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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 investigate the protective effect and the mechanism of amitriptyline on sepsis-induced kidney injury.Methods 48 male Sprague Dawley (SD) rats were randomly divided into six groups:sham group,sham + saline group,sham + amitriptyline group,sepsis group,sepsis + saline group,sepsis + amitriptyline group.Sepsis model was induced by cecal ligation and puncture (CLP).Sham group did not undergo CLP.Amitriptyline were injected subcutaneously to rats with amitriptyline 3 mg/kg before treatment for 10 d,2 times/d,while the control group was injected with the same amount of normal saline.Blood sample was obtained to detect serum level of acid sphingomyelinase (ASMase),ceramide,blood urea nitrogen (BUN),creatinine and interleukin-1β.Malondialdehyde in kidney tissue was determined,and Kidney hematoxylin eosin (HE) staining was performed to observe pathologic change.Another 48 rats were randomly divided into four groups:sham group,sepsis group,sepsis + saline group,sepsis + amitriptyline group.The intervention to each group were same as above description.Rats of the four groups were observed for seven days to determine the survival rate.Results The survival rate of rats in sham group were 100％,the survival rate of the sepsis group and the sepsis + physiological saline group was less than 20％,while the survival rate of the sepsis + amitriptyline group was ＞ 50％.The serum concentration of IL-1β,ceramide,BUN,SCr,the activity of ASMase and the level of malondialdehyde (MDA) in kidney tissue were respectively increased in sepsis group,sepsis + saline group and sepsis + amitriptyline group at the 4 time points were significantly higher than the sham groups (P ＜0.01),these indexes in sepsis plus amitriptyline group were significantly decreased compared to sepsis group (P ＜ 0.01).There was no pathologic change in sham group,sham + saline group and sham + amitriptyline group.In sepsis group and sepsis + saline group,general degeneration and necrosis of renal proximal tubular epithelial cells,marked dilatation of renal tubular capsular space,extensive infiltration of inflammatory cells and hemorrhage in renal interstitium were observed.These pathologic changes were obviously reduced in sepsis + amitriptyline group compared with sepsis group.Conclusions Amitriptyline can alleviate sepsis-induced kidney injury,through inhibiting the activity of ASMase and then restraining inflammatory response and oxidative stress in renal tissues.