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Objective To establish a method focusing on regulation of CNN3 gene in the rat hippocampus and help to explore the role of CNN3 gene played in the brain physiology and pathology.Methods One cDNA sequence and three shRNAs targeting CNN3 gene were designed and synthesized.The recombinant lentivirus-mediated expressing and three short hairpin RNA ( shRNA) vectors targeting CNN3 gene in the rats were constructed with engineering technology.All recombinant vectors were intravenously injected into rats hippocampi guided by stereotaxic apparatus.Western blot was performed to explore the best shRNA and to study the changes of CNN3 gene in the rat hippocampus after transfection with the silence and over-expressed vectors.Results The lentivirus-mediated vector expressing CNN3-OE and three shRNA vectors targeting CNN3 gene were successfully constructed.Within eight weeks after transfection, the vectors of CNN3-OE and three CNN3-shRNAs changed the expression of CNN3 gene in the rat hippocampus, in particular, all the protein levels of calponin-3 encoded by CNN3 gene were significantly down-regulated along with the time, with the highest inhibitory rate of 73.6%in the CNN3-shRNA2 group.Significant up-regulation of calponin-3 protein level by 93.88%, was found only on the 14th day after transfection.Conclusions Lentivirus-mediated vectors of CNN3-OE and CNN3-shRNAs may regulate in vivo the CNN3 gene level in the local brain region of rats via stereotactic injection.The study lays a foundation for disease prevention and treatment in the future.
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Objective To explo the antioxidant effect and molecular mechanism of gastrodin (Gas) in epilepsy (EP) rats induced by LiCl-pilocarpine (PILO) . Methods Eighty male SD rats were randomly divided into 5 groups: sham group, EP group, therapy groups (pretreated with 60 mg/kg, 90 mg/kg, 120 mg/kg of gastrodin respectively) . The EP model was esteblished by peritoneal injection of LiCl-PILO. Therapy groups were pretreated with various concerntration of Gas. The control group was given the same dosage of normal saline. The alteration of behavior was observed, the concentration of catalase (CAT), glutathion (GSH), superoxide dismutase (SOD), glutathion reductase (GR), total antioxidtion (T-AOC) and malondialdehyde (MDA) in rats brain cortex were detected by chemical colorimetric method, phosphorylation of p38 was determined by western blot. Results There was no EP seizure in sham group,and the EP seizure degree in therapy groups (gas pretreated groups) was significantly decreased,and had statistically significant difference with EP group (P<0.05) . The EP model rats exhibited a significant decrease in the concentration of endogenous antioxidants (CAT, GSH, SOD, GR and T-AOC), while an increase of the concentration of MDA and phosphorylation p38 protein as compared to sham group (P<0.05) . After treatment of the Gas,treatment group rats attenuated the seizure degree,exhibited a significant increase of the concentration of endogenous antioxidants (P<0.05),while a decrease in concentration of MDA and phosphorylation of p38 as compared to model group (P<0.05) . Conclusion Gas may have a neuroprotective role in central nervous system of epileptic rats modle by down-regulateing the seizure degree and the activity of p38 kinase and up-regulateing the content of endogenous antioxidants.
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Objective To explore the activition and polarization of microglia in the epileptic rats induced by lithium chloride-pilocarpine. Methods One hundred male SD rats were randomly divided into five groups: control group and different time points model groups including 1d,3d,7d and 14d. Epilepsy models were established by lithium chloride-pilocarpine intraperitoneal injection. The control group was given the same dosage of normal saline. The morphology change was detected by immunofluorescence,and the expressions of iNOS and Arg-1 were determined by IHC at respective time points. Results Compared the model groups with control group,microglia was activated,synapsis was shorten,volume got bigger,most of them seemed as amoebocyte,the expression of iNOS increased and Arg-1 decreased,especially at 3d.ConclusionThe results from this study indicated that microglia was activated and polarized in epileptic rats induced by pilocarpine.
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Objective To explore the effect and injury mechanism of reactive oxygen species (ROS) after spinal cord injury (SCI) through detecting the dynamic changes of malonyldialdehyed (MDA)content in spinal cord and observing neurocyte apoptosis and correlation apoptosis factor expression after SCI. Methods Totally 132 adult SD male rats were randomly divided into three groups: sham group, SCI group, methylprednisolone (MPSS) group. The SCI of SD rats was performed by Allen's weight dropping way to impact on the posteriors of spinal cord T_(10). The contents of MDA were determined by chromatometry, the expression of Caspase-3 and Bcl-2 family in the injured spinal cord was detected by immunohistochemical staining;Apoptotic cells were detected by using fluorometric terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (fluorometric TUNEL) staining. Results The content of MDA in the injured cord increased significantly after SCI;R3eached the peak at 6 hours and 3 days post-injury, then dropped down gradually, then was back to the normal level after 7 days. The number of TUNEL labeling positive cells of SCI group increased at 6 hours post-injury;R3eached the peak at 3 days, then dropped down gradually;Bcl-2, Bax protein began to increase at 6 hours post-injury;R3eached the peak at 5 days after injury, then dropped down gradually. Caspase-3 protein began to increase at 6 hours post-injury;R3eached the peak at 3 days after injury, then dropped down gradually. The content of MDA, the number of TUNEL labeling positive cells, the expression of Caspase-3 and Bax of MPSS group decreased significantly than that of SCI group at the same time;R3espectively, while Bcl-2 protein was up-regulated after administration of MPSS.Conclusion ROS could promote the expression of Caspase-3 and degrade the ratio of Bcl-2/Bax to induce apoptosis of neurocyte, which might play significantly role in the process of secondary SCI. In addition, MPSS exerts neuroprotective effects against ROS toxicity, which might be of importance and might contribute to their clinical efficacy for the treatment of SCI.
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ObjectiveTo examine the protective effects of 17-β estradiol on the experimental model of spinal cord injury (SCI) rats. Methods One hundred and eighty male Sprague Dawley (SD) rats, after Allen' s model, SD rats were divided into three groups: the sham group, the acute spinal cord injury (control groups) and the acute spinal cord injury supplying with 17-β estradiol treatment group. SCI was made by Allen's weight dropping, impacting on the posteriors of spinal cord T10. The content of malonyldialdehyed (MDA), glutathione (GSH), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were determined by chromatometry. The expressions of Caspase-3 and Bcl-2 family in the injured spinal cord were detected by immunohistochemical staining. Results The BBB scores at each time point in 17-β estradiol treatment group were significantly higher than that in SCI group (P<0.05). The contents of GSH, SOD, GSH-Px and the expression of Bcl-2 protein at the majority of time point in 17-β estradiol treatment group were significantly higher than that in SCI group(P<0.05), however, the MDA, Caspase-3 and Bax were markedly decreased (P<0.05). Conclusions This study suggests that 17-β estradiol administration might prevent the cells from SCI-induced apoptosis by triggering to reduce the oxidative stress.