AIM: To verify the neuroprotective effect and optimize the therapeutic dose and time window of picroside Ⅱon cerebral ischemic injury in rats .METHODS:The forebrain ischemia model was established by the method of bilateral common carotid artery occlusion ( BCCAO ) .The successful model rats were randomly divided into 16 groups according to orthogonal design and treated by intraperitoneal injection of picroside Ⅱat different ischemic time poinis and different doses .The changes of the nerve fiber myelin were observed by fast green staining .The immunohistochemical assay and Western blotting were used to quantitatively and qualitatively determine the expression of myelin basic protein (MBP). The mRNA level of MBP in the brain tissues was tested by reverse transcription polymerase chain reaction (RT-PCR).RE-SULTS:Picroside Ⅱ increased the expression of MBP and decreased demyelination after cerebral ischemic injury .The best therapeutic time window and dose were:(1) ischemia for 2.0 h with picrosideⅡat dose of 10 mg/kg according to the results of fast green staining;(2) ischemia for 2.0 h with the dose of 10 mg/kg according to the results of immunohisto-chemical assay;(3) ischemia for 2.0 h with the dose of 10 mg/kg according to the analysis of Western blotting;(4) is-chemia for 1.5 h with the dose of 20 mg/kg according to the detection of RT-PCR.CONCLUSION:Given the principle of the lowest therapeutic dose with the longest time window , the optimized therapeutic dose and time window for rat cerebral ischemic injury is intraperitoneal injection of picroside Ⅱat the doses of 10~20 mg/kg and the time window of ischemia for 1.5~2.0 h.

Objective To study the time relationships between apoptosis of neurons and endotheliocytes with the expression of Bcl 2 and Bax after reperfusion of focal cerebral ischemia in rats. Methods Coronal sections of brain were analyzed using an in situ terminal deoxynucleotidyl transferase mediated biotinylated deoxyuridine triphosphate nick end labeling(TUNEL)and immunohistochemical staining methods to observe apoptosis of neurons and endotheliocytes,and expression of Bcl 2 and Bax after reperfusion(2,6,12,24hours and 2,3,7,14,21 days)of focal cerebral ischemia. Results 1.In the ischemic penumbra,apoptotic cells were increased at 2h after reperfusion,peaked at 12 24?h,then decreased successfully for 7 14?days.The time of apoptotic endothelial cells was 12?h later than that of apoptotic neurons.2.The expression of Bcl 2 protein began at 2?h after reperfusion,peaked at 12 24?h,and decreased for 7 14?days.3.Bax protein expressed from 6?h after reperfusion;peaked at 24 48?h,and lasted for 14days.4.The time phase of Bcl 2 expression was similar to the Bax is but later than it. Conclusion\ Apoptosis was a pattern of cell death after cerebral ischemia/reperfusion.The time of apoptotic endothelial cells was later than that of apoptotic neurons.Bcl 2 and Bax play a regulatory role in the apoptotic process.\;[

Objective To explore the neuroprotective effect and mechanism of picroside II on ERK1/2 signal transduction pathway after cerebral ischemia injury in rats.Methods The focal cerebral is-chemic models were established by inserting a monofilament threads into middle cerebral artery occlusion (MCAO) in 100 Wistar rats and treated by injecting picroside II (20 mg/kg) intraperitoneally.The neu-robehavioral function was evaluated by modified neurological severity score points ( mNSS) test.The cerebral infarct volume was measured by tetrazolium chloride ( TTC) staining.The apoptotic cells were counted by terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL) assay.The expression of pERK1/2 in cortex was determined by the immunohistochemistry ( IHC) and Western Blot ( WB) .Results mNSS test showed that severe neurological dysfunction was found in model and LPS groups,and the scores of mNSS were significantly increased;meanwhile the scores of mNSS in treatment group and U0126 group were signifi-cantly lower than that in model and LPS groups (P<0.05).TUNEL assay showed that the apoptotic cell inde-xes (ACI) in different groups were (0.06±0.02),(0.27±0.03),(0.07±0.02),(0.26±0.03)and(0.09± 0.05) ,and the ACI in treatment and U0126 groups was obviously lower than that in model and LPS groups (P<0.05) .With IHC and WB,pERK1/2 level in model group was the highest,which was slightly higher than that of LPS group,and pERK1/2 expression in treatment and U0126 groups was significantly decreased com-pared with that in model and LPS groups (P<0.05) .Conclusion The activation of ERK1/2 by cerebral is-chemia could induce the cell apoptosis.Picroside II might reduce cell apoptosis by inhibiting the activation of ERK1/2 in ischemic brain injury.

Objective To study the apoptosis of endothelial cells and the relation between it and the expression of P53 protein after focal cerebral ischemia reperfusion in rats.Methods The apoptosis of endothelial cell 2,6,12 h and 1,2,3,7,14,21 d after cerebral ischemic reperfusion were observed using an in situ end labeling method,and the expression of P53 protein was detected by immunohistochemical staining methods.Results Apoptotic endothelial cells in ischemic penumbra were observed 2h after cerebral ischemic reperfusion, they peaked at 12~24 h, and decreased gradually.There was no remarkable difference between it and the sham operative group at 21 d.The P53 protein began to express 6h after cerebral ischemic reperfusion, it peaked at 1~2 d, and then declined gradually to controlled level at 7d. The expression peak time of P53 was 24 h later than that of cell apoptosis.Conclusion Apoptosis was a pattern of endothelial cell death after reperfusion of MCAO. P53 protein played an important role in the process of apoptosis of endothelial cells.

Objective To study the neuroprotective effects of neuregulin-1?(NRG-1?) on the nervous behavioral function,cerebral infarction volume,brain water content(BWC),neuronal apoptosis and aquaporin-4(AQP-4) expression in astrocytes after cerebral ischemic reperfusion and the related mechanism in mice.Methods Intraluminal thread methods were applied to establish the middle cerebral artery occlusion reperfusion models in the mice.Neuregulin-1?(2?g / kg) was injected into the internal carotid artery for treatment.The nervous behavioral function was evaluated with Bederson's test.The cerebral infarction volume was observed with tetrazolium chloride staining.The BWC was measured by dry-wet weight comparing.The apoptosis positive cells were counted by immunofluorescence assay.The expression of AQP-4 was determined by immunohistochemical assay.Results Nervous behavioral malfunction appeared in all the mice with left middle cerebral artery occlusion and/or reperfusion.The infarction focus showed in the ischemic hemisphere after the injury.The BWC,the number of neuronal apoptosis cells and AQP-4 expression in astrocytes were higher than those in the sham group. In the NRG-1? treatment group,the nervous behavioral function was improved 24 hours after ischemia,the number of apoptosis positive cells reduced and the infarction volume decreased significantly compared with the control group(P0.05).In the groups of reperfusion for 22,46 and 70 hours,the five indexes mentioned above were significantly different from those in the corresponding control groups(P

Objective To investigate the regulating effects and mechanism of Laminaria japonica (L.japonica) on serum lipid of hyperlipidemia rats.Methods Forty healthy female Wistar rats were used to establish hyperlipidemia models by feeding fat-rich forage,and the powder of L.japonica was applied as a supplement in forage for test groups.The levels of serum lipid including the triglyceride(TG),total cholesterol(TC),low-density lipoprotein (LDL),high-density lipoprotein(HDL) and the activities of lipoproteinesterase(LPL) and hepatic lipase(HL) were detected by biochemical assay.Results The levels of serum TG and TC in test group decreased significantly than those in pre-treated and model group (P

SIRT1 (Sirtuin type 1 ), a member of histone deacetylase, dependents on nicotinamide adenine dinucleotide ( NAD + ). It involves in the covalent modification of histones, participates in tumor development and progression through transcription, translation and post-translational modification and so on. Therefore, the expression of SIRT1 in tumor cells or abnormal function could be one of the important mechanisms of tumor development, and may become a new potential therapeutic target for neoplasms.

HIC1 (hypermethylated in cancer 1) encodes a transcriptional repressor,and extensively resides in various kinds of normal tissue.HIC1 gene is located in chromosome 17p13.3,in which loss of heterozygote or super-methylation is frequently found in a variety of human cancers.As a new tumor marker,HIC1 has been confirmed down-regulated in a wide variety of solid cancers because of HIC1 promoter hypermethylation,and may be associated with tumor prognosis.As a tumor suppressor,HIC1 participates in the development of tumor process through various ways,and is involved in cell proliferation,tumour growth,and angiogenesis.Therefore,the abnormal hypermethylation or the loss of expression of HIC1 in tumor cells or abnormal function could be one of the important mechanisms of tumor development,and may become a new potential therapeutic targets for cancer.

Objective To study the association between CYP2C9 gene polymorphism and warfarin maintenance dosage in anticoagulation therapy.Methods 200 Han patients admitted to our hospital for heart valve replacement were included in this study.CYP2C9 * 2,CYP2C9 * 3,CYP2C9 *c65 in CYP2C9 gene were sequenced using the CAPS technique and conventional DNA sequencing method.Dosages of warfarin used in patients carrying different genes were analyzed.Results No mutation of CYP2C9 * 2 but only one kind of allele C was detected in 200 patients.The genotype of CYP2C9 * 2 was C/C wild type.Allelic gene was detected at CYP2C9 * 3 A and C,with A/A wild type detected in 171 patients,A/C heterozygote mutation type detected in 18 patients,and C/C heterozygote mutation type detected in 11 patients respectively.The frequency of allelic genes A and B was 94.3 ％ and 5.7 ％ respectively.A significant difference was found between CYP2C9 * 3 mutation and warfarin dosage (P＜0.05).The dosage of warfarin reduced 18.46％ and 76.0％ respectively in patients carrying A/C heterozygote mutation type and in those carrying C/C heterozygote mutation type.Two kinds of allelic gene were detected at CYP2C9 * c65 G and C,with G/G wild type detected in 182 patients and G/C heterozygote mutation type detected in 18 patients respectively.No significant association was found in warfarin maintenance dosage for patients carrying G/G wild type and G/C heterozygote mutation type.Conclusion CYP2C9 gene polymorphism is associated with warfarin maintenance dosage in anticoagulation therapy.

BACKGROUND: It has been demonstrated that insulin-like growth factor-1 (IGF-1) is a kind of neurotrophic factor and protects from cerebral ischemia/reperfusion injury, the expression of IGF-1 is associated with the attack of ischemic stroke. The effects of IGF-1 and its receptor (IGF-1R) on neurobehavioral function are to be further studied.OBJECTIVE: To observe the effects of IGF-1 and IGF-1R on neurobehavioral function in rat models of cerebral ischemia/reperfusion injury.DESIGN: A randomized controlled observation.SETTING: Institute of Cerebrovascular Diseases, Affiliated Hospital of Qingdao University Medical College.MATERIALS: The experiments were carried out in Shandong Key Laboratory for Prevention and Treatment of Brain diseases. Twenty-eight healthy adult Wistar rats of clean degree, weighing 220-260 g, were provided by the experimental animal center of Shandong University.METHODS: The rats were randomly divided into experimental group (n =24) and sham-operated group (n =4). The middle cerebral artery occlusion/reperfusion (MCAO/R) models were established by inserting a thread through left external-internal carotid arteries. The sham-operated rats were given the same treatments except inserting thread. ①Neurologic deficit test: The rats in the experimental group were assessed according to Bederson standard after 1-hour ischemia and 6, 12-hour, 1, 3, 7 and 14-day reperfusion respectively. The sham-operated rats were assessed at corresponding time points; Without neurologic deficit was marked as 0 point; flexion of anterior claws as 1 point; unable to act against the pushing from the contralateral side as 2 points; circling while walking as 3 points; shaking as 4 points;unconscious mind as 5 points. ② Sample collection and treatment: The samples in the experimental group were collected after 1-hour ischemia and 6, 12-hour, 1, 3, 7 and 14-day reperfusion, and those in the sham-operated group ere collected at 24 hours postoperatively. The rats were anesthetized, brain samples were got at about 5 mm posterior to optic chiasma after brains were removed completely, then serial coronal sections (5 μm) were prepared, and 1 from 10 sections was stuck to the cover glasses treated with poly-L-lysine. ③ Morphological observation of neurons: The neurons in brain were observed by toluidine blue staining. ④ Detection of IGF-1 and IGF-1R: The expressions of IGF-1 and IGF-1R in cortex and striatum were detected with immunohistochemical technique, 4 fields were randomly selected to count the positive cells under high-power microscope (×400).MAIN OUTCOME MEASURES: ① The neurologic deficit; ② Morphological changes of neurons in brain; ③ Expressions of IGF-1 and IGF-1R in cortex and striatum.RESULTS: All the 28 rats were involved in the analysis of results. ① The neurologic deficit: The scores of neurologic deficit were (1.50±058) and (1.50±0.78) in rats after 7 and 14-day reperfusion, which were lower than that in rats after 6-hour reperfusion [(3.00±0.00), P ＜ 0.05]. ② Morphological changes of neurons in brain: The neurons in ischemic area appeared as paryopyknosis and became irregular in shape, there were obvious gaps around the cells, also deeply stained as purplish blue, nucleolus disappeared, and there were many scattered cellular fragments. ③ Expressions of IGF-1 and IGF-1R in cortex and striatum: The numbers of IGF-1 positive cells in cortex were (8.75±2.06), (11.13±1.14),(19.75±3.18), (17.38±3.11 ) and (11.23±2.28) respectively in rats after 6, 12-hours and 1, 3, 7-day reperfusion, which all were higher than that in sham-operated rats [(3.88±1.46), P ＜ 0.05], the numbers of IGF-1 positive cells in striatum were(8.25±2.21), (11.34±2.21), (18.23±2.64), (18.56±2.34) and (11.31±2.14) respectively in rats after 6, 12 hours and 1, 3, 7days reperfusion , which were also higher than that in sham-operated rats [(4.12±2.24), P ＜ 0.05]. The numbers of IGF-1R positive cells in cortex were (7.63±1.50), (10.50±2.34), (15.55±3.12), (15.37±3.01), (8.86±2.75) respectively in rats after 6, 12-hours and 1,3,7-day reperfusion, which all were higher than that in sham-operated rats [(4.13±1.81), P ＜0.05]. Those in striatum were (8.33±2.31), (10.24±2.09), (14.72±2.17), (14.24±2.77), (8.38±2.05), which were also higher than that in sham-operated rats [(3.76±2.35), P ＜ 0.05].CONCLUSION: The neurological function is damaged after cerebral ischemia/reperfusion, but it has a trend of self-recovery. The expressions of IGF-1 and IGF-1R are mainly distributed in cortex and striatum. Higher expressions of IGF-1 and IGF-1R maintain during 12 hours to 7 days after reperfusion and have a peak value at 1-3 days, which suggests that early expression of IGF-1 and IGF-1R are certain related to the recovery of neurological function.