Western blot assay when KIBRA was precipitated.The results showed that the relative enzyme activity of PAK3 in 2VO hippocampal tissue (0.64±0.04) was significantly lower than that in sham group (1.02±0.07), while the relative enzyme activity of PAK3 in 2VO+ AAV-KIBRA group (0.86±0.03) was significantly higher than that in 2VO group.Golgi staining showed that the density of dendritic spines in 2VO hippocampal neurons((6.85±0.43)/10 μm) was significantly lower than that in sham group((11.83±0.58)/10 μm), while the density of dendritic spines in 2VO+ AAV-KIBRA group((10.22±0.39)/10 μm) was significantly higher than that in 2VO group. Conclusion:The down-regulated of KIBRA after chronic cerebral hypoperfusion plays a key role in cognitive dysfunction and is also involved in the decrease of synaptic functional plasticity.The downregulation of KIBRA is involved in the structural plasticity of dendrites through the regulation of PAK3 activity.Therefore, KIBRA may be an important target for the prevention and treatment of cognitive function of chronic cerebral hypoperfusion.
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Objective:To explore the protective effect and mechanism of diammonium glycyrrhizinate (DAG) on cognitive dysfunction caused by chronic cerebral hypoperfusion.Methods:Seventy-three male Sprague Dawley rats in SPF degree were divided into sham group, chronic cerebral hypoperfusion group(2VO group), chronic cerebral hypoperfusion with DAG treatment group(2VO+ DAG group), and DAG treatment group(DAG group). During one-month chronic cerebral hypoperfusion models reproduced by the occlusion of bilateral common caroid artery, the rats were injected intraperitoneally with 2.917 mmol/L(20 mg·kg -1·d -1) DAG or saline for 15 days.Then the ability of learning and memory were tested by Morris water maze.Elisa, Western blot and Golgi staining were employed to test the spatial cognition, the changes of inflammatory factors, and inflammatory signal pathway molecules in hippocampus.The distribution of dendritic spines were observed and counted. Results:Morris water maze test showed that the learning latency of rats in 2VO group (3rd -7th day ) ((50.70±2.01)s, (43.53±3.22)s, (35.41±2.13)s, (25.26±1.85)s, (17.92±2.24)s) was significantly longer than that of sham group((40.28±1.94)s, (31.51±3.23)s, (24.7±2.25)s, (13.23±2.51)s, (9.42±1.91)s) (all P<0.01), while that of 2VO+ DAG group ((46.27±1.64)s, (38.54±1.51)s, (28.74±2.52)s, (19.73±2.13)s, (13.26±1.71)s) was significantly shorter than that of 2VO group ( P<0.05, P<0.01). After removing the platform to detect the memory of rats, the results showed that the latency of 2VO group (18.56±1.72)s) was significantly longer than that of sham operation group (11.25±2.11)s) ( P<0.01), while the time of 2VO+ DAG group was shorter than that of 2VO group (14.26±1.51)s ( P<0.01). In terms of the time of staying in the platform quadrant, the times of crossing through the platform area, the rats in the 2VO group were significantly less than those in the sham group ( P<0.01), while the rats in the 2VO+ DAG group were significantly more than those in the 2VO group ( P<0.01). Elisa data showed the levels of TNF-α, IL-1β and IL-6 in 2VO group (TNF-α: (27.42±1.91) pg/mg; IL-1β: (18.21±1.56)pg/mg; IL-6: (17.94±1.61)pg/mg)) were higher than those in sham group (TNF-α: (8.11±1.27)pg/mg; IL-1β: (6.78±1.12)pg/mg; IL-6: (5.67±0.91)pg/mg)) ( P<0.01), while the levels of three inflammatory factors in 2VO+ DAG group (TNF-α: (12.25±2.38)pg/mg; IL-1β: (9.93±0.96)pg/mg; IL-6: (8.72±0.65)pg/mg)) were significantly lower than those in 2VO group ( P<0.01). Western blotting data showed that the relative level of NF-κB in the nucleus of 2VO group (1.82±0.15) was significantly higher than that of sham group (1.00±0.09)( P<0.01), while that of 2VO+ DAG group (1.42±0.10) was significantly lower than that of 2VO group ( P<0.01). Golgi staining showed that the density of dendritic spines in CA1 area of hippocampus in 2VO group ((5.00±1.41)/10 μm) was significantly lower than that in sham group ((12.86±1.12)/10 μm) ( P<0.01), while that in 2VO+ DAG group was significantly higher than that in 2VO group ((9.23±1.65)/10 μm) ( P<0.01). Conclusion:DAG can effectively inhibit the neuroinflammatory response of hippocampus in chronic cerebral hypoperfusion, improve the damage of synaptic plasticity, and then improve the cognitive dysfunction caused by chronic hypoperfusion.DAG may be a potential effective drug for the treatment of chronic cerebral ischemia and vascular dementia.
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Objective:To explore the application effect of BOPPPS teaching model and "Internet + WeChat" platform in the teaching of Clinical Pharmacology.Methods:Undergraduate students from two consecutive batches of clinical medicine in Wuhan University were selected as the control group and the experimental group. The control group adopted the traditional teaching method, and the experimental group adopted the BOPPPS teaching model combined with the "Internet + WeChat" platform. SPSS 12.0 was used for t test. Results:The scores of the final test in the experimental group and the control group were (86.34±7.36) and (80.77±9.21), respectively, with statistical significance ( P<0.05). For the questions of clinical case analysis, with a total score of 20 points, the scores of the experimental group students (13.31±2.25) were significantly higher than those of the control group (10.58±3.04), with significant difference ( P<0.001). Conclusion:The application of BOPPPS model and "Internet + WeChat" platform in the undergraduate teaching of Clinical Pharmacology could improve students' examination performance, and especially the ability of solving clinical problems comprehensively.
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Objective To explore the effect and molecular mechanism of CC chemokine ligand 2 (CCL2) on epithelial-mesenchymal transition in human breast cancer cells.Methods Breast cancer Michigan cancer foundation-7 (MCF-7) cells were treated with 50 ng/ml CCL2.The abilities of invasion were detected by Transwell assay.The expression of epithelial-mesenchymal transition (EMT)-associated markers,E-cadherin and vimentin were detected by Western blot and quantitative real-time polymerase chain reaction (qRT-PCR).The expressions of Snail,protein kinase B (AKT),phosphorylated protein kinase B (p-AKT),phosphorylated glycogen synthase kinase 3β (p-GSK3β3) and GSK3β were detected by Western blot.Snail nuclear localization was detected by immunoflurescence staining.Results We found that CCL2 treatment could induce morphological alteration of MCF-7 cells from epithelial morphology to mesenchymal morphology.CCL2 significantly increased the migration of MCF-7 cells,and increased the expression of mesenchymal maker vimentin and decreased epithelial marker E-cadherin.More important,treatment of CCL2 significantly increased the expression of Snail,and promoted the nuclear localization of Snail.Knockdown of Snail significantly reverse the effects of CCL2 on the EMT in MCF-7 cells.Moreover,treatment of CCL2 significantly increased the phosphorylation levels of p-AKT and p-GSK3β,and AKT inhibitor LY294002 significantly inhibited CCL2-induced Snail and p-GSK3β expression.Conclusion CCL2 might induce EMT in MCF-7 cells,by which mechanism is related to activate AKT/GSK3β Snail pathway.
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OBJECTIVE:To systematically evaluate the efficacy and safety of modified infusion(2-4 h infusion or continuous 24 h infusion)versus traditional infusion(0.5-1 h infusion)of meropenem in the treatment of severe infectious,and to provide evi-dence-based reference for clinic treatment. METHODS:Retrieved from Medline,CJFD,VIP database and Wanfang database, modified infusion(test group)versus traditional infusion(control group)of meropenem in the treatment of severe infections were collected,and Mata-analysis was performed by using Rev Man 5.0 statistical software after extracting data and evaluating quality. RESULTS:A total of 13 studies were included,involving 1 012 patients. Results of Meta-analysis showed the effective rate [RR=1.25,95%CI(1.10,1.43),P<0.001] and bacterial eradication rate [RR=1.25,95%CI(1.05,1.48),P=0.01] in test groups were sig-nificantly higher than those of control group,and there were no significant differences in the mortality rate [RR=0.74,95%CI (0.46,1.18),P=0.21] and incidence of adverse reactions [RR=0.81,95%CI(0.48,1.39),P=0.45]. CONCLUSIONS:Compared with traditional infusion of meropenem,extended or continuous infusion can improve efficacy in the treatment of severe infections, with similar safety. Due to methodology limit of included studies,large-scale and high quality RCT are required for further valida-tion of the conclusions.