Objective To evaluate the role of 1-phosphatidylinositol 3-kinase (PI3K) signaling pathway in nerve growth factor-beta (NGF-β)-produced mitigation of cell apoptosis induced by endoplasmic reticulum stress during hypoxia-reoxygenation (H/R) in rat cardiomyocytes.Methods H9c2 cells were seeded in 96-well plates at a density of 5× 105 cells/ml (100 μl/well).The wells were randomly divided into 5 groups (n =15 each) using a random number table: control group (group C);group H/R;NGF-β group (group N);NGF-β+NGF receptor trkA antagonist K252a group (group N+K);NGF-β+ PI3K inhibitor LY294002 group (group N+L).The cells were exposed to 95％ N2-5％ CO2 for 4 h in an anaerobic incubator, followed by reoxygenation in a standard incubator for 4 h in H/R, N, N+K and N+L groups.In addition, the cells in N, N + K and N + L groups were incubated in a standard incubator containing NGF-β, the mixture of NGF-β and K252a and the mixture of NGF-β and LY294002, respectively, during reoxygenation, and the final concentrations of NGF-β , K252a and LY294002 were 50 ng/ml, 100 nmol/L and 50 μmol/L, respectively.The cell viability was detected by using CCK-8 assay, and the cell survival rate was calculated.The cell apoptosis was examined by flow cytometry, and apoptosis rate was calculated.The expression of glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), caspase-12, Akt and phosphorylated Akt (p-Akt) was detected by Western blot.The ratio of p-Akt to Akt was calculated.Results Compared with group C, the cell survival rate was significantly decreased, and the apoptosis rate was increased in H/R and N groups, and the expression of GRP78, CHOP and caspase-12 was significantly up-regulated in group H/R, and p-Akt/Akt was significantly increased in group N (P＜0.05).Compared with group H/R, the cell survival rate was significantly increased, the apoptosis rate was decreased, the expression of GRP78, CHOP and caspase-12 was up-regulated, and p-Akt/Akt was increased in group N (P＜0.05), and no significant change was found in the parameters mentioned above in N+K and N+L groups (P＞0.05).Compared with group N, the cell survival rate was significantly decreased, the apoptosis rate was increased, the expression of GRP78, CHOP and caspase-12 was up-regulated, and p-Akt/Akt was decreased in N+K and N+L groups (P＜0.05).Conclusion NGF-β can mitigate the cell apoptosis induced by endoplasmic reticulum stress during H/R, and activation of PI3K signaling pathway is involved in the mechanism.

Objective To evaluate the effects of nerve growth factor-beta (NGF-β) pretreatment on cell apoptosis induced by endoplasmic reticulum stress during ischemia-reperfusion in isolated rat hearts.Methods Male Sprague-Dawley rats,weighing 180-220 g,were anesthetized with intraperitoneal 10％ chloral hydrate 300 mg/kg.The hearts were excised and perfused in a Langendorff apparatus with K-H solution aerated with 95％ O2 and 5％ CO2 at 37 ℃.Thirty-two isolated rat hearts were randomly divided into 4 groups (n =8 each) using a random number table:control group (group C),I/R group,NGF-β pretreatment group (group N) and NGF-β combined with K252a (trkA receptor antagonist) pretreatment group (group N + K).In group C,the hearts were continuously perfused with K-H solution for 195 min.The hearts were perfused with K-H solution for 45 min in group I/R.In N and N + K groups,the hearts were perfused with K-H solution for 15 min,and then with K-H solution containing 0.1 μg/ml NGF-β and 0.1 μg/ml NGF-β mixed with 100 nmol/L K252a,respectively,for 30 min.The perfusion was suspended for 30 min followed by 120 min of reperfusion with K-H solution in I/R,N and N + K groups.HR,left ventricular end-diastolic pressure (LVEDP),left ventricular developed pressure (LVDP) and + dp/dtmax were measured at the end of 15 min equilibration (baseline) and at 5,30,60 and 120 min of reperfusion.Myocardial specimens were obtained at 120 min of reperfusion for detection of myocardial apoptosis (by TUNEL) and expression of glucose-related protein 78 (GRP78),CCAAT/enhancer-binding protein homologous protein (CHOP),and caspase-12 (by Western blot analysis).Apoptosis index was calculated.Results Compared with group C,HR,LVDP and + dp/dtmax were significantly decreased,and LVEDP,apoptosis index and expression of GRP78 and CHOP were increased in I/R and N groups,and the expression of caspase-12 was upregulated in I/R group.Compared with group I/R,HR,LVDP,and + dp/dtmax were significantly increased,and LVEDP,apoptosis index and expression of GRP78,CHOP and caspase-12 were decreased in group N,and the expression of GRP78 was down-regulated in group N + K.There was no significant difference in cardiac function indexes between group I/R and N + K.Compared with group N,HR,LVDP and + dp/dtmax were significantly decreased,and LVEDP,apoptosis index,and expression of GRP78,CHOP and caspase-12 were increased in group N + K.Conclusion NGF-β pretreatment can protect the isolated rat hearts against ischemia-reperfusion injury,and inhibition of the endoplasmic reticulum stress-triggered cell apoptosis after activating trkA receptors is involved in the mechanism.

Purpose To prepare a novel fusion protein of CREKA and tTF as a universal carrier targeting to cancer,and to analyze its activities.Methods CREKA and tTF gene were acquired by PCR,and inserted into plasmid pET22b(+)to construct recombinant plasmid CREKA/tTF/pET22b(+),and the fusion gene was expressed in E.coli BL21.The fusion protein Wag purified through Nickel-affinity chromatography column.After purifying,the fusion protein was refold by subsequent dialysis.The activities of the fusion proteins were measured by coagulation timing and quantitative fluorescence test in vitro.Results The recombinant plasmid CREKA/tTF/pET22b(+)with correct sequence was obtained.The fusion protein was highly expressed in E.coli BL21.The coagulation of the fusion protein Was determined by the coagulation test.And the capability of the fusion protein effectively binding to clotted plasma proteins is identified in quantitative fluorescence test.Conclusion The recombinant plasmid CREKA/tTF/pET22b(+)with correct sequence was built.The fusion protein CREKA/tTF with both TF and CREKA activity was successfully obtained.

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Objective To evaluate the effect of small-dose ketamine on the onset time and course of modified electroconvulsive therapy (MECT) in mentally depressed rats.Methods Sixty SPF adult male SpragueDawley rats,aged 2-3 months,weighing 220-250 g,were randomly divided into 6 groups (n =10 each) using a random number table:normal control group (group C),depression group (group D),ECT group,propofol + ECT group (group PE),ketamine + ECT group (group KE) and ketamine + propofol + ECT group (group KPE).The depression model was established by chronic unpredictable mild stress (CUMS).Mter CUMS,C,D and ECT groups received intraperitoneal normal saline 8 ml/kg,group PE received intraperitoneal propofol 100 ml/kg,group KE received intraperitoneal ketamine 10 ml/kg,and group KPE received intraperitoneal ketamine 10 ml/kg + propofol 80 ml/kg.All the groups received ECT once a day for 7 consecutive days starting from the time point when righting reflex was lost except C and D groups.Open-field test was performed before CUMS,at 1 day after CUMS and at the end of each ECT (T0 8).The total distance and the number of standing on the back legs were recorded.Morris water maze test was performed at 2 days after CUMS and 1 day after the end of therapy,and the escape latency and time of staying at the original platform quadrant were recorded.Results Compared with group C,the total distance was shortened and the number of standing on the back legs was reduced,the escape latency was prolonged,and the time of staying at the original platform quadrant was shortened at T1-8 in D,ECT,PE and KE groups and at T1 5 in KPE group,and no significant was found in KPE group in the total distance,number of standing on the back legs,escape latency,and time of staying at the original platform quadrant at T6-8.Compared with group D,the total distance was prolonged and the number of standing on the back legs was increased at T6-8 in ECT and PE groups and at T4-8 in KE and KPE groups,the escape latency was prolonged,and the time of staying at the original platform quadrant was shortened in ECT group,and the escape latency was shortened,and the time of staying at the original platform quadrant was prolonged in KPE group.Compared with ECT and PE groups,the total distance was prolonged and the number of standing on the back legs was increased at T4-7 in group KE and at T4-8 in group KPE,and the escape latency was shortened,and the time of staying at the original platform quadrant was prolonged in KPE group.Compared with group KE,the total distance was prolonged and the number of standing on the back legs was increased at T6.7,the escape latency was shortened,and the time of staying at the original platform quadrant was prolonged in KPE group.Conclusion Small-dose ketamine can shorten the onset time and course of MECT in mentally depressed rats.

Objective To understand the pattern of and influencing factors on leisure time physical exercises among adults in Zhejiang. Method Totally 17 437 residents aged 18 years and over selected by stratified cluster random sampling in Zhejiang in 2010 were investigated. Result Overall 21.82%(95%CI:17.12%-27.38%) of adults took part in leisure time physical exercises in 2010. The proportion was higher in urban (29.04%,95%CI:23.46%-35.33%) than in rural (18.81%,95%CI:12.89%-26.61%) (χ2=222.06, P<0.05) areas, and also higher in females (22.91%,95%CI:17.66%-29.15%) than in males (20.56%,95%CI:16.39%-25.48%) (χ2=13.94, P<0.05). Adults aged 18-24 and 55-74 years were more likely to take apart in physical exercises (χ2=266.73, P<0.05), and the lowest proportion was found among farmers (5.33%,95%CI:3.99%-7.11%) (χ2=2 078.40, P<0.05). These proportions both increased along with education level and family income increase (χ2=444.87, P<0.05;χ2=332.20, P<0.05). Overall 83.04%and 90.08%of physical exercisers took part in exercises at least 3 times per week and 30 minutes per time, only 30.67% of those reached moderate or vigorous intensity, and overall 5.38% (95%CI: 3.85%-7.48%) of adults took part in regular leisure time physical exercises in 2010. Multiple logistic regression showed that age, education level, occupation and chronic disease history have influence on regular physical exercises. Conclusion The leisure time physical exercises level was generally low among adults in Zhejiang. Young and middle-aged adults with less education and low income especially farmers should be put on emphasis.

Objective To evaluate the effect of electroconvulsive therapy (ECT) on the expression of phosphorylated glutamate receptor 1 (p-GluR1) and Ca2+/calmodulin-dependent protein kinase Ⅱ α (p-CaMK Ⅱ α) under small dose ketamine combined with propofol anesthesia in the depressed rats.Methods Forty healthy adult male Sprague-Dawley rats,weighing 200-250 g,aged 2-3 months,were used in this study.Mental depression was induced by exposing the animals to chronic unpredictable mild stress (CUMS).Forty mentally depressed rats were divided randomly into 5 groups (n =8 each) using a random number table:M0-4 groups.Propofol 80 mg/kg and ketamine 10 mg/kg were injected intraperitoneally in M0-4 groups.After disappearance of righting reflex,M1-4 groups received ECT of 60,120,180 and 240 mC once a day for 7 consecutive days,respectively,by means of a current (frequency 50 Hz,sine-wave,pulse width 0.7 ms,1-s duration) delivered via ear-clip electrodes,while group M0 received ECT of no quantity of electric charge via ear-clip electrodes.Before CUMS,at 1 day after CUMS and at 1 day after ECT,sucrose preference test was applied to evaluate the depressive behavior.The sucrose preference percentage (SPP) was calculated.At 4 days after CUMS and 4 days after ECT,the learning and memory function was assessed using Morris water maze test.The rats were then sacrificed,and hippocampi were isolated to detect the expression of GluR1,p-GluRl,CaMK Ⅱ α and p-CaMK Ⅱ α by Western blot.Results The SPP was significantly lower after CUMS than before CUMS in M0-4 groups (P＜0.05).Compared with that after CUMS,the SPP was significantly increased,the escape latency was shortened,and the space exploration time was prolonged after ECT in M1-4 groups (P＜0.05).There was no significant difference in SPP after ECT between M1-4 groups (P＞0.05).Compared with group M0,the SPP was significantly increased,and the expression of pGluR1 and p-CaMK Ⅱ α was up-regulated in M1-4groups (P＜0.05).Compared with group M2,the escape latency was significantly prolonged,the space exploration time was shortened,and the expression of pGluR1 and p-CaMK Ⅱ α was down-regulated after ECT in the other groups (P＜0.05).There was no significant difference in GluR1 and CaMK Ⅱ α expression after ECT between the five groups (P＞ 0.05).Conclusion ECT can induce cognitive decline when applied for anti-depression under small dose ketamine combined with propofol anesthesia,and the mechanism is related to increased phosphorylation of GluR1 and CaMK Ⅱ α expression in rats.

Objective To investigate the inhibitory effect of the cytochrome P450 3A4(CYP3A4) gene expression and function in CHL-3A4 cells lines by vector-ecpressed small hairpin interfering RNA(shRNA).Methods The shRNA expression vectors targeting CYP 3A4 gene(CYP3A4Ⅰ,CYP3A4Ⅱ,CYP3A4Ⅲ) were designed and constructed.The inhibitory effect of shRNA was detected by Western blot and RT-PCR analysis.The inhibitory effect of cytotoxic of cyclophosphamide using shRNA was measured by MTT assay.Results CYP3A4Ⅲ shRNA expressing vector significantly reduced the CYP3A4 mRNA(70%) and protein expression levels(75%) of the CYP3A4 gene in CHL3A4 cells suppression of CYP3A4 gene expression by CYP3A4Ⅲ largely reversed cyclophosphamide cytotoxicity(75%) in CHL-3A4 cells.Conclusion Vector-based RNAi could suppress CYP3A4 gene expression and function,and the use of RNAi to inhibit CYP3A4 gene expression in mammalian cells was a promising new tool for the study of cytochrome P450 gene function.

Objective To explore the characteristics of emotion and mental health of soldier under field training stress. Methods Self-rating anxiety scale,self-rating depression scale and stressful psycho-behavior questionnaire were used to evaluate soldiers’ state under field training. Results At 3 months after training,there were 21.7% soldiers with SAS score higher than 50,and 6.4% soldiers higher than 60,but the scores were 11.6% and 4.2% respectively 2 months before field training; There were 45.7% soldiers with SDS score higher than 50 and 19.2% higher than 60 at 3 months after training,and only 25.3% and 11.6% at 2 months before training. Score of stressful psycho-behavior was (24.1?6.1) in 3 months after training,significantly higher than that of 2 months before training (18.3?4.7,P