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Objective To investigate the preventive effect and mechanisms of Naoshuantong capsule in stroke induced by artificial cold wave in hypertensive rats.Methods A total of 130 SD rats were randomly divided into a sham operation group (n =30),a model control group (n =50) and a Naoshuantong treatment group (n=50;intragastric administration of Naoshuantong,0.5 g/kg,2/d for 7 days).Renovascular hypertensive rats model were established by two-kidney,two clip method.At 13th week after operation,rats were exposed to artificial cold wave for 3 days (12 h light of 22 ℃ and 12 h dark of 4 ℃,3 cycles).The brain tissue samples were extracted at the end of the experiment.Differential protein proteomic techniques were used for the identification,functional classification and preliminary analysis of the differentially expressed protein spots,and Western blot was used for the validation of some key proteins.Results There was no occurrence of stroke in the sham operation group,and the incidence of stroke in the model control group (36.00％,18/50) was significantly higher than that in the Naoshuantong treatment group (18.00％,9 / 50;x2 =4.110,P =0.043).With the two-dimensional gel electrophoresis analysis,6 different proteins were identified from 14 protein spots.Among them,the up-regulated superoxide dismutase 2 (SOD2) and the down-regulated B-cell lymphoma 10 (Bcl-10) were found to be at the central location of protein interactions,which has been verified by Western blot.Conclusion Naoshuantong can reduce the occurrence of stroke induced by artificial cold wave in renovascular hypertensive rats.SOD2 up-regulating and Bcl-10 downregulating may be involved in the mechanisms of of Naoshuantong in the prevention of cold wave-induced stroke in hypertensive rats.
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AIM: To clarify the modulation of autophagy in ischemic penumbra by hydroxysafflor yellow A ( HSYA) after cerebral ischemia/reperfusion ( I/R) injury.METHODS:Male SD rats subjected to transient middle cere-bral artery occlusion for 90 min were randomly divided into 4 groups:sham-operation (sham) group, cerebral I/R (I/R) group, I/R+HSYA group and sham+HSYA group.Modified neurological severity score (mNSS) was used to evaluate the neurological deficits of the rats at 6 h, 1 d, 3 d and 7 d after reperfusion , accompanied by the detection of autophagy , apoptosis and mRNA expression of IFN-βin ischemic penumbra .RESULTS:Compared with sham group , upregulation of LC3-Ⅱand degradation of SQSTM1/P62 were observed in I/R group, indicating the activation of autophagy after I/R.The activation of autophagy in I/R+HSYA group was significantly enhanced by HSYA on day 1 and day 3, and inhibited on day 7, as compared with I/R group (P<0.05).Besides, the mRNA expression of IFN-βin I/R group was significantly upregulated at 6 h, then downregulated on day 1 and day 3, and returned to basal level on day 7, as compared with sham group (P<0.05).In I/R+HSYA group, the mRNA expression of IFN-βwas significantly upregulated on day 1 and day 3, accompanied by the inhibition of apoptosis on day 3 and the significantly decreased mNSS from day 4, as compared with I/R group (P<0.05).CONCLUSION:HSYA alleviates cerebral I/R injury by dynamically modulating the activation of autophagy and the expression of IFN-βin ischemic penumbra .
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AIM:To observe the Toll-like receptor 9 (TLR9) activation in microglia BV-2 cells after oxygen-glucose deprivation and reoxygenation ( OGDR) , and its effects on neuronal apoptosis.METHODS:The BV-2 cell super-natants were collected after the corresponding treatment and added to mouse primary cortical neurons after OGDR for 4 h, followed by normal culture for 24 h.The cells were divided into normal BV-2 group, NC-siRNA group, TLR9-siRNA group, OGDR group, OGDR+NC-siRNA group, OGDR+TLR9-siRNA group and control group (without adding BV-2 cell supernatant) .The changes of the neuronal morphology were observed under an inverted phase-contrast microscope, and the neuronal apoptosis was detected by TUNEL.The protein expression of cleaved caspase-3 was detected by Western blot-ting.RESULTS:After OGDR, the axon turned thin, twisted and broken, and neuronal swelling, decrease in refraction and vacuolar degeneration were observed.The green-stained apoptotic bodies in the neurons in all groups were positive. Compared with control group, the caspase-3 protein levels in other groups were increased.Compared with the normal BV-2 group, the caspase-3 protein in OGDR group and TLR9-siRNA group was increased.Compared with OGDR+TLR9-siRNA group, the caspase-3 protein in TLR9-siRNA group and OGDR group was decreased.CONCLUSION: After OGDR, TLR9 activation in BV-2 cells induces neuronal apoptosis with the increase in caspase-3 protein level.Inhibition of TLR9 expression reduces neuronal damage.
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BACKGROUND: Looking for effective measures to ensure the survival of the implanted stem cells against ischemia-induced hypoxia becomes the major concern in the research of cell transplantation therapy for cerebral infarction.OBJECTIVE: To study the effects of human Persephin gene transfer on hypoxia-induced apoptosis of neural stem cells.DESTGN: A randomized controlled basic study on cells.SETTTNG: Department of Neurology, the Second Affiliated Hospital of Sun Yat-sen University.MATERTALS: This study was completed in the Lin Baixing Laboratory Center of the Second Affiliated Hospital of Sun Yat-sen University from July to December in 2006. Recombinant adenovirus pAdCMV persephin was constructed in our lab. C17.2 neural stem cells were kindly provided by Prof. Snyder, Harvard Medical University, USA. Trypsin and DMEM/F12 were purchased from Gibco Company (USA), fetal bovine serum (FBS) from Sijiqing Biological Engineering Materials Co. Ltd (Hangzhou, China); Poly-lysine from Sigma Company (USA), TUNEL assay kit and FuGENE kit from Roche Molecular Biochemicals Company (Swiss), and S-P immunohistochemical detection kit and DAB reaction kit from Mycine Biological Engineering Company (Fujian). Rat anti-human monoclonal Nestin antibody and rabbit anti-human polyclonal persephin antibody were manufactured by Santa Cruz Company (USA), and persephin anti-senseoligodeoxynucleotide (ODN) was synthesized by Shanghai Biological Engineering Company.METHODS: ① Interventions: C17.2 neural stem cells cultured in vitro were infected by recombinant adenovirus containing persephin gene, and they were divided into four groups: blank control group (Group A, in which the C17.2 neural stem cells were not treated with hypoxia), hypoxic group [Group B, in which the cells were cultured at 37 ℃ in anaerobic incubation containing N2 (0.95 in volume fraction) and CO2 (0.05 in volume fraction)], hypoxia + pAdCMV persephin infection group [Group C, where the cells were cultured under the conditions as in group B after pAdCMV persephin infection for 48 hours], and hypoxia + pAdCMV persephin infection + anti-sense persephin ODN group (Group D, where the cells were infected by pAdCMV persephin and anti-sense persephin ODN. ② Evaluation: The expression of Persephin protein was analyzed using Western blotting; Apoptotic index was detected with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay; The changes of apoptotic rate was determined with flow cytometry.MAIN OUTCOME MEASURES: Expression of Persephin protein; Apoptotic index; Apoptotic rate.RESULTS: ① Expression of Persephin protein: A specific band (relative molecular mass of 24 000) was detected by Western blotting in pAdCMV persephin infected cells, suggesting the successful expression of persephin gene.Interestingly, the cells infected with both pAdCMV persephin and anti-sense persephin ODN also showed the specific band of about 24 000, but with much less density, indicating that anti-sense persephin ODN could effectively inhibit the expression of pAdCMV persephin. However, this band was not presented in the blank control groups. ② Apoptotic index:The apoptotic index in group C was significantly lower than those in groups B and D (P＜0.01), but still higher than that of group A (P＜0.01), suggesting that persephin gene transfer could attenuate apoptosis to some extent. ③ Apoptotic rate: The apoptotic rate in groups B and D were obviously higher than that in group A (P ＜ 0.01), and it was lower in group C than in groups B and D (P＜0.01).CONCLUSION:Recombinant adenovirus can efficiently mediate Persephin gene transfer into C17.2 neural stem cells,resulting in high expression of the exogenous Persephin in vitro, which effectively reduces C17.2 neural stem cell apoptosis induced by hypoxia.
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ObjectiveTo investigate the therapeutic effects of insulin o n acute cerebral infarction.MethodsEighty-six non-diabetes mel litus patients with acute middle cerebral artery (MCA) infarction were divided i nto two groups in random fashion:42 cases (control group) were treated by routin e methods and 44 cases (insulin group) by regular insulin on the basis of routin e methods.12～16U regular insulin per diem was administered by intravenous dripp n g in 7 days.Plasma glucose,insulin sensitivity indice (ISI) and score values of the European Stroke Scale (ESS) were observed.ResultsThere wer e no statistically significant differences between the 2 groups at baseline.The everyday plasma glucose levels of the insulin group.were within the normal range but were significantly lower than those of the control group (P