AIM:To look for a suitable signal peptide which may effectively conduct hepatopoietin(HPO)secretion,various recombinant eukaryotic expression vectors were constructed.METHODS:Different exogenous signal sequences were spliced with HPO cDNA by PCR,and the spliced genes were cloned into eukaryotic expression plasmids.The different recombinants were respectively tansfected into COS-7 cells by Lipofectamine 2000 method and the secretion of HPO was analyzed by Western blotting.RESULTS:Western blotting analysis indicated that the signal peptides from interleukin-1 receptor antagonist(IL-1ra)and an artifical signal peptide did not secret HPO directly and effectively,but the signal peptide from murine Ig kappa secreted HPO directly with great efficiency.The molecular weight of the secreted HPO was 30 kD,which means that the secreted HPO existed in homodimer.CONCLUSION:Secreted recombinant expression plasmid is successful constructed.The result may pave the way for the gene therapy of hepatic fibrosis.

Objective To evaluate the effect of rhubarb on intestinal mucosal damage in septic rats by comparing with ulinastatin.Methods A total of 84 healthy Sprague-Dawley rats (half male,half female),aged 3 months,weighing 200-330 g,were randomly divided into 5 groups according to the random number table:control group (group C,n =6),sham operation group (group S,n =6),sepsis group (group Sep),rhubarb group (group R,n=24) and ulinastatin group (group U,n=24).Sepsis was induced by cecum ligation and puncture.In group R,rhubarb 1.2 g/100 g was dissolved in normal saline at room temperature,3 and 4 h later the filtrate about 2-3 ml was obtained and injected through a gastric tube into stomach once every 12 h,and 72 h later sepsis was induced.In group U,ulinastatin 50 000 U/kg (in 2 ml of normal saline) was injected once every 24 h,and 72 h later sepsis was induced.In Sep,R and U groups,at 6,12,24 and 48 h after ligation (T1 4),blood samples were collected from the orbital venous plexus for determination of plasma diamine oxidase (DAO) activity.Results The activity of plasma DAO was significantly higher at T1-4 in Sep,R and U groups than in C and S groups.The activity of plasma DAO was significantly lower at T3,4 in R and U groups than in Sep group.There was no statistical difference in the plasma DAO activity between R group and U group.Conclusion Rhubarb can reduce intestinal mucosal damage in septic rats,which is similar to that of ulinastatin.

Objective To investigate the prognostic value of decreased vasopressin (VP)modulation in the late-phase of septic shock. Methods A prospective study was conducted. Fifty-five septic shock patients hospitalized in intensive care unit (ICU)of the First Hospital of Hebei Medical University from January 2012 to February 2014 were enrolled. All patients received 3% hypertonic saline solution infusion. Serum concentrations of sodium and VP were measured before and after hypertonic saline solution infusion. Patients with ratio of difference in sodium and VP before and after infusion of 3%hypertonic saline (△VP/△Na)≤0.5 pg/mmol were defined as non-responders,and who>0.5 pg/mmol were defined as responders. The levels of lactic acid,C-reactive protein (CRP),and vasoactive drug〔dopamine(DA)and norepinephrine(NE)〕usage between the two groups were compared. The 28-day mortality,live time in the dead,and ICU day in survivors were analyzed between the two groups. The receiver operating characteristic curve (ROC curve)was drawn to assess prognostic value of VP. Results There were 30 cases (54.5%) in non-responsive group,and 25 (45.5%)in responsive group. There were no significant differences in the age,acute physiology and chronic health evaluationⅡ (APACHEⅡ)score,central venous pressure (CVP),blood pressure, plasma albumin level,sodium level before and after hypertonic saline solution infusion between the two groups. The baseline level of VP in the non-responsive group was markedly lower than that of the responsive group (ng/L:10.66± 1.57 vs. 17.13 ±5.12,t=6.091,P<0.001). After hypertonic saline solution infusion,the VP level was also significantly decreased compared with that in the responsive group(ng/L:11.65±1.74 vs. 22.50±5.31,t=9.758,P<0.001). The non-responders showed higher lactic acid (mmol/L:3.04±0.55 vs. 2.28±0.38,t=-5.881,P<0.001) and CRP (mg/L:117.9±23.0 vs. 94.9±17.0,t=-4.143,P<0.001),and received larger dosage of vasoactive drugs〔DA(μg·kg-1·min-1):14.8±3.9 vs. 8.9±1.6,t=-5.725,P<0.001;NE(μg·kg-1·min-1):0.96±0.42 vs. 0.40± 0.09,t=-5.625,P<0.001〕for maintaining blood pressure compared with those in responders. The non-responsive group showed higher 28-day mortality(66.7%vs. 40.0%,χ2=3.911,P=0.048)and longer ICU day(days:9.9±2.3 vs. 6.7±1.7,t=-4.044,P<0.001),but the live time in the dead showed no difference between non-responsive group and responsive group(days:5.8±1.9 vs. 6.1±2.3,t=0.384,P=0.704). ROC curve showed that the area under ROC curve(AUC)forΔVP/ΔNa predicting the outcome was 0.828,and theΔVP/ΔNa threshold value of 0.5 pg/mmol had the sensitivity of 66.7%and specificity of 64.0%for prediction of the outcome(95%confidence interval:0.722-0.934). Conclusion Osmotic pressure-regulated VP secretion was impaired and decreased in the late-phase of septic shock, and made the sense in prognosis.

Objective To evaluate the accuracy of vasopressin (VP) secretion in the late phase of septic shock for predicting patient outcomes and further investigate its relationship with the prognosis of septic shock.Methods Fifty-five patients presented at late phase of septic shock,who were admitted to the intensive care unit of our hospital,were enrolled.Their VP secretion was measured.The method for measurement was as follows:3％ sodium chloride solution 600 ml was infused over 2 h,serum concentrations of VP and sodium were measured before and after infusion,the difference in VP before and after infusion (△VP) and in Na before and after infusion (△Na) was calculated,and △VP/△Na was used to reflect VP secretion.The patients were divided into either abnormal secretion of VP group (△ VP/△ Na ≤ 0.5 ng/mmol) or normal secretion of VP group (△VP/△Na＞0.5 ng/mmol) according to △VP/△Na ratio.Immediately before testing VP secretion,venous blood samples were collected for determination of serum lactic acid and C-reactive protein concentrations.The consumption of vasoactive drugs at the moment of enrollment and 28-day fatality rate were recorded.Results There were 30 cases in abnormal group (54％) and 25 cases in normal group (46％).Compared with normal group,the serum lactic acid,C-reactive protein concentrations and consumption of dopamine or norepinephrine were significantly increased,and the 28-day fatality rate was increased (67％ vs 40％) in abnormal group.ROC curve analysis showed that when △VP/△Na 0.5 ng/mmol was used as the criteria for determining prognosis,the sensitivity was 66.7％,specificity was 64.0％,and the area under the ROC curve was 0.828.Conclusion VP secretion in the late phase of septic shock may affect patient prognosis.

Objective To investigate the vasopressin (VP) response to increasing osmotic pressure in the late-phase of septic shock patients.Methods Thirty-seven septic shock patients hospitalized in intensive care unit (ICU) of the First Hospital of Hebei Medical Unive~ity from January 2012 to September 2013 were enrolled.All patients received 3％ hypertonic saline solution infusion.Serum concentrations of VP and sodium were measured before and after hypertonic saline solution infusion.Patients with ratio of difference in VP and sodium before and after infusion of 3％ hypertonic saline (△VP/△Na) ≤0.5 pg/mmol were defined as nonresponders,and who ＞0.5 pg/mmol defined as responders.The age,acute physiological and chronic health evaluation Ⅱ (APACHE Ⅱ) score,blood pressure,albumin level,vasoactive drug between the two groups were also analyzed.Results VP level in the nonresponsive group (n=20,54.05％) was markedly lowered before (ng/L:10.41 ± 1.70 vs.18.25 ± 5.90,t=5.29,P＜0.01) and after (ng/L:11.36 ± 1.90 vs.24.33 ± 5.46,t=9.33,P＜0.01) 3％ hypertonic saline solution infusion,compared with that in the responsive group (n =17,45.95％).All patients in the two groups were given dopamine (DA) or norepinephrine (NE) for maintaining blood pressure,and the dose in the nonresponsive group were higher than those in the responsive group [DA (μg· kg-1· min-1):14.91 ± 3.78 vs.8.64 ± 1.69,t =-5.02,P＜ 0.01 ; NE (μg· kg-1· min-1):1.03 ± 0.48 vs.0.38 ± 0.12,t=-3.12,P＜0.01].Three patients were given DA plus NE in the nonresponsive group while patients in the responsive group received only single drug therapy.The age,APACHE Ⅱ score,blood pressure,albumin level,sodium level before and after hypertonic saline solution infusion between the two groups were not statistically different.Conclusion VP secretion to osmotic challenge was impaired and decreased in the late-phase of septic shock,prompting dysfunction in VP synthesis.

Objective: Numerous studies have shown that bone marrow-derived mesenchymal stem cells (MSCs) enhance neurological recovery after cerebral ischemia. However, the mechanisms are still not clear. The present study aimed to investigate the beneficial effects of MSCs on global cerebral ischemia induced by cardiac arrest (CA) and the underlying mechanisms. Methods: Rats subjected to asphyxial CA were injected intravenously with MSCs (5×106 ) at 2 hours after resuscitation. Whole brain histopathologic damage scores (HDS) were assessed by histopathology at 3 and 7 days after resuscitation. The distribution of donor MSCs in the brain was evaluated. The expression of tumor necrosis factor-α-induced protein 6 (TSG-6) and pro-inflammatory cytokines in cerebral cortex was assayed. After intravenous infusion of TSG-6 siRNA-MSCs, HDS and pro-inflammatory cytokines were reevaluated at 7 days after resuscitation. Results: Intravenously administered MSCs significantly reduced whole brain HDS after global cerebral ischemia. Immunofluorescence microscopy revealed that donor MSCs were primarily found in cerebral cortex and expressed TSG-6. MSCs treatment significantly increased the expression of TSG-6 and reduced the expression of pro-inflammatory cytokines in cerebral cortex. In addition, intravenous infusion of TSG-6 siRNA-MSCs failed to attenuate brain inflammation. Conclusion: Systemically administered MSCs reduced inflammatory damage to brain in rats with global cerebral ischemia via secretion of TSG-6.
Heart Arrest ; Mesenchymal Stromal Cells

Objective To investigate the effect of propofol on human renal tubule epithelial cell (HK-2 cells) fibrosis induced by ATP depletion/recovery and the role of transforming growth factor β activated kinase 1 (TAK1) in it.Methods HK-2 cells were seeded in 96-well plates,and randomly divided into 4 groups (n =36 each) using a random number table:control group (group C),ATP depletion/recovery group (group D/R),propofol group (group P),and TAK1 over-expression group (group T).HK-2 cells were exposed to antimycin A for 1 h and then returned to normal culture medium to establish the model of ATP depletion/recovery-induced injury.At 1 h before ATP depletion,the cells were incubated for 1 h in the DMEM liquid culture medium containing propofol with the final concentration of 20 μmol/L in group P,and the cells were incubated for 1 h in the DMEM liquid culture medium containing propofol with the final concentration of 20 μmol/L and TAK1 with the titer of 2× 107 TU/ml in group T,and the other treatments were similar to those previously described in group D/R.At 12 h after ATP recovery,the cell viability was evaluated by methyl thiazolyl tetrazolium assay,and cell apoptosis was detected using TUNEL and scored.The expression of TAK1 was detected using Western blot at 12,24 and 48 h after ATP recovery.The expression of α-smooth muscle actin (αSMA),fibronectin (FN),and collagen protein 1 (COL1) was measured at 48 h after ATP recovery.Results Compared with group C,the cell viability was significantly decreased,the apoptosis score was increased,and the expression of TAK1,COL1,αSMA and FN was up-regulated after ATP recovery in D/R,P and T groups (P＜0.05).Compared with group D/R,the cell viability was significantly increased,the apoptosis score was decreased,and the expression of TAK1,COL1,αSMA and FN was down-regulated after ATP recovery in P and T groups (P＜0.05).Compared with group P,the cell viability was significantly decreased,the apoptosis score was increased,and the expression of TAK1,COL1,αSMA and FN was up-regulated after ATP recovery in group T (P＜ 0.05).Conclusion Propofol can reduce HK-2 cell fibrosis induced by ATP depletion/recovery,and the mechanism may be related to down-regulation of TAK1 expression.

Objective To investigate the effects of bone marrow mesenchymal stem cells (MSCs)treatment on TSG-6 in a rat model of cardiopulmonary resuscitation (CPR).Methods Sprague Dawley (SD) rats were randomly (random number) divided into sham group,phosphate buffer solution (PBS)-treated group and MSCs-treated group.Animals were subjected to asphyxial cardiac arrest followed by CPR.In PBS-treated group or MSCs-treated group,animals were injected intravenously with PBS or MSCs at 2h after resuscitation.Neurological deficit scores (NDS) were assessed at 1,3 and 7 d after CPR.Serum S-100B was assayed using enzyme linked immunosorbent assay (ELISA).Immunofluorescence was performed to detect donor MSCs and the expression of TSG-6 in brain.TSG-6 and proinflammatory cytokines in brain were assayed using real time reverse transcription-polymerase chain reaction (RT-PCR).Western blot analysis was performed to measure the levels of neutrophil elastase (NE) in brain.Multiple comparisons were made by analysis of variance.Results At 3d and 7d,MSCs-treated group demonstrated higher NDS than PBS-treated group (P ＜ 0.01),and serum S-100B levels significantly reduced in MSCs-treated group compared with PBS-treated group (P ＜ 0.01).DAPI-labeled MSCs migrated into the ischemic brain and some DAPI + cells colocalized with TSG-6.Compared with PBS-treated group,MSCs treatment significantly up-regulated the expression of TSG-6 and reduced the expression of NE and proinflammatory cytokines in brain at 3 d and 7 d after CPR (P ＜ 0.05).Conclusion Systemically administered MSCs suppressed inflammatory responses in brain after CPR and improved neurological function in rats possibly via induction of TSG-6.

Objective: Asphyxia and ventricular fibrillation are the two most prevalent causes of cardiac arrest. The study investigated the differences in brain damage after cardiac arrest between asphyxial and ventricular fibrillation cardiac arrests in rats. Methods: Male healthy Sprague-Dawley rats were randomly assigned to the asphyxial group (cardiac arrest of 6 min, n=15), ventricular fibrillation group (cardiac arrest of 6 min, n=15) and sham group (n=5). Neurologic deficit scores and tape removal test were evaluated at 1, 3 and 7 days after cardiopulmonary resuscitation from three groups. Serum S-100B and brain histopathologic damage scores were also examined. Results: There were no differences in neurologic performance at 1, 3 and 7 days after cardiopulmonary resuscitation between the asphyxial group and ventricular fibrillation group (P>0.05, respectively). Serum S-100B level was higher in the asphyxial group at 1, 3 and 7 days, compared with the ventricular fibrillation group (P<0.05, respectively). There were significantly higher histopathologic damage scores at 1, 3 and 7 days in the asphyxial group compared with the ventricular fibrillation group (P<0.05, respectively). Conclusion: Asphyxial cardiac arrest has worse morphologic brain damage compared with ventricular fibrillation cardiac arrest, but the functional brain damage caused by asphyxial cardiac arrest is similar to that caused by ventricular fibrillation cardiac arrest.

Hepatitis B virus ; drug effects ; physiology ; Hodgkin Disease ; drug therapy ; Humans ; Immunoconjugates ; therapeutic use ; Virus Activation ; drug effects