Influencing factors of vascular endothelial function in patients with non-obstructive coronary atherosclerosis: a 1-year observational study.

BACKGROUND: Endothelial dysfunction may play a key role in non-obstructive coronary artery atherosclerosis. Our study aimed to evaluate the vascular endothelial function and its influencing factors in patients with non-obstructive coronary artery atherosclerosis. METHODS: A total of 131 consecutive patients with non-obstructive coronary artery atherosclerosis were enrolled. Flow-mediated dilatation (FMD) was measured at baseline and 1-year follow-up. Endothelial progenitor cells (EPCs) were counted by staining the fasting venous blood with antibodies against CD34 and vascular endothelial growth factor receptor 2. RESULTS: Systolic blood pressure, pulse pressure and the levels of HbA1c in participants with baseline FMD < 6% (n = 65) were significantly higher than those with baseline FMD ≥ 6% (n = 66). Baseline FMD was negatively associated with EPC counts (r = - 0.199, P < 0.05) and systolic blood pressure (r = - 0.315, P < 0.01). The 1-year FMD was significantly increased compared to the baseline FMD [(9.31 ± 5.62) % vs (7.31 ± 5.26) %, P < 0.001]. Independent predictors of FMD improvement included elevated EPC counts (OR = 1.104, 95% CI: 1.047-1.165, P < 0.001) and decreased levels of serum creatinine (OR = 0.915, 95% CI: 0.843-0.993, P = 0.034). CONCLUSIONS: Family history of premature cardiovascular diseases, hypertension, elevated systolic pressure, and HbA1c > 6.5% are independent risk factors for endothelial dysfunction in non-obstructive atherosclerotic patients. Elevated peripheral blood EPC counts and decreased levels of serum creatinine are independent predictors of endothelial function improvement.

Characterization and Hypoglycemic Activity of a Rhamnan-Type Sulfated Polysaccharide Derivative.

Polysaccharide chromium (III) derivatives are gaining increasing attention in improving type 2 diabetes. In this study, the sulfated polysaccharide from Enteromorpha prolifera (SPE) with 4.8 kDa was prepared by specific enzymatic hydrolysis. The obtained SPE was used to prepare a rhamnan-type sulfated polysaccharide derivative (SPED). Results indicated that O-H, C=O, and S=O were effectively involved in the chelation of SPED (chromium content 20.26%). Acute (half lethal dose > 2.38 g/kg) and sub-acute toxicity showed that SPED had no damaging effects on mice. Anti-diabetic experiment demonstrated that SPED improved glucose metabolism. Moreover, SPED promoted the PI3K/PKB/GSK-3ß signaling pathway by regulating mRNA expression of insulin receptors (IR), insulin receptor substrate 2 (IRS-2), phosphatidylinositol 3 kinase (PI3K), protein kinase B (PKB), and glycogen synthase kinase 3ß (GSK-3ß). In conclusion, the SPED might represent a novel marine-derived candidate against hyperglycemia, which may undergo further pharmaceutical development as a hypoglycemic agent.

[Clinical Manifestations and Complications in Patients with Anti-NMDA Receptor Encephalitis].

OBJECTIVE: To investigate the clinical manifestations and complications in the patients of anti-N-methyl-D-aspartate (anti-NMDA) receptor encephalitis. METHODS: We retrospectively collected clinical data of 143 patients diagnosed with anti-NMDA receptor encephalitis. According to the complexity of clinical symptoms, the patients were divided into two groups: ≥4 clinical symptoms group (96 cases, 67.1%) and <4 group (47 cases, 32.9%). There were 109 patients with complications and 34 patients without any complicaitons, The related factors to symptom complexity and complications were analyzed by univariate and multivariate analysis method. RESULTS: In the 143 patients of anti-NMDA receptor encephalitis, 120 (83.9%) exhibited seizures, 130 (90.9%) presented with psychiatric symptoms. Pulmonary infections (58.7%) and gastrointestinal disorders (49.0%) were the most common complications. Univariate analysis showed that the complexity of clinical symptoms was related to abnormal electroencephalography (EEG), positive anti-NMDA receptor antibody in cerebrospinal fluid(CSF) and serum. There were more frequent seizures, disorders of consciousness, abnormal movements, central hypoventilation in the patients with complications than those in patients without complications, all the differences were signifcant (P<0.05). Multivariate analysis showed that abnormal EEG was an independent risk factor for symptom complexity 〔odds ratio(OR)=2.620, P<0.05〕. The abnormal movements and the activities of daily living (ADL) on admission were predictor factors for the incidence of complications (OR=4.338, 0.980, respectively, P<0.05). CONCLUSIONS: In the patients of anti-NMDA receptor encephalitis, abnormal EEG may related to more complex clinical symptoms. Abnormal movements and low ADL on admission seems to be independent risk factors related to the incidence of complications.

Prearrest hypothermia improved defibrillation and cardiac function in a rabbit ventricular fibrillation model.

BACKGROUND: Hypothermia when cardiopulmonary resuscitation begins may help achieve defibrillation and return of spontaneous circulation (ROSC), but few data are available. OBJECTIVE: The objective of this study was to determine whether prearrest hypothermia improved defibrillation and cardiac function in a rabbit ventricular fibrillation (VF) model. RESULTS: Thirty-six New Zealand rabbits were randomized equally to receive normothermia (Norm) (~39°C), post-ROSC hypothermia (~33°C), or prearrest hypothermia (~33°C). Ventricular fibrillation was induced by alternating current. After 4 minutes of VF, rabbits were defibrillated and given cardiopulmonary resuscitation until ROSC or no response (≥30 minutes). Hemodynamics and electrocardiogram were monitored; N-terminal pro-brain natriuretic peptideand troponin I were determined by enzyme-linked immunosorbent assay. Myocardial histology and echocardiographic data were evaluated. First-shock achievement of perfusion rhythm was more frequent in prearrest than normothermic animals (7/12 vs 1/12; P=.027). After ROSC, dp/dtmax was higher in prearrest than normothermic animals (P<.001). Left ventricular end-systolic pressure was higher in prearrest than normothermic animals (P=.001). At 240 minutes after ROSC, troponin I and N-terminal pro-brain natriuretic peptide were lower in prearrest than normothermic animals (15.74±2.26 vs 25.09±1.85 ng/mL and 426±23 vs 284±45 pg/mL, respectively), the left ventricular ejection fraction and cardiac output were lower in the Norm group than other 2 groups (P<.01). Myocardial histology was more disturbed in normothermic than post-ROSC and prearrest animals, but was not different in the latter 2 groups. CONCLUSIONS: Induction of hypothermia before VF led to improved cardiac function in a rabbit VF model through improving achievement of perfusing rhythm by first-shock defibrillation and facilitating resuscitation.

Identification of repellent and insecticidal constituents of the essential oil of Artemisia rupestris L. aerial parts against Liposcelis bostrychophila Badonnel.

The aim of this research was to determine the chemical composition and insecticidal and repellent activity of the essential oil of Artemisia rupestris L. aerial parts against the booklice Liposcelis bostrychophila Badonnel and isolation of insecticidal and repellent constituents from the essential oil. The essential oil of A. rupestris was obtained by hydrodistillation and analyzed by GC-MS. A total of 30 components of the essential oil of A. rupestris was identified and the principal compounds in the essential oil were α-terpinyl acetate (37.18%), spathulenol (10.65%), α-terpineol (10.09%), and linalool (7.56%), followed by 4-terpineol (3.92%) and patchoulol (3.05%). Based on bioactivity-guided fractionation, the four active constituents were isolated from the essential oil and identified as α-terpineol, α-terpinyl acetate, 4-terpineol and linalool. The essential oil of A. rupestris exhibited contact toxicity against L. bostrychophila with LD50 value of 414.48 µg/cm². α-Terpinyl acetate (LD50 = 92.59 µg/cm²) exhibited stronger contact toxicity against booklice than α-terpineol (LD50 = 140.30 µg/cm²), 4-terpineol (LD50 = 211.35 µg/cm²), and linalool (LD550 = 393.16 µg/cm²). The essential oil of A. rupestris (LC50 = 6.67 mg/L air) also possessed fumigant toxicity against L. bostrychophila while the four constituents, 4-terpineol, α-terpineol, α-terpinyl acetate and linalool had LC50 values of 0.34, 1.12, 1.26 and 1.96 mg/L air, respectively. α-Terpinol and α-terpinyl acetate showed strong repellency against L. bostrychophila, while linalool and 4-terpinol exhibited weak repellency. The results indicate that the essential oil of A. rupestris aerial parts and its constituent compounds have potential for development into natural insecticides or fumigants as well as repellents for control of insects in stored grains.

[The effects of activated protein C on the von Willebrand factor and von Willebrand factor cleaving protease of rat aortic endothelial cell induced by lipopolysaccharide].

OBJECTIVE: To investigate the activated protein C (APC) on the von Willebrand factor antigen (vWFAg) and von Willebrand factor cleaving protease (ADAMTS-13) protein expression in rat aortic endothelial cells (RAECs) induced by lipopolysaccharide (LPS). METHODS: RAECs from Wistar rats were cultured with the tissue explants adherence method. RAECs were cultured for one week, After one week culture, RAECs in 4-5 generations were divided into control group, LPS stimulation groups (1 mg/L) and APC intervention groups (0.1, 1 and 10 mg/L APC was added after LPS stimulation). The supernatants were obtained at 12, 24, 48, and 72 hours after LPS stimulated to determine the vWFAg and protein of ADAMTS-13 expression by enzyme-linked immunoadsorbent assay (ELISA). RESULTS: In the control group, RAECs expressed little vWFAg and protein of ADAMTS-13. With stimulation of LPS, the vWFAg was significantly increased at 12 hours, and reached the peak at 48 hours [(285.45±30.13)%], and the level of ADAMTS-13 (µg/L) was gradually decreased, and reached the nadir at 72 hours (13.32±2.37), there was significant difference compared with control group [vWFAg: (94.53±7.83)%, ADAMTS-13: 115.76±2.36, both P<0.01). The effects on vWFAg promoting and ADAMTS-13 inhibition after LPS stimulation could be dose-dependently reversed by APC. 10 mg/L of APC could decrease the peak of vWFAg at 48 hours of LPS stimulation [(198.43±17.92)% vs. (285.45±30.13)%], and increase the minimize of ADAMTS-13 (µg/L) at 72 hours of LPS stimulation (125.25±2.70 vs. 13.32±2.37), with significant difference (both P<0.01). CONCLUSIONS: After stimulation with LPS, the level of vWFAg was time-dependent increased, as the protein of ADAMTS-13 was decreased. APC could attenuate the effect of LPS on vWFAg and protein of ADAMTS-13 with dose-dependent and time-dependent patterns.

A Novel Mitochondrial-Related Gene Signature for the Tumor Immune Microenvironment Evaluation and Prognosis Prediction in Lung Adenocarcinoma.

Lung adenocarcinoma (LUAD) remains the most common deadly disease and has a poor prognosis. More and more studies have reported that mitochondrial-related genes (MTRGs) were associated with the clinical outcomes of multiple tumors solely. In this study, we aimed to develop a novel prognostic model based on MTRGs. Differentially expressed MTRGs were identified from TCGA-LUAD and GSE31210 cohorts. Univariate Cox regression analysis was utilized to screen differentially expressed MTRGs that were related to prognosis of LUAD. Then, LASSO Cox regression analysis was used to develop a prognostic signature. ESTIMATE was used for estimating the fractions of immune cell types. In this study, we identified 44 overlapping differentially expressed MTRGs in TCGA-LUAD and GSE31210 cohorts. Among 44 overlapping differentially expressed MTRGs, nine genes were associated with prognosis of LUAD. When the penalty parameter lambda was the minimum, there were six genes meeting the conditions of constructing the signature, including SERPINB5, CCNB1, FGR MAOB, SH3BP5, and CYP24A1. The survival analysis suggested that prognosis of patients in the high-risk group was significantly worse than that in the low-risk group. Cox regression analyses showed that the risk score was an independent predictor of LUAD prognosis. As with the results of ESTIMATE score, the degree of immune cell infiltration in the low-risk group was higher than that in the high-risk group, such as TIL, Treg, and B cells. In addition, TMB and cancer stem cell infiltration were higher in the low-risk group than the high-risk group. In conclusion, we developed a novel MTRG signature acting as a negative independent prognostic factor. In the future, individualized treatments and medical decision-making may benefit from using the predicted model.

IFN-gamma, as secreted during an alloresponse, induces differentiation of monocytes into tolerogenic dendritic cells, resulting in FoxP3+ regulatory T cell promotion.

IFN-gamma has been shown to inhibit monocyte (Mo) differentiation into mature dendritic cells (DC). Because IFN-gamma also plays a role in tolerance induction, we asked whether this could be related to generation of tolerogenic DC (Tol-DC). Toward this aim, we cultured Mo with GM-CSF plus IL-4 in the presence or absence of IFN-gamma for 6 days and induced their maturation with TNF-alpha for 2 additional days. We showed that IFN-gamma deviated Mo differentiation from mature DC toward Tol-DC. Indeed, IFN-gamma-generated DC 1) expressed moderate levels of costimulatory molecules, but high levels of Langerin and CD123 molecules, 2) were maturation resistant, and 3) were unable to efficiently present alloantigen to T cells. More interestingly, naive CD4(+) T cells primed with IFN-gamma-generated DC expressed FoxP3 mRNA at high levels and exerted regulatory functions upon secondary stimulation with alloantigen. To address whether endogenously secreted IFN-gamma mediates a similar effect, we used the alloreaction as a model. We showed that cell-free supernatant harvested from an HLA-mismatched, but not HLA-identical, alloresponse induced differentiation of Mo into Tol-DC able to promote regulatory T cell generation. Moreover, when supplemented with GM-CSF plus IL-4, HLA-mismatched cell-free supernatant inhibited differentiation of Mo into mature DC. Finally, by adding Abs directed against inflammatory cytokines, we demonstrated that IFN-gamma plays a preponderant role in this inhibition. In conclusion, our results clearly demonstrate that exogenous or endogenous IFN-gamma, as well, induces differentiation of Mo toward Tol-DC, which results in FoxP3(+) regulatory T cell promotion.

[The effects of mild hypothermia on cardiac function and myocardial structure in a rabbits model of ventricular fibrillation after restoration of spontaneous circulation].

OBJECTIVE: To examine the impact of mild hypothermia on cardiac function, myocardial tissue integrity, and 48 hours mortality in a rabbits model of ventricular fibrillation after restoration of spontaneous circulation (ROSC). METHODS: The rabbits were randomly divided into four groups: normothermic post ROSC (NTPR, n = 10), mild hypothermia post ROSC (HTPR, n = 10), normothermic control (NTC, n = 8) and mild hypothermia control (HTC, n = 8). Ventricular fibrillation was induced by trans-epicardium electric-shook with alternating current in all the animals and ROSC was achieved through administration of adrenaline (i.v.) and artificial ventilation in group NTPR and HTPR. The body temperature of the animals was kept either at (39.0 ± 0.5) centigrade (NTPR and NTC) or (33.5 ± 0.5) centigrade (HTPR and HTC) for 4 hours after surgery for hemodynamic index data collection 0.5, 1, 2, 3 and 4 hours after surgery, 48 hours later, the mortality in the animals was recorded, and myocardial tissue samples were collected from survived animals for morphological examination by light and electric microscopy and analysis of apoptosis by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining. The content of ATP, ADP and AMP in the tissue samples was measured by high performance liquid chromatography (HPLC) for the calculation of energy charges (EC). RESULTS: (1)Hemodynamic indexes: as compared to the NTC group, HTC group exhibited significantly lower levels of heart rate (HR) and -dp/dt max in all the time points. No significant difference between the two groups in the levels of +dp/dt max and mean artery pressure (MAP) was found in all the time points but 0.5 hour. There was no significant difference in the levels of left ventricular end-diastolic pressure (LVEDP), left ventricular end-systolic pressure (LVESP), and femoral artery blood pressure between the two groups.(2) In comparison with NTPR group, HTPR group exhibited significantly (all P < 0.05) lower levels of HR (bpm) and -dp/dt max in all time points (ROSC 0.5, 1, 2, 3, 4 hours: HR 216.5 ± 33.3 vs. 292.9 ± 38.4, 218.2 ± 28.0 vs. 294.3 ± 37.0, 227.5 ± 25.4 vs. 291.4 ± 25.3, 232.4 ± 27.4 vs. 278.1 ± 30.8, 230.6 ± 22.0 vs. 285.1 ± 38.2; -dp/dt max 1847.1 ± 241.2 vs. 2383.3 ± 470.9, 1860.7 ± 167.8 vs. 2154.6 ± 319.5, 1822.3 ± 389.7 vs. 2239.7 ± 379.0, 1950.6 ± 412.9 vs. 2229.6 ± 392.4, 1875.7 ± 555.6 vs. 2396.7 ± 420.1). There was no significant difference between the two groups in the levels of LVEDP, +dp/dt max, LVESP, and femoral artery blood pressure. (3)Optical and electron microscopy revealed myocardium injury in samples from animals underwent ROSC. However, in comparison with the NTPR group, samples from HTPR group exhibited less damage to the myocardium structure. (4) Apoptosis index (AI) of myocardium was significantly (P < 0.05) higher in NTPR group (42.02%) than in HTPR group (26.39%). (5) Tests of myocardial energy: ATP level (µmol/g) in HTPR was significantly (P < 0.05) higher than NTPR (0.97 ± 0.26 vs. 0.65 ± 0.16). EC in NTPR was significant lower than it in two control groups [(0.33 ± 0.13)% vs. (0.52 ± 0.12)%, (0.55 ± 0.06)%, both P < 0.05], whereas no such difference was found between HTPR [(0.41 ± 0.12)%] and two control groups. (6) 48 hours survival rate in HTPR group was significantly higher (P = 0.043) as compared to NTPR group (100% vs. 60%). CONCLUSIONS: Myocardial dysfunction and myocardium tissue injury both develop in post-resuscitation rabbits with ventricular fibrillation. In these animals, reducing body temperature to the level of mild hypothermia after ROSC may improve the 48 hours survival rate, probably via mechanisms that suppress myocardial cell apoptosis. In our study, such intervention produced no obvious negative impact neither on the cardiac function nor hemodynamics.

[Induction of expression of von Willebrand factor cleaving protease of rat aortic endothelial cell by lipopolysaccharide challenge].

OBJECTIVE: To investigate the dose response effect and time response effect of lipopolysaccharide(LPS) on von Willebrand factor cleaving protease (ADAMTS-13, Vwf-cp) mRNA expression and protein in rat aortic endothelial cells (RAECs). METHODS: RAECs were grown by culturing of aortic tissue. When ARECs were cultured for one week, it was co-cultured by 1:3 to reach 4-5 generations. ARECs were randomly divided into five groups: control group and four LPS stimulation groups (0.01, 0.1, 1 and 5 µg/ml) . The RAECs and supernatants were obtained at 12, 24, 48, and 72 hours after being stimulated by LPS. ADAMTS-13 mRNA expression of RAECs was assessed by quantitation reverse transcription-polymerase chain reaction (RT-PCR), and protein of ADAMTS-13 in supernatants was determined by enzyme linked immunoadsorbent assay (ELISA). RESULTS: In the control group RAECs were shown to express ADAMTS-13 at both protein and mRNA levels. With the increase of concentration of LPS, or increase in stimulus duration, expression of ADAMTS-13 mRNA and protein were gradually lowered. Compared with the control group (25.22 ±1.41), the level of ADAMTS-13 mRNA in 0.01 µg/ml LPS stimulation group was markedly decreased at 48 hours (18.78±0.86, P<0.01). At 24 hours, the levels of ADAMTS-13 mRNA (23.43±0.63, 22.41±0.76) were markedly decreased in 0.1 µg/ml and 1 µg/ml LPS stimulation groups (P<0.05 and P<0.01). The level of ADAMTS-13 mRNA (20.01±2.47) in 5 µg/ml LPS stimulation group was markedly decreased at 12 hours (P<0.01). Compared with the control group [(115.76±2.36) ng/ml], protein level of ADAMTS-13 [(113.43±1.07) ng/ml] was markedly decreased at 12 hours in 0.01 µg/ml LPS stimulation group (P<0.05). The protein level of ADAMTS-13 [(7.63±2.64) ng/ml] was lowest in 5 µg/ml LPS stimulation group at 72 hours (P<0.01). CONCLUSION: Normal RAECs can express ADAMTS-13 at both mRNA and protein to certain extent. The expression of ADAMTS-13 mRNA and protein are decreased after LPS challenge in different concentrations for different duration in dose dependent and time dependent manner.

Challenges of patients with epilepsy and measures for improving epilepsy care in western China: A qualitative study.

OBJECTIVE: The aim of this qualitative study was to explore the challenges that patients with epilepsy (PWEs) face and the opportunities or areas where changes in nursing care may improve epilepsy care in western China. METHODS: Semi-structured interviews with open-ended questions based on a review of the literature were conducted at the epilepsy center of a tertiary hospital in western China. A total of 18 PWEs, 18 caregivers and 11 neurology nurses were interviewed by using purposive sampling. The data were transcribed verbatim, and a content analysis was used to conduct the framework analysis. RESULTS: Three key themes were identified, namely, the impact of epilepsy, barriers to epilepsy management, and measures in nursing care for improving epilepsy care. Psychological stress, the side effects of drugs and accidental injury related to seizures were reported to be the main negative impacts on patients. Limited knowledge about epilepsy, poor adherence to therapy, and a lack of effective communication between patients and medical staff were the major barriers to epilepsy management. Strengthening health education, assessing the frequency and type of seizures, screening for psychological disorders and mental intervention, and maintaining continuity of care were identified as crucial measures for nurses to improve epilepsy care. CONCLUSIONS: This study highlights the challenges among PWEs and opportunities for improving the quality of epilepsy care in western China. Limited knowledge and poor drug adherence are the main barriers to epilepsy management, which might be improved by more health education and continuing care provided by nurses. Assessing seizures, screening for psychological disorders and providing appropriate psychological care would help improve epilepsy care.

Dexamethasone attenuated endotoxin-induced acute lung injury through inhibiting expression of inducible nitric oxide synthase.

Application of glucocorticoids in sepsis or severe infection is disputable in clinic. In this experiment, we studied the effect of dexamethasone on nitric oxide synthases and whether dexamethasone could attenuate endotoxin-induced acute lung injury (ALI). SD rats received 5 mg/kg lipopolisaccharide (LPS) injection. Then arterial oxygen partial pressure (PaO2), lung histology, lung tissue nitric oxide (NO) production and expression of nitric oxide synthases (NOS) were detected at 0.5, 1, 2, 3 or 4 h after LPS injection. PaO2 and lung injury deteriorated upon time. Production of NO in lung tissue increased significantly particularly in the first two hours, and this change was mainly due to the over-expression of inducible NOS (iNOS), but not endothelial NOS (eNOS). Furthermore, a tight positive correlation was observed between lung injury score (LIS) and NO production level in lung tissue. Dexamethasone could ameliorate PaO2 and lung damage evidently, which were paralleled by significant decreases in the production of NO and in the expression of iNOS mRNA. In conclusion, dexamethasone could effectively attenuate endotoxin-induced lung injury through inhibiting iNOS expression and activation in the very early stage of ALI.

[Determination of trace elements in hollyhock by microwave digestion-FAAS].

Hollyhock powder was digested by microwave digestion before determination. The eight trace elements, Fe, Cu, Zn, Mn, K, Ni, Ca and Mg, in hollyhock were determined by microwave FAAS. Standard curves method was used for the determination. The samples were digested with HNO3. The conditions of digestion were optimized. The effect of different microwave digestion conditions on the analysis results was reviewed, and the optimal condition for using atomic absorption spectrophotometry was determined. The experiments indicated that the content of trace elements of human needs, Mg, Ca, Fe, K etc. are highly enriched, the content of Mn and Zn is middling, and the content of Cu and Ni is low. The method is highly sensitive, convenient and speedy. The recovery (n = 6) is 92.25%-110.50%, and the RSDs (n = 6) are lower than 5%. The results are satisfactory.

[Effect of Xuebijing injection on expression of endothelial protein C receptor and protease activated receptor 1 mRNA and protein in endothelial cells induced by lipopolysaccharide].

OBJECTIVE: To investigate the effect of Xuebijing injection on expression of endothelial protein C receptor (EPCR) and protease activated receptor 1 (PAR1) mRNA and protein in rat aortic endothelial cells (RAECs) after lipopolysaccharide (LPS) challenge. METHODS: RAECs were cultured for one week, and the purity was determined with flow cytometry. ARECs were randomly divided into four groups: control group, LPS stimulation group (1 mg/L LPS), Xuebijing injection treatment group (LPS: 1 mg/L, Xuebijing injection ; 10 g/L) and activated protein C (APC) treatment group (LPS: 1 mg/L, APC: 0.1 mg/L). The RAECs were collected at 12, 24, 48, and 72 hours after being stimulated by LPS. EPCR and PAR1 mRNA of RAECs were assessed by reverse transcription-polymerase chain reaction (RT-PCR). EPCR and PAR1 protein were assessed by flow cytometry. RESULTS: At 12 hours, EPCR and PAR1 protein expressions were not significantly different among groups (all P>0.05). The level of EPCR and PAR1 mRNA were decreased in LPS stimulation group, but they were elevated in both APC and Xuebijing injection treatment groups (P<0.05 or P<0.01). From 24 to 72 hours, compared to control group, the levels of EPCR mRNA and protein expression were significantly decreased after LPS stimulation (P<0.05 or P<0.01). The levels of EPCR expression were decreased and negatively correlated with the time of LPS treatment. Also, compared to LPS stimulation group, treatment with Xuebijing markedly elevated the levels of EPCR (P<0.01). The levels of PAR1 expression were significantly decreased by LPS stimulation compared with those of control group (P<0.05 or P<0.01). After the treatment with Xuebijng, the expression of PAR1 was gradually increased (all P<0.01). Compared with APC treatment group, Xuebijing could increase the PAR1 expression better. CONCLUSION: Xuebijing could raise EPCR and PAR1 mRNA and protein expression of RAECs after LPS challenge, and it may be related to its protection of endothelial cell from undergoing apoptosis.

Efficient generation of CD34+ progenitor-derived dendritic cells from G-CSF-mobilized peripheral mononuclear cells does not require hematopoietic stem cell enrichment.

As a result of their potent antigen-presentation function, dendritic cells (DC) are important tools for cell therapy programs. In vitro-generated DC from enriched CD34+ hematopoietic stem cells (HSC; enriched CD34 DC) have already proven their efficiency in Phase I/II clinical trials. Here, we investigated whether enrichment of CD34+ HSC before the onset of culture was absolutely required for their differentiation into DC. With this aim, we developed a new two-step culture method. PBMC harvested from G-CSF-mobilized, healthy patients were expanded for 7 days during the first step, with early acting cytokines, such as stem cell factor, fetal liver tyrosine kinase 3 ligand (Flt-3L), and thrombopoietin. During the second step, expanded cells were then induced to differentiate into mature DC in the presence of GM-CSF, Flt-3L, and TNF-alpha for 8 days, followed by LPS exposure for 2 additional days. Our results showed that the rate of CD34+/CD38+/lineageneg cells increased 19.5+/-10-fold (mean+/-sd) during the first step, and the expression of CD14, CD1a, CD86, CD80, and CD83 molecules was up-regulated markedly following the second step. When compared with DC generated from enriched CD34+ cells, which were expanded for 7 days before differentiation, DC derived from nonenriched peripheral blood stem cells showed a similar phenotye but higher yields of production. Accordingly, the allogeneic stimulatory capacity of the two-step-cultured DC was as at least as efficient as that of enriched CD34 DC. In conclusion, we report herein a new two-step culture method that leads to high yields of mature DC without any need of CD34+ HSC enrichment.

Perinatal nicotine exposure increases obesity susceptibility by peripheral leptin resistance in adult female rat offspring.

Maternal nicotine (NIC) exposure causes overweight, hyperleptinemia and metabolic disorders in adult offspring. Our study aims to explore the underlying mechanism of perinatal NIC exposure increases obesity susceptibility in adult female rat offspring. In our model, we found that adult NIC-exposed females presented higher body weight and subcutaneous and visceral fat mass, as well as larger adipocytes, while no change was found in food intake. Serum profile showed a higher serum glucose, insulin and leptin levels in NIC-exposed females. In adipose tissue and liver, the leptin signaling pathway was blocked at 26 weeks, presented lower Janus tyrosine kinase 2 and signal transducer and activator of transcription 3 gene expression, higher suppressor of cytokine signaling 3 gene expression (in adipose tissue) and lower leptin receptors gene expression (in liver), indicating that peripheral leptin resistance occurred in NIC-exposed adult females. In female rats, the expression of lipolysis genes was affected dominantly in adipose tissue, but lipogenesis genes was affected in liver. Furthermore, the glucose and insulin tolerance tests showed a delayed glucose clearance and a higher area under the curve in NIC-exposed females. Therefore, perinatal NIC exposure programed female rats for adipocyte hypertrophy and obesity in adult life, through the leptin resistance in peripheral tissue.

[Construction of recombinant adenovirus vector of mouse keratinocyte growth factor].

OBJECTIVE: To construct a recombinant adenovirus vector expressing keratinocyte growth factor (KGF) gene in mouse. METHODS: KGF gene amplified from mouse cDNA by polymerase chain reaction (PCR). It was reverse transcript from RNA that had been harvested from C57BL/6 mouse, and then inserted into the plasmid pShuttle-CMV to construct the shuttle plasmid pShuttle-CMV-KGF (pKGF). After linearized by restriction enzyme, the plasmid was transformed into E.Coli BJ5183 containing adenovirus backbone. The homologous recombinant pAdeasy-1-pShuttle-CMV-KGF (pAd-KGF) was identified, linearized, and then transfected into HEK293 cells using the lipofectamine TM 2000 to package the adenovirus, Adeasy-1-pShuttle-CMV-KGF (Ad-KGF), followed by further amplification, caesium chloride density gradient centrifugation purification and measurement of virus titer. Ad-GFP was used as control, and its transfection efficacy was observed. RESULTS: (1) The shuttle plasmid pKGF was proved to be successfully constructed by gene sequencing and restriction enzyme, as well as the recombinant adenovirus plasmid. (2) The cytopathic effects of HEK293 cells observed under the microscope suggested that the duplication of the virus was successful. (3) The plague titration of HEK293 cells showed virus titers were 3.0 x 10(10) pfu/ml,the concentration of which was adequate for future test in vivo or in vitro. CONCLUSION: The harvest of recombinant adenovirus vector of Ad-KGF, is the first step for the future test to investigate the effects of KGF in pulmonary diseases, and the possible gene therapy to treat pulmonary fibrosis.

[Research of gene expression profile of liver tissue in rat sepsis model].

OBJECTIVE: To screen differentially expressed genes of liver tissue in rat sepsis model and analyze them in terms of functions. METHODS: Thirty male Wistar rats were randomly divided into model group and blank control group with 15 rats in each group. Cecal ligation and puncture (CLP) was used to reproduce rat sepsis model, gene expression profile microarray that contains 4 096 rat cDNA clones was used to detect the change in gene expression pattern of rat liver tissue 24 hours after CLP, then differentially expressed genes that high correlated to sepsis were screened, and the functions of these genes were analyzed by means of related computer software. RESULTS: Compared to the controls, gene expression of 522 genes in rat sepsis model were changed 24 hours after CLP, accounting for 12.7%, among them 244 gene expression down-regulated, and 278 gene expression up-regulated. CONCLUSION: Multiple organ dysfunction syndrome (MODS) induced by sepsis involves to a series of gene differential expressions, such as cell cycle and control related genes, cell apoptosis genes, immunity related genes, genes concerning energy metabolism, blood system related genes, cancer related genes, growth factor genes, acute stress reaction related genes etc. Gene microarray technique can be used to comprehensively study gene expression profile in rat sepsis model, in order to find new research objectives and gene therapy strategies for sepsis.

[Effects of Xuebijing injection on thrombomodulin and endothelial cell protein C receptor in septic rats].

OBJECTIVE: To investigate effects of the integrated traditional Chinese medicine Xuebijing injection on thrombomodulin (TM) and endothelial cell protein C receptor (EPCR) in septic rats. METHODS: Ninety-six healthy Wistar rats were randomly divided into four groups: control group, sham operation group, cecal ligation and puncture (CLP) model group, and Xuebijing-treated group. Sepsis was reproduced by CLP. The two latter groups were divided into five subgroups of 2, 8, 24, 48 and 72-hour with 8 rats in each subgroup. Tissue samples from liver and lung were collected to determine tissue TM and EPCR mRNA expression. RESULTS: TM and EPCR mRNA expressions were observed in liver and lung in control group and sham operation group, while with no significant differences at 2 hours post-CLP (both P>0.05). TM and EPCR gene expression levels in tissues were significantly increased to certain extent at 8-48 hours (all P<0.01), and were dramatically decreased following Xuebijing injection at 72 hours post-CLP (both P>0.05). Also, treatment with Xuebijing injection markedly decreased TM and EPCR mRNA levels to certain extents at 8 and 24 hours, and markedly increased at 48 and 72 hours compared with those of model group. CONCLUSION: These data suggest that Xuebijing injection could raise TM and EPCR mRNA expression, thereby it might be effective in prevention of development of severe sepsis.

[Effects of Xuebijing injection on protein C and tumor necrosis factor-alpha mRNA in rats with sepsis].

OBJECTIVE: To investigate the effects of the integrated traditional Chinese medicine Xuebijing injection on protein C (PC) and tumor necrosis factor-alpha (TNF-alpha) mRNA in rats with sepsis. METHODS: Sepsis was induced in Wistar rats by cecal ligation and puncture (CLP). Ninety-six healthy animals were randomly divided into four groups: normal group, sham-operation group, CLP model group, and Xuebijing-treated group. The two latter groups were divided into 2, 8, 24, 48, and 72-hour subgroups with 8 rats in each subgroup. Platelet count of blood obtained from abdominal aorta was determined and tissue samples from liver and lungs were collected to measure tissue PC and TNF-alpha mRNA expression. RESULTS: PC gene expression levels in lung tissues were significantly lowered (all P<0.01), but they were dramatically raised by Xuebijing injection during 8-72 hours post-CLP (all P<0.01). Compared with normal group, TNF-alpha mRNA levels in liver and lungs were significantly elevated at 2 hours post-CLP (P<0.05 or P<0.01). However, treatment with Xuebijing injection markedly reduced TNF-alpha mRNA both in liver and lungs at 2-24 hours (P<0.05 or P<0.01). In CLP group, blood platelet count was significantly decreased to certain extent at different intervals within 8-72 hours, and it was markedly elevated in the Xuebijing-treated group (P<0.05 or P<0.01). CONCLUSION: The current study suggests that Xuebijing injection could exert preventing effect on the development of severe sepsis by suppressing PC and TNF-alpha mRNA.