ABSTRACT
<p><b>OBJECTIVE</b>To construct effective RNA-interference plasmids targeting mouse HIF-1alpha gene and testify their effects and specificities in interfering HIF-1alpha expression.</p><p><b>METHODS</b>Three RNA-interference plasmids targeting mouse HIF-1alpha gene, pBS/U6/HIF-1alpha-siRNAI~III, were constructed and identified using double digestion method in the present study. RT-PCR, immunostaining and western blotting were employed to detect the expression alterations of HIF-1alpha in 293T cells following transfections of the three plasmids, respectively. The interference effect of pBS/U6/HIF1alphai-II in SH-SY5Y cell line was further investigated.</p><p><b>RESULTS</b>All the three RNA-interference plasmids, especially pBS/U6/HIF1alphai-II, showed significant inhibition in HIF-1alpha expression in 293T cell line. pBS/U6/HIF1alphai-II could also inhibit HIF-1alpha expression in SH-SY5Y cell line, in a dose-dependent way.</p><p><b>CONCLUSION</b>Plasmid pBS/U6/HIF1alphai-II constructed in our study can effectively and specifically inhibit HIF-1alpha expression, and its role in neural tube development and dysfunction will be further investigated. Construct of pBS/U6/HIF1alphai-II plasmid will provide a useful tool to study the role of HIF-1 pathway in embryogenesis, oncogenesis and ischemia development.</p>
Subject(s)
Animals , Humans , Mice , Analysis of Variance , Cell Line, Tumor , Dose-Response Relationship, Drug , Gene Expression , Gene Silencing , Physiology , Green Fluorescent Proteins , Genetics , Hypoxia-Inducible Factor 1, alpha Subunit , Genetics , Metabolism , Plasmids , Genetics , RNA, Small Interfering , Genetics , Pharmacology , Transfection , MethodsABSTRACT
Objective To evaluate the effect of Naogongfude (NGFD, a traditional Chinese medicine orally administrated) on learning and memory and the expressions of synaptophysin (SY) and Tau-protein in cerebral cortex and hippocampus in aged rats and to explore the possible mechanism. Methods Rats were divided into normal and neural disturbance groups according to the outcomes of active avoidance reaction (AAR) test, and then each group was randomly divided into control and NGFD-treated experimental groups. Animals were orally fed with NGFD for 2 months (5 ml/d) in experimental group or routinely fed in control and taken AAR and passive avoidance reaction (PAR) tests. After the rats were sacrificed, the synaptosome count, the expression of SY and Tau-protein, and the neuron apoptosis in cerebrum were examined. Results The rats after 2-month NGFD administration had an increased AAR acquisition, obviously delayed AAR extinction and prolonged step through latency (STL) of PAR. The number of synaptosomes was raised and the immunoreactive intensity of synaptophysin was increased remarkedly, while Tau-protein immunoreactivity and apoptotic cells were decreased in cerebrum. Conclusion NGFD does have the effect of improving brain function and putting off the aging of rat brain according to the results of behavior study and morphological observation.
ABSTRACT
Objective To evaluate the effect of Naogongfude (NGFD, a traditional Chinese medicine orally administrated) on learning and memory and the expressions of synaptophysin (SY) and Tau-protein in cerebral cortex and hippocampus in aged rats and to explore the possible mechanism. Methods Rats were divided into normal and neural disturbance groups according to the outcomes of active avoidance reaction (AAR) test, and then each group was randomly divided into control and NGFD-treated experimental groups. Animals were orally fed with NGFD for 2 months (5 ml/d) in experimental group or routinely fed in control and taken AAR and passive avoidance reaction (PAR) tests. After the rats were sacrificed, the synaptosome count, the expression of SY and Tau-protein, and the neuron apoptosis in cerebrum were examined. Results The rats after 2-month NGFD administration had an increased AAR acquisition, obviously delayed AAR extinction and prolonged step through latency (STL) of PAR. The number of synaptosomes was raised and the immunoreactive intensity of synaptophysin was increased remarkedly, while Tau-protein immunoreactivity and apoptotic cells were decreased in cerebrum. Conclusion NGFD does have the effect of improving brain function and putting off the aging of rat brain according to the results of behavior study and morphological observation.