ObjectiveTo observe the relationship between neuronal function score and pathological changes of fluid percussion brain injury in rats and to explore their clinical significances.MethodsThe fluid percussion models of brain injury in rats were established by using the improved device with three kinds impact pressure such as 0.1 MPa,0.2 MPa,0.3 MPa,and vital signs and mortality rate were observed.Behavior changes,brain water content,histological changes were observed by Shapira and Wahld method,dry-wet measure,light microscopy at 1 h,6 h,12 h,24 h,3 d and 7 d after operation respectively.ResultsThe animals accepted impact pressure of 0.1 MPa showed temporary hypopnea with mortality rate of 2.08％,those of 0.2 MPa suffered apnoea of ( 10.88 ±2.69 ) s with mortality rate of 4.17％ and those of 0.3 MPa suffered apnoea of ( 20.60 ± 3.02 ) s with mortality rate of 16.67％.As the impact pressure increased,nervous function score minimumly decreased to (7.17 ±0.75) of 0.1 MPa group,(4.83 ± 0.75 ) of 0.2 MPa group and (2.67 ± 0.52) of 0.3 MPa group respectively,and recovered more slowly.Brain water content maximumly reached to (81.12 ± 0.03 )％,(82.74 ± 1.11 )％ and (83.89± 0.04) ％ at time point of 24 h respectively.The brain injury was involved in the outer layer of cerebral cortex,hippocampal formation and brain stem respectively and histological observation verified above findings.Conclusion Light,moderate and heavy fluid percussion brain injury in rats have more and more low nervous function scores,which have positive relationship with more and more serious pathological changes.

Objective To explore the mechanism by which tumor necrosis factor alpha(TNF-α) induces RIP1 kinase-dependented apoptosis in L929-A fibroblastoma cells.Methods The sub-mitochondrial localization of receptor-interacting protein 1(RIP1),caspase-8 and Bid proteins was detected by dose-gradient trypsin digestion and Western blotting.The levels of reactive oxygen species (ROS),intracellular calcium concentration,mitochondrial membrane potential (MMP),and cellular adenosine triphosphate(ATP) content were determined by fluorescent probe labeling and flow cytometry assay.The mitochondrial respiratory chain complex Ⅰ and Ⅲ activities were detected by commercial kits.Nec-1,A RIP1 kinase specific inhibitor,and RIP1-/-or Bid-/-L929-A cells were used to detect the roles of RIP1 kinase and Bid protein in cell death.Results RIP1,caspase-8 and Bid proteins were co-located in the outer membrane of mitochondrial.TNF-α exposure for 3 h could induce Bid cleavage,inhibit mitochondrial respiratory chain complex Ⅲ activity and reduce MMP.Following these changes and after TNF-α exposure for 6-12 h,the intracellular calcium concentration and ROS were increased,whereas the ATP concentration was decreased,and the cells were killed.Inhibiting RIP1 kinase or knockdown RIP1 or Bid protein could suppress all the cytotoxic effects of TNF-α.Conclusion TNF-α treatment can result in RIP1 kinase-mediated Bid cleavage and inhibit mitochondrial respiratory chains and cell energy metabolism,which ultimately leads to the death of L929-A cells.

Objective To investigate the expression of Toll-like receptor 4 (TLR4),and explore its relationship with neurological function after fluid percussion brain injury in rats.Methods 56 adult rats were randomly divided into traumatic brain injury group(TBI group,n=48) and sham operation group(SO group,n=8).The experimental models were established.The water content of edematous brain and the expression of TLR4 were measured with dry-wet measure,immunohistochemistry and Western Blot at 1 h,6 h,12 h,24 h,3 d,7 d after shock respectively.Results Compared with SO group,neuronal function score decreased in TBI group from 6 h(3.86±0.42),reached to the lowest level at 24 h(2.65±0.32),and gradually rose at 3rd day (3.25±0.17).TLR4 immunoreactive expression increased from 6 h,reached its maxmum at 24 h,lasted to 3rd day,and then began to drop at 7th day.The linear regression analysis indicated that expression of TLR4 had negative correlation with change of neuronal function score (r 1 =-0.824,r w =-0.867,P＜0.05).Conclusion TLR4 expression is upregulated following fluid percussion injury in rats and involved in neurological function impairment by inducing secondary inflammatory brain injury.

Arg-Gly-Asp (RGD) is a unique minimal integrin-binding sequence found within several glycoprotein ligands and also in snake-venom disintegrins. Adinbitor, a protein with 73 amino acid residues including 12 cysteins and an RGD motif, was cloned from Gloydius blomhoffi brevicaudus in my laboratory. As a new member of disintegrin family, adinbitor can inhibit both human platelet aggregation induced by ADP and angiogenensis in vivo and in vitro, the typical characters of disintegrin family. To separate the effect of inhibiting platelet aggregation from that of inhibiting angiogenensis, the motif KGD was introduced into adinbitor cDNA to replace RGD by site-directed and PCR-based mutagenesis. The recombinant protein (recombinant adinbitor (KGD)) was expressed in E. coli BL21 and purified through the His· Bind affinity chromatography. Recombinant adinbitor (KGD) could inhibit ADP-induced platelet aggregation with IC50 value of 85 nmol/L. Considerably, it was a more effective inhibitor on platelet aggregation than recombinant adinbitor (RGD), which has an IC50 of 150 nmol/L. Interestingly, recombinant adinbitor (KGD) has no potency in inhibiting angiogenesis in vivo compared with recombinant adinbitor (RGD). These findings showed that KGD containing adinbitor was more suitable for inhibiting ADP-induced human platelet aggregation as a potential and specific inhibitor of human platelet aggregation, which might have promising therapeutic potential as an antithrombotic agent.

Objective To evaluate the effect of military performance stress on the changes of concentrations in salivary chromogranin A ( CgA) ,β-endorphin (β-EP) and salivary IgA ( sIgA) of submariners.Methods Twenty-nine submarine soldiers were selected, and their saliva samples were collected separately at the end of a long dive trip and nine months after relaxation ashore.In addition,the saliva samples of twenty-eight graduate students were collected as the normal control.The method of ELISA was used to detect the level of salivary CgA,β-EP and sIgA.Results After long-term dive training, the submariners showed significantly decreased CgA,β-EP and sIgA.Conclusion After a long term dive trip, chronic military performance stress is associated with the decline of salivary CgA,β-EP and sIgA, indicating that the function of sympathet-ic adrenal medulla is suppressed.The biological significance of these changes needs to be assessed in the future.

Objective To investigate the effects of rapamycin on the expression of glioma patients tumor helicase RECQ1.Methods 50 glioma patients admitted to the department of neurosurgery in second hospital of hebei medical university were selected and randomly divided into 2 groups,25 patients in control group,were treated with routine admission surgical treatment;25 cases in the experimental group,firstly were given rapamycin capsule 1 mg,1 times/day orally,took 14 days in a row,and had surgical treatment after stopping drug a week.Glioma tissue samples were taken during the operation,mRNA and protein expression of tumor helicase RECQ1 were detected by reverse transcriptase polymerase chain reaction(RT-PCR)and Western blot.Results Glioma tumor helicase RECQ1 mRNA expression in the control group increased more significantly than experimental group,the optical density value in control group was(1.657 ±0.748),while the experimental group optical density value was(1.059 ±0.894),and the difference was statistically significant(P<0.05 );all organizations had the expression of tumor helicase RECQ1 protein,but gliomas tumor helicase RECQ1 protein expression in the experimental group patients(0.952 ±0.021)was significantly lower than that in the control group(1.211 ±0.024),and the difference was statistically significant(P<0.05).Conclusion Rapamycin capsule could reduce the expression of mRNA helicase RECQ1,inhibit DNA glial tumor cells of brain replication,effectively kill cancer cells,control the the progress of brain glioma,and improve prognosis,worth clinical promotion.

Objective To observe dynamically the influence of Insulin-like growth factor-1 (IGF-1) on the nerve function and expression of bFGF protein and Basic fibroblast growth factor(bFGF) mRNA after cerebral ischemic reperfusion in rats.Methods Seventy-two SD rats were randomly divided into sham-operated group,cerebral ischemia group,and IGF-1 treated group.The rat model of middle cerebral artery occlusion (MCAO) and reperfusion was performed.The evaluation of etiology was performed with mNSS at 12 h,24 h,3 d,7 d after ischemia-reperfusion,expression of bFGF protein was determined with immunohistochemical technique and expression of bFGF mRNA was determined with RT-PCR.Results The ratings of mNSS in IGF-1 treated group((8.67± 1.21),(7.50± 1.52),(4.33± 1.03),(3.67± 1.37)) were lower than those in ischemia group((11.0±1.26),(9.83±1.33),(7.83±1.17),(7.17±1.72) at 12 h,24 h,3 d or7 d after reperfusion(P＜0.05).For the IGF-1 treated group,the expression level of bFGF protein was higher than that of the cerebral ischemia group (P＜0.05),especially at 12 h after reperfusion (P＜0.01).The expression level of bFGF mRNA in the IGF-1 treated group was higher than that of the cerebral ischemia group (P＜ 0.05),especially at 24h after reperfusion (P＜ 0.01).Conclusion IGF-1 treatment has a protective effects on cerebral ischemia injury,which may contribute to its action on regulating expression of bFGF protein and bFGF mRNA.

ObjectiveTo observe the relationship between neuronal function score,brain edema and aquaporin4(AQP4) expression of fluid percussion brain injury in rats.MethodsThe fluid percussion models of brain injury of rats were established by using the improved device.Nervous function score,brain water content,histological changes,AQP4 expression were observed by Shapira and Wahld method,dry-wet measure,light microscopy,immunohistochemistry and western blot at 1 h,6 h,12 h,24 h,3 d and 7 d after operation respectively.ResultsNervous function score in TBI group decreased at 12 h( 11.17 ± 1.32),reached its minimum at 24 h( 10.17± 0.75),and recoved partially at 3rd day( 10.66 ± 1.37 ).The water content obviusly increased in those of TBI group at 12h( (80.27 ±1.47)％ ),reached its peak at 24h( (82.19 ±0.97)％ ),and then began to drop at 3d ( (8 1.74 ± 1.69 ) ％ ),while Western blot showed that AQP4 immunoreactive expression obviusly increased at 12 h (OD:0.65 ±0.05),reached its maximum at 24h( OD:0.72 ±0.08),and decreased at 3d( OD:0.56 ±0.07),and immunohistochemistry showed the same trendency of AQP4 expression as that of Western blot.The linear regression analysis indicated that nervous function score had a negtive correlation with expression of AQP4 in edematous brain and change of brain water content respectively ( r =- 0.615,P ＜ 0.01 ; r =- 0.605,P ＜ 0.05 ).ConclusionNervous function score of fluid percussion brain injury in rats decrease,has negative relationship with brain edema and AQP4 expression,and possible mechanisms is that AQP4 is indirectly involed in nerve function impairment by mediating brain edema.

Objective To investigate the significance of salivary cortisol , dehydroepiandrosterone sulfate (DHEA-S) and cortisol/DHEA-S ratio changes for evaluation of military performance stress .Methods Forty submarine soldiers were selected, whose saliva samples were collected separately at the end of long-term dive training and after nine months of relaxation break.In addition, the saliva samples of thirty-four graduate students were collected the moment they finished a three-hour final examination and one week later .The method of ELISA was used to detect the levels of salivary cortisol and DHEA-S and to count their ratio .Results After long-term dive training , the submarine soldiers showed significantly decreased DHEA-S and an increased cortisol/DHEA-S ratio, but the cortisol level did not change very much .In contrast, the final examination stress did not change the level of cortisol , DHEA-S or the cortisol/DHEA-S ratio among these students.Conclusion This is the first study to show that long-term, chronic military performance stress is associated with the salivary DHEA-S and cortisol/DHEA-S ratio changes .The increase in the cortisol/DHEA-S ratio may be used as an important and useful biomarker to evaluate chronic stress .In addition , it is relatively simple and sensitive to detect stress biomarkers by using saliva samples .

Objective To study the effects of verussurinine (VSRN), an alkaloid isolated from Veratrum nigrum var. ussuriense alkaloids (VnA), against thrombosis and its platelet aggregation inhibitory activity in rats in order to find out whether VSRN is the antithrombotic active ingredient of VnA. Methods The electrically induced rat carotid artery thrombosis and stasis-induced rat inferior vena cava thrombosis models were used to evaluate the anti-arterial and anti-venous thrombosis effect of VSRN, respectively. Borns turbidimetric method was used to examine the in vivo and in vitro anti-platelet effect so as to investigate the antiplatelet aggregation of VSRN. Results In comparison with saline, VSRN in five different doses (1.25—20.00 ?g/kg) showed significantly and dose-dependently prolonged occlusion time (OT) of carotid artery injured by electrical stimulation and reduced thrombus dry weight of inferior vena cava ligated for 4 h to cause stasis. Platelet aggregation was found to be inhibited by VSRN in the doses of 1.25—5.00 ?g/kg and at the concentration of 6.25—50 ?g/L in both in vivo and in vitro test. Conclusion VSRN has powerful arteriovenous antithrombosis and antiplatelet aggregation of rats. The antithrombotic effect of VSRN is related to its platelet aggregation inhibitory activity. The above findings indicate that VSRN is an antithrombotic active ingredient of VnA.