ABSTRACT
Aim To assess the effects of Trillium Tschonoskii Maxim ( TTM ) decoction on Tau protein phosphorylation and synaptic development in AD model rats induced by high activity GSK-3β. Methods The SD rats were divided into five groups of ten animals, named sham-operated group ( blank group) , AD model group, TTM group (0. 5, 0. 25, 0. 125 g·kg-1 · d-1 ) . Treatment group received gavage once a day for seven days with TTM decoction, while other groups by gavage once a day for seven days with drinking water. On 2nd day by gavage, Morris water maze test was used to assess the spatial learning and memory ability of the rats. After five days' training, rats in the treat-ment groups and AD model group were injected wort-mannin ( WT, PI3K specific inhibitor ) and GF-109203X (GFX, PKC specific inhibitor) (100 μmol ·L-1 of each, total volume of 10 μL) into the right lateral ventricle. Western blot was used to detect the levels of phosphorylation Tau protein at multiple sites and the expression level of PI3K, Akt, PKC, GSK-3β(S9, T216) and synapse-associated proteins. Immu-nohistochemical method was used to detect the hyper-phosphorylation of Tau protein in hippocampus of rats. Golgi staining was applied to detect the number and morphological changes of synaptic development and dendritic spines. Nissl' s staining was employed to ob-serve the development of neonatal neurons in hippo-campus and cortex. Results Western blot showed that the phosphorylation level of Tau in hippocampus increased in model group, and the activity of GSK-3βwas up-regulated. Among them, however, in middle dose TTM group, the phosphorylation level of Tau in hippocampus decreased and the activity of GSK-3βde-creased. The expression levels of p-PKC and p-Akt in low and middle dose treatment group were higher than those in model group, thus increasing the activity of PKC and Akt to inhibit the activity of GSK-3β kinase. Immunohistochemistry also indicated that TTM could decrease the biological effects of Tau phosphorylation in hippocampus of AD rats. Western blot showed that TTM could increase the expression levels of synapsin-1 , syn-aptophysin and GluR-1 in hippocampus of AD rats. Nissl staining showed that the number of Nissl bodies in hippocampal neurons of AD model group were signif-icantly fewer than those of sham operation group, which could be increased by TTM middle and high dose group, and the complexity and dendritic spine density of hippocampal neurons in AD rats could be en-hanced as well. Conclusion TTM can effectively im-prove the cognitive function of AD rats induced by the increase of GSK-3β activity, and its possible mecha-nism may be via down-regulating the activity of GSK-3β and inhibiting the phosphorylation of tau protein and promoting the development of neurons.
ABSTRACT
Objective: To develop and validate methods for determination of cholesteryl-phosphoryl zidovudine(CPZ)and its metabolite zidovudine(AZT)in rat plasma and tissues,and use the methods to investigate the pharmacokinetics of CPZ in rats. Meth- ods: An HPLC-UV method was applied for the determination of CPZ in biological samples. The biological samples were precipitated with acetonitrile at first,and then CPZ was separated on a Diamonsil C 18 column(200 mm×4.6 mm I.D.,5 μm)with a gradient elution system comprising 90% methanol and 10% isopropanol(containing 2 mmol/L cetyl trimethylammonium bromide,2% acetic acid and 0.1% ammonium hydroxide). The eluent was monitored at 266 nm by UV detector. The determination of AZT in biological samples was performed by LC-MS/MS after it was extracted from the biological samples by liquid-liquid extraction with methyl tertbutyl ether. Chro- matographic separation was performed on a Poroshell 120 EC-C 18 column(50 mm×2.1 mm I.D.,2.7 μm). Using the mobile phase con- sisted of 35% methanol and 65% water containing 0.2% acetic acid,the column was eluted by an isocratic elution with the flow rate of 0.3 ml/min. Detection of AZT and the internal standard(IS)acetaminophen was achieved by ESI MS/MS in the positive ion mode using m/z 268.2→m/z 127.0 and m/z 152.1→m/z 110.0 transitions,respectively. Results: The linear ranges for the quantitative determina- tion of CPZ and AZT were 5-400 μg/ml and 2-500 ng/ml,respectively,when 50 μl plasma was analyzed. The lower limit for the quan- tification of CPZ and AZT was 5 μg/ml and 2 ng/ml,respectively. The inter-and intra-day precision values were below 15%,and the accuracy(relative error)was within ± 13.8% in all quality control samples. CPZ was quickly metabolized in rats,while AZT was oppo-site. The half-life of AZT was about 8 h. The distribution of CPZ and AZT was more in the liver,spleen and lungs of rats,and less in the heart and kidney. Conclusion: The methods completely meet the requirements for pharmacokinetic study of CPZ in rats. The phar- macokinetic results show that CPZ could release AZT targeting liver,spleen,and lungs in rats.
ABSTRACT
Objective To investigate the effect of extracellular signal-regulated kinases(ERK) signal transduction in the process of NSCs differentiating into neurons in the fimbria-transected hippocampi's extracts. Methods Twelve Sprague-Dawley rats'right fimbrias were transected. The extracts were gained from the fimbria-transected hippocampi at the 14th day normal rat, and the extracts supernatant fluid was collected after centrifugal process, then the protein concentration in the extracts was determined. In the serum-free medium,NSCs from the fetal hippocampus were planted on 24 well culture plate, then were divided into three group and eight wells for each group as follows: the transected group contained the extracts of the fimbria-transected hippocampi;the normal group contained the extracts of the normal hippocampi;the pure control group have no extracts. After cultured for 14 days,the cells were detected by using MAP-2 and p-ERK immunofluorescence. Result The number, area, perimeter of MAP-2 positive neurons were all declined in transected group, the normal group and the control group orderly. Statistic results showed significant difference between every two groups. The number of MAP-2/p-ERK double-positive neurons were decreased in transected group, the normal group and the control group orderly, but the percentage of double-labeled neurons in total MAP-2 positive neurons were increased in turn. In these two aspect, there were also significant difference between every two group. And most of the MAP-2/p-ERK double-positive neurons were immature. Conclusion The extracts of the fimbria-transected hippocampi had obvious effects on promoting NSCs differentiating into neurons and speeding up the maturation of neurons than those of the normal hippocampi. The morphological results showed that ERK signal transduction might be related to the differentiation of NSCs into neurons.
ABSTRACT
The X-ray structure determination of yeast phosphoglycerate kinase and subsequent substrate binding studies have helped to define the binding sites for the triose and nucleoside phosphate substrates. This communication deals with one feature of the binding site—the location of an aspartic acid residue close to the phosphoryl binding site of the nucleotide substrate—and relates this and other structural features of the active site to the properties of this enzyme as deduced from nuclear magnetic resonance studies.