Objective:To investigate whether deletion of Gpr174 alleviates gut injuries during sepsis and identify the differential gut microbiota, metabolites and related metabolic pathways between C57BL/6 and Gpr174 -/- mice in physiological condition and sepsis. Methods:Twelve 8-week-old male C57BL/6 and Gpr174 -/- mice were randomly (random number) divided into the following four groups: Wildtype (WT) group, Gpr174 -/- (KO) group, Wildtype + CLP (WT+CLP) group and Gpr174 -/- + CLP (KO+CLP) group. Sepsis mice model was established by cecal ligation and puncture (CLP) as previously described. Feces were collected from C57BL/6 and Gpr174 -/- mice in normal condition and 3 days after CLP. The nucleic acid of stool was extracted and then amplified the V3 V4 region using TransStart FastPfu DNA Polymerase; 16S rDNA gene amplicons were sequenced on an Illumina MiSeq instrument. After demultiplexing and quality filtering, differential microbiota was identified. Metabonomics was determined by liquid chromatography (LC-MS). Endogenous metabolites were identified by the Feihn metabonomics database. Then, metabolic pathways were further analyzed via KEGG. Results:The principal component analysis (PCA) showed a cluster type distribution between the WT group and KO group . The difference between the WT+ CLP group and KO + CLP group was significantly reduced after CLP. The differential gut microbiota included Lactobacillus, Akkermansia, Bacteroides, Allobaculum, Bifidobacterium, Rikenella, Olsenella, Barnesiella, and the differential metabolites included L-Alanine, Hydroxypropionic acid, Oxoglutaric acid. The related signal pathways of differential metabolites were Glucose-Alanine Cycle, Alanine Metabolism and Propanoate Metabolism. Conclusions:Gpr174 deletion could alleviate gut injuries during sepsis and change the composition of gut microbiota and metabolites.

Objective To investigate the changes of circulaling progenitor cells and endothelial progenitor cells(EPCs)in non-septic and septic shock patients using flow cytometry.Method A total of 27 sepsis patients hospitalized in emergency department of Zhongshan hospital during August 2007 to February 2008 were enrolled in this study.The patients were dividedinto septic shock group(n=12)and non-septic shock group(n=15).Ten healthy individuals and ten non-sepsis ICU patients were collected as controls.Peripheral blood mononuclear cells(PBMCs) were isolated by Ficoll density gradient centrifugation,and EPCs labelled with antibodies against CDl33,CD34 and VEGFR-2 were identified and isolated by three-color fluorescence flow cytometry.Differences within the groups were analyzed using One way ANOVA.Results The percentages ofprogenitor cells and EPCs in the PBMC fraction in healthy controls were(0.25%4-0.14%),(0.09%-I-0.02%),respectively,and those in ICU controls were(O.38%.4-0.29%),(0.12%.4-0.02%).The percentages of progenitor cells and EPCs were significantly higher in栅sel如c shock patients(0.57%±0.12%),(0.22%.4-0.10%)than in heathy and non-sepsis ICU controls(P<0.05).However.the percentages of progenitor cells and EPC8 in septic shock pa.tienta(O.20%±0.12%,0.04%-t-O.01%)was obviousely lower than those in healthy,ICU controls and ilionseptic shock patients(P<0.05).Sptic shock survivors had significantly higher numbers of cEPCs than nonsur.vivors(P<0.05).Conclusions The level of progenitor cells and EPC8 in peipheral blood of sepsis patients might be the valuable markers to as.se88 the severity and outcome ofthese ptienS.

Objective To investigate the genetic variants in the protein C (PC) and endothelial protein C receptor (EPCR) genes associated with the risk and outcome of acute respiratory distress syndrome (ARDS) patients in Chinese Han race.Methods Five tagSNPs (single nucleotide polymorphism,SNP) in the PC and EPCR genes were genotyped in patients with ARDS (n =275) and non-ARDS (n =337) in order to find the association between them in this case-control study.The SNPs were genotyped by SNPstream Beckman platform.Then,the correlation between the associated SNPs and plasma levels of activated protein C (APC) in patients with ARDS was investigated.The APC levels were measured using enzyme linked immunosorbent assay (ELISA) method.Results Association analysis rcvealed that two PC SNPs in perfect linkage disequilibrium,rs1799809 and rs1158867,were significantly associated with susceptibility to ARDS.T allele frequency of rs1799809 in ARDS patients was significantly higher than that in non-ARDS patients (OR =1.569,95％ CI:1.192-2.066).And the genotype frequencies of rs1799809 were also significantly different between these two groups (P =0.007).The association remained significant after adjustment for multiple comparisons.Haplotype consisting of three SNPs in the PC gene was also associated with susceptibility to ARDS.The frequency of haplotype CCC in the ARDS samples was significantly lower than that in the non-ARDS group (P ＜ 0.01).Moreover,ARDS patients canrying rs1799809 TT genotype showed lower serum levels of APC than patients with TC and CC genotypes (Padj =0.02).However,genotype and allele analyses of EPCR did not show any significant difference between ARDS and non-ARDS patients.Conclusions These findings indicated that common genetic variation in the PC gene was significantly associated with susceptibility to ARDS in Chinese Han race.The PC genetic variation influenced plasma concentration of APC in patients with ARDS.

Objective To investigate the possible association of IRAK4 polymorphisms with susceptibility to and prognosis of severe sepsis.Methods A total of 192 patients hospitalized in emergency department of Zhongshan Hospital from February 2006 to December 2009,and another 192 healthy volunteers were enrolled in this case-control study.Patients were excluded if they had metastatic tumors,autoimmune diseases,AIDS or received immunosuppressive drugs.This study was approved by the ethical committee of Zhongshan Hospital,Fudan University.Sepsis patients were divided into survival group(n =124)and non-survival group(n =68)according to the 30-day mortality.Primer 3 software was used to design the PCR and sequencing primers.Genomic DNA was extracted from peripheral blood mononuclear cells.Seven tagSNPs were selected based on the data of Chinese Han in Beijing from the Hapmap projectand genotyped by direct sequencing.We used x2 analysis to evaluate the significance of differences in genotype and allele frequencies between different groups.Results The distributions of all tagSNPs were consistent with Hardy-Weinberg equilibrium.The allele and genotype frequencies of rs4251545(G/A)were significantly different between severe sepsis and healthy control groups(P =0.015,P =0.035,respectively).Carriers of the rs4251545A had a higher risk for severe sepsis compared with carriers of the rs4251545G(OR =1.69,95％ CI:1.10-2.58).The allele and genotype frequencies of all SNPs were not significantly different between survivor group and non-survivor group.Conclusions These findings indicated that the variants in IRAK4 are significantly associated with severe sepsis susceptibility in the Chinese Han population.

Objective To investigate the possible association of TLR4 polymorphisms with susceptibility and prognosis of SCAP.Method A total of 360 CAP patients hospitalized in emergency department of Zhongshan hospital from May 2005 to April 2008 were enrolled in this case-control study.Patients were excluded if they had metastatic tumors,autoimmune diseases,AIDS or received immunosuppressive drugs.This study was approved by the ethical committee of Zhongshan hospital,Fudan University.Patients were divided into SCAP group(n = 180)and NSCAP group(n = 180)according to the illness severity,and were divided into survival group(n = 300)and death group(n = 60)according to the 30-day mortality.Hapmap database of Han Chinese population was used to select the Tag SNPs.Primer 3 software was used to design the PCR and sequencing primers.Genomic DNA was extracted from peripheral blood mononuclear cells.Genotyping was performed by sequencing the PCR products.We used X2 analysis to evaluate the significance of differences in genotype and allele frequencies between different groups.Results The distributions of three TagSNPs(rs2149356,rs11536879,rs1927907)were consistent with Hardy-Weinberg equilibrium.The allele and genotype frequencies of three TagSNPs in the SCAP group did not differ from the NSCAP group.Also,no significant difference was found between survivor group and non-survivor group.The haplotype frequencies of CA,TA and TG were not significantly different between SCAP group and NSCAP group.And no significant difference of haplotype frequency was existed between survivor group and nonsurvivor group.Conclusions This study suggested that TLR4 gene polymorphisms were not significantly associated with the susceptibility and prognosis of SCAP.

Objective To investigate the characteristics of inanune status change in sepsis and severe sepsis patients by quantitative analysing the serum concertrations of pro-and anti-inflammatory cytokines. Method Serum of 38 sepsis patients, 32 severe sepsis patients were collected and 15 health individuals were as controls.ELJSA method was used to quantify the serum levels of inflammatory cytokines. The severity of patient's condition was assessed according to the APACHE Ⅱ system, Retolts The serum concentrations of inflammatory cytokines were different among sepsis and severe sepsis patients. In the serum of sepsis patients the pro-inflammatory eytokine were dominant. But in the serum of severe sepsis patients the anti-inflammatory cytokine were dominant.The serum levels of TNF-α, IL-6, IL-1, IL-10 were obviously different among control, sepsis and severe sepsis groups ( P<0.05). The serum levels of IL-1 and IL-10 were significantly correlated with APACHE II scores. The multiple linear regression eqution was APACHE Ⅱ=- 9.393 + (IL-10 x 0.550) + (IL-1 x 0.305) (F =26.198,P<0.001) Conclusions The serum levels of pro-and anti-inflammatory cytokines were significantly different among patients with different stages of sepsis, and the immune status were different. The activation of inflatrmmtory reaction were constant in sepsis patients, while the expression of anti-inflammatory cytokines increased in severe sepsis patients.

AIM: To investigate the molecular mechanism by which hypoxia affect the endothelial nitric oxide synthase (eNOS) expression in cerebral artery endothelial cells (CAECs). METHODS: Primary cultured porcine CAECs were exposed to hypoxia for 2 h, 6 h, 12 h, 24 h and 48 h. The eNOS mRNA level was determined by RT-PCR. The level of eNOS protein was detected by Western blotting. After specific PKC inhibitors BIM Ⅰ(1 ?mol/L) and G6983 (1 ?mol/L) were added, CAECs were exposed to hypoxia for 24 h. The effect of hypoxia on eNOS mRNA stability was analyzed after actinomycin D was added. RESULTS: After exposure to hypoxia for 2 h, the levels of eNOS mRNA and protein in CAECs were increased. The levels of eNOS mRNA and protein reached peak after 12 h of hypoxia (about 2 5 fold and 2 0 fold, respectively, compared to control), and remained at higher level even after 48 h of hypoxia. Moreover, hypoxia did not change the stability of eNOS mRNA. The specific PKC inhibitors BIM Ⅰ and G6983 attenuated significantly the effects of hypoxia on eNOS gene expression. CONCLUSION: These results suggest that hypoxia may enhance the expression of eNOS gene in CAECs through PKC signaling pathway, which might be one of the mechanisms of cerebral artery dilation and neuroprotection during cerebral hypoxia.

Objective To investigate the effect of miR-132-3p on the proliferation of endothelial progenitor cells and its regulatory mechanism in order to provide a new theoretical basis for the treatment of deep venous thrombosis. Methods Real-time quantitative PCR (qPCR) was used to detect the expression of miR-132-3p in the plasma and endothelial progenitor cells of 27 healthy volunteers and 22 thrombus patients, and in endothelial progenitor cells under normoxic and hypoxic conditions. The miR-132-3p analogue and the specific siRNA were transferred into endothelial progenitor cells by the electroporation method. The effect of miR-132-3p on the proliferation of endothelial progenitor cells was detected using MMT and Cell Counting Kit-8 (CCK-8) methods. The effects of miR-132-3p on the expression of FOXO1 in endothelial cells were analyzed using the luciferase assay and western blots. Results The expression of miR-132-3p in clinical patients with thrombosis was significantly decreased to 0.45 ± 0.05 times of that of the healthy volunteers (P＜0.05). The expression of miR-132-3p in endothelial progenitor cells under hypoxia was down-regulated to (0.23 ± 0.13) times of that of under normoxia (P＜0.05). The expression of miR-132-3p of experiment group under hypoxia was up-regulated to (15.72 ± 2.06) times of that of control group (P＜0.05). MMT assay showed that the proliferation of cells in the experimental group under hypoxic condition was up-regulated to (7.79 ± 1.37) times of that in the control group (P＜0.01). CCK-8 assay showed that cell proliferation in experimental group was up-regulated to (6.46 ± 0.38) times of that in the control group (P＜0.01). Software analysis showed that FOXO1 was a direct target of miR-132-3p. Luciferase activity of miR-132-3p mimics transfected endothelial progenitor cells under hypoxic conditions were 0.47 times of that in siRNA treatment group. Western blot showed that the expression of FOXO1 protein in endothelial progenitor cells transfected with miR-132-3p mimics in hypoxia was 0.18 times of that in siRNA treatment group. Conclusions Compared with healthy volunteers, miR-132-3p expression in the blood of patients with thrombosis was significantly reduced that can promote transcription of the FOXO1 gene (and protein expression) and inhibit the proliferation of endothelial progenitor cells. It could be closely related to the formation of venous thrombosis.

Objective:Evaluation of combined inflammatory and coagulation markers for early identification of DIC in septic patients.Methods:This study was a single-center, retrospective, observational study involving 356 patients with sepsis. Sepsis was defined by the diagnostic criteria of Sepsis version 3.0. Definition of DIC was from the International Society on Thrombosis and Hemostasis (ISTH) DIC Score. Inflammatory biomarkers, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β,2R,6,8,10, etc. and biomarkers of coagulation, like platelet (PLT), international normalized ratio (INR), D-dimer, fibrinogen (Fib), etc. were included in this study.Results:Among 356 patients with sepsis, 301 patients did not develop DIC (non-DIC) during hospitalization, 32 patients had DIC on the day of admission (overt-DIC), and 23 patients developed DIC within 1 week of admission (pre-DIC). Compared to non-DIC patients, pre-DIC patients had lower platelet counts and fibrinogen ( P < 0.05), higher levels of INR and D-dimer ( P < 0.05), higher levels of cytokines (TNF-α、IL-1β、IL-2R、IL-8、IL-10) and procalcitonin ( P < 0.05), higher APACHEⅡ and SOFA scores ( P < 0.05). Using receiver operating characteristics (ROC) analysis, we found that some biomarkers of coagulation and inflammation could discriminate pre-DIC from non-DIC patients. The area under the curve (AUC) of INR in the ROC analysis was 0.773 (95% CI: 0.696-0.851), the AUC of IL-2R was 0.700 (95% CI: 0.599-0.798) which is highest among inflammation markers, the highest AUC was obtained from the combination of platelets, INR, Fib, D-dimer and IL-2R (AUC = 0.843; 95% CI: 0.758-0.928). Kaplan-Meier survival curve suggested that high level of IL-2R (> 1064.5 U/mL) was a valuable predictor of 28-day mortality in septic patients. Conclusion:Inflammatory marker, IL-2R, is related to the occurrence of DIC in septic patients and has predictive value for pre-DIC. Combination of coagulation (platelets, INR, Fib, D-dimer) and inflammatory markers (IL-2R) can help to identify pre-DIC state in septic patients.

Objective To measure the reads numbers of Human Herpes Virus in blood sample from patients with sepsis by using Next Generation sequencing (NGS) and explore the relationship between read number of virus and the severity, prognosis, immune status of septic patients.Methods Blood sample and clinical information from 150 patients with sepsis were enrolled in this study. All patients' blood samples were sent to perform NGS pathogenic test. According to the results of NGS, septic patients were divided into HHV-detected group and HHV-undetected group. Besides, patients were scored with Sequential Organ Failure Assessment (SOFA) and Acute Physiology and Chronic Health Evaluation Ⅱ(APACHE Ⅱ ) on the day of blood collection. The counts of total leukocytes, lymphocytes and the levels of cytokines were also measured. Results 51.3 percent of septic patients were detected with HHV nucleic acid. The APACHE Ⅱ and SOFA scores were significantly higher in HHV-detected patients compared with patients in HHV-undetected group. Besides, patients who had a higher SOFA score might lead to a higher detection rate of HHV. Moreover, the 28-day and 90-day mortality rates were higher in detected group (P< 0.01). The detection of HHV nucleic acid was positively correlated with a high 90-day mortality rate (P= 0.0056). One-way analysis of variance revealed that the counts of total lymphocyte and different types of lymphocyte (CD19+B、CD4+T、CD8+T、CD56+ lymphocyte) were significantly less in detected group than that in undetected group. Furthermore, both the levels of pro-inflammatory cytokines (TNF-α、IL-2R、IL-6、IL-8) and anti-inflammatory cytokines (IL-10) in detected group were significantly higher than those in undetected group. Gender, age, APACHE Ⅱ , SOFA, IL-2R, IL-10, CD19+B lymphocyte and T cells, were still significant even after multivariate logistic analyses. Conclusions The detection rate of HHV nucleic acid in patients with sepsis was high. The detection of HHV was a high-risk factor of death in patients with sepsis. The cut-off value which is more than 100 had a significant clinical value. The infection of HHV could be conducted by dysfunction of immunity.