Neutrophilic granule protein (NGP) attenuates lipopolysaccharide-induced inflammatory responses and enhances phagocytosis of bacteria by macrophages.

Neutrophilic granule protein (NGP) belongs to the cystatin superfamily. Even though this superfamily is critically involved in cancer biology and adaptive immunity, the relationship of macrophage NGP to inflammation and phagocytosis remains poorly understood. In this study, we observed a significant increase of NGP in peritoneal macrophages (PMs) isolated from mice challenged with E. coli or lipopolysaccharide (LPS), as judged by NGP mRNA microarray. We also found changes in NGP to be mainly Toll-like receptor 4 (TLR4)-dependent. By western blot and electrophoretic mobility shift assay, we demonstrated NGP overexpression to reduce TNF-α and IL-1ß production by LPS-induced RAW264.7 cells (RAW) via suppression of the NF-κB (p65 and p50) signalling pathway, rather than the JNK1/AP-1 (fos and jun) signalling pathway. NGP overexpression by LPS-induced RAW also induced IL-10, an anti-inflammatory cytokine, which was partially involved in the anti-inflammatory effect produced by NGP overexpression. Moreover, upregulated NGP enhanced the phagocytosis of E. coli by RAW. Taken together, these results demonstrated NGP to be an important host defense component that regulates inflammatory responses and phagocytosis by activated macrophages. As such, NGP may be useful for the treatment of inflammatory based disease.

Effects of small interfering RNA targeting TLR4 on expressions of adipocytokines in obstructive sleep apnea hyponea syndrome with hypertension in a rat model.

We explored the effects of RNA interference-mediated silencing of TLR4 gene on expressions of adipocytokines in obstructive sleep apnea hyponea syndrome (OSAS) with hypertension in a rat model. Systolic blood pressure of caudal artery and physiological changes were observed when establishing rat models of OSAS with hypertension. Mature rat adipocytes were induced from separated and cultured primary rat adipocytes. To transfect rat mature adipocytes, TLR4 siRNA group and negative control (NC) siRNA group were established. Expressions of TLR4 mRNA of adipocytes were examined after silenced by siRNA by quantitative real-time polymerase chain reaction (qRT-PCR). By enzyme-linked immunosorbent assay (ELISA), expressions of inflammatory cytokines, and adipocytokines of adipocytes were detected. Blood pressure in rat caudal artery was higher in the intermittent hypoxia group than that of the blank control group by 29.87 mmHg, and cardiocytes in the former group showed physiological changes, which indicated successful establishment of rat models of OSAS with hypertension. Red particles could be seen in mature rat adipocytes when stained with Oil Red O. Transfection of TLR4 mRNA was significantly suppressed in the TLR4 siRNA group, which didn't happen in the untransfected control group. Rats in the TLR4 siRNA group had significantly reduced expressions of such inflammatory cytokines as interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) and such adipocytokines as visfatin, adiponectin (ADN), and leptin than those in the untransfected control group. RNA interference-mediated silencing of TLR4 gene could regulate occurrence and development of OSAS with hypertension in rats by downregulating expressions of adipocytokines.

Soluble Egg Antigen Activates M2 Macrophages via the STAT6 and PI3K Pathways, and Schistosoma Japonicum Alternatively Activates Macrophage Polarization to Improve the Survival Rate of Septic Mice.

Sepsis is one of the most challenging health problems worldwide. Our previous study showed that chronic schistosoma japonica (SJ) infection might increase serum anti-inflammatory factors to play a protective role, thus improving the survival rate of septic mice. Further research revealed that SJ infection promoted J774A.1 macrophage differentiation into M2 macrophages; suppressed LPS-induced activation of M1 macrophages; up-regulated CD163, IL-10, and TGF-ß1 expression; inhibited TNF-α and iNOS expression; and blocked the effect of LPS-promoted TNF-α and iNOS expression. Furthermore, adoptive transfer of ex vivo programed M2 macrophages significantly increased the survival rate of septic mice. In vitro studies suggested that soluble egg antigen (SEA) from SJ played the same role as worm infection but had no impact on M1 macrophages. SEA reduced LPS-induced TNF-α and iNOS expression, decreased the inhibitory effect of LPS on IL-10 and TGF-ß1 expression, increased STAT6 phosphorylation, and up-regulated PI3K and Akt expression but inhibited SOCS1 expression. When PI3K inhibitors were added, SEA-induced expression of CD163, IL-10, and arg1 might be reduced. Therefore, worm infection has a protective effect in septic mice in which SEA may play a key role via the STAT6 and PI3K pathways. This finding may provide a favorable solution for the treatment of sepsis, especially early cases. J. Cell. Biochem. 118: 4230-4239, 2017. © 2017 Wiley Periodicals, Inc.

Micrometam C Protects against Oxidative Stress in Inflammation Models in Zebrafish and RAW264.7 Macrophages.

Micrometam C is a core of novel marine compound isolated from the mangrove associates Micromelum falcatum. In this study, we investigated the protective effects of micrometam C in inflammation models in the transgenic zebrafish line Tg (corola: eGFP) and RAW264.7 macrophages. We found that micrometam C significantly suppressed the migration of immune cells in tail-cutting-induced inflammation in transgenic zebrafish and reduced lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) in both zebrafish and macrophages. In addition, micrometam C also restored LPS-induced reduction of endogenous antioxidants, such as catalase (CAT), glutathione (GSH) and superoxide dismutase (SOD). The protective effects of micrometam C were in parallel to its inhibition of NADPH oxidase and nuclear factor-kappa-binding (NF-κB) activity. Thus, the present results demonstrate that micrometam C protects against LPS-induced inflammation possibly through its antioxidant property.

[The research of establishing discriminant function for patients with angina pectoris by stepwise analysis based on serum inflammatory factors].

OBJECTIVE: To improve cost-efficiency, discriminant functions in stepwise method was founded for the differential diagnosis of angina pectoris by detecting the serum level of high-sensitivity C-reactive protein (hs-CRP), macrophage migration inhibitory factor (MIF), interleukin-4 (IL-4) and interleukin-10 (IL-10) in patients with stable angina pectoris (SAP) and unstable angina pectoris (UAP). METHODS: Thirty-nine SAP patients and 47 UAP patients were enrolled into the study, while 39 healthy volunteers were enrolled into the controlled group forming the entire set of training samples. The serum levels of hs-CRP, MIF, IL-4 and IL-10 were measured by enzyme linked immunosorbent assay (ELISA). Data was analyzed by software to define discriminant functions in the ways of "entering" and "stepwise". Both functions were evaluated by the results of validation. RESULTS: By the way of "enter independent together", the following discriminant functions were defined based on the data of training samples' age, hs-CRP, MIF, IL-4, IL-10: healthy control group =-129.858 + 2.869×age -2.451×hs-CRP + 1.393×MIF + 6.001×IL-4 + 4.848×IL-10; SAP group=-161.037 + 2.896×age-2.022×hs-CRP + 1.662×MIF + 6.703×IL-4 + 6.287×IL-10; UAP group=-199.087 + 2.468×age-1.440×hs-CRP + 3.404×MIF-13.875×IL-4 + 7.752×IL-10. Retrospective validation showed 4.8% of total miss-grouping, while cross-validation showed 5.6% of total miss-grouping. By the way of "stepwise", the above data was screened by software and training samples' age, MIF and IL-10 were suggested to define the following functions: healthy control group = - 125.218 + 2.659 × age + 0.599×MIF + 5.040 × IL-10; SAP group=-157.864 + 2.721×age + 1.008×MIF + 6.468×IL-10; UAP group=- 197.327 + 2.360×age + 2.932×MIF + 7.640×IL-10. Both retrospective and cross validation showed 6.4% of total miss-grouping. Both sets of discriminant functions had the same efficiency (100%) for differential diagnosis of SAP and UAP. CONCLUSION: The discriminant functions based on samples' age, MIF and IL-10, which were screened and suggested by stepwise method, may contribute to the differential diagnosis of atypical SAP and UAP, and therefore demonstrate better cost-efficiency.

[Expression and effects of microRNA-155 in the livers of septic mice].

OBJECTIVE: To evaluate the effects of microRNA-155 (miR-155) on liver injury in mice with sepsis. METHODS: One hundred and twenty BALB/c mice were randomly divided into two groups of equal number according to random number table. Sepsis was induced by intraperitoneal injection of lipopolysaccharide (LPS,20 mg/kg). The mice were sacrificed at the time-points of 0, 2, 6, 12, 24, 48 hours. Blood and liver tissue were collected, and the levels of tumor necrosis factor- α (TNF- α ), interleukin (IL-6, IL-10) in serum and liver homogenate and alanine transaminase (ALT) in serum were determined by enzyme linked immunosorbent assay(ELISA). The injury of liver tissue was evaluated by histopathology. The expression of miR-155 in liver tissue was assessed by fluorescent quantitation reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The levels of TNF- α , IL-6 and IL-10 in serum and liver homogenate of septic mice increased with passage of time, and then the levels of TNF-α and IL-6 lowered after reaching the peak value, but remained higher than that of control group.TNF-α (ng/L) reached the peak value at 2 hours post-LPS-injection (serum: 1538.46 ± 102.12 vs. 64.52 ± 18.44,liver homogenate: 255.26 ± 41.23 vs. 60.21 + 13.55, both P<0.05). The level of IL-6 (µg/L) reached the peak value at 6 hours post-LPS-injection (serum: 875.33 ± 102.37 vs. 153.72 ± 20.67, liver homogenate: 9.22 + 0.82 vs. 3.35 ±0.36, both P<0.05), and that of IL-10 (ng/L) reached the peak value at 48 hours post-LPS-injection (serum: 520.13 ± 88.52 vs. 23.43 3.01, liver homogenate: 260.12 + 50.38 vs. 16.37 ± 3.71, both P<0.05). There were significant differences in above indexes between septic and control group (all P<0.05). The serum level of ALT (U/L) rose with passage of time, reaching the peak value at 48 hours post-LPS-injection (603.26 + 70.21 vs. 45.84 + 5.64, P<0.05). The values showed significant differences between septic and control group (P<0.05). A large number of leucocytic infiltration was found in liver. Hepatic tissue showed architectural distortion. Hepatocyte vacuolation and nodular necrosis were obvious at 12 hours post-LPS-injection. Relative expression of miR-155 was found to be increased at 2 hours post-LPS-injection, reaching its peak value at 12 hours post-LPS-injection [(72.96 ± 9.34)-fold of control group, P<0.05]. CONCLUSION: The increase in miR-155 expression might play an important role in the mechanism of liver injury during sepsis.

The expression of oxidative stress genes related to myocardial ischemia reperfusion injury in patients with ST-elevation myocardial infarction.

BACKGROUND: We aimed to investigate the gene expression of myocardial ischemia/reperfusion injury (MIRI) in patients with acute ST-elevation myocardial infarction (STEMI) using stress and toxicity pathway gene chip technology and try to determine the underlying mechanism. METHODS: The mononuclear cells were separated by ficoll centrifugation, and plasma total antioxidant capacity (T-AOC) was determined by the ferric reducing ability of plasma (FRAP) assay. The expression of toxic oxidative stress genes was determined and verified by oligo gene chip and quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, gene ontology (GO) enrichment analysis was performed on DAVID website to analyze the potential mechanism further. RESULTS: The total numbers of white blood cells (WBC) and neutrophils (N) in the peripheral blood of STEMI patients (the AMI group) were significantly higher than those in the control group (WBC: 11.67±4.85 ×109/L vs. 6.41±0.72 ×109/L, P<0.05; N: 9.27±4.75 ×109/L vs. 3.89±0.81 ×109/L, P<0.05), and WBCs were significantly associated with creatine kinase-myocardial band (CK-MB) on the first day (Y=8.945+0.018X, P<0.05). In addition, the T-AOC was significantly lower in the AMI group comparing to the control group (12.80±1.79 U/mL vs. 20.48±2.55 U/mL, P<0.05). According to the gene analysis, eight up-regulated differentially expressed genes (DEGs) included GADD45A, PRDX2, HSPD1, DNAJB1, DNAJB2, RAD50, TNFSF6, and TRADD. Four down-regulated DEGs contained CCNG1, CAT, CYP1A1, and ATM. TNFSF6 and CYP1A1 were detected by polymerase chain reaction (PCR) to verify the expression at different time points, and the results showed that TNFSF6 was up-regulated and CYP1A1 was down-regulated as the total expression. GO and kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis suggested that the oxidative stress genes mediate MIRI via various ways such as unfolded protein response (UPR) and apoptosis. CONCLUSIONS: WBCs, especially neutrophils, were the critical cells that mediating reperfusion injury. MIRI was regulated by various genes, including oxidative metabolic stress, heat shock, DNA damage and repair, and apoptosis-related genes. The underlying pathway may be associated with UPR and apoptosis, which may be the novel therapeutic target.

[The protective effect of chronic schistosoma japonica infestation against sepsis in mice].

OBJECTIVE: To preliminarily study the protective effect of chronic schistosoma japonica (SJ) infestation against sepsis in mice and its mechanism. METHODS: BALB/c male mice were used, and the experiment was divided into three parts. Experiment 1: chronic SJ infestation model was reproduced by SJ cercaria inoculation through abdominal skin for 8 weeks. Twenty mice were randomly grouped into normal group (n=10) and SJ group (n=10). The levels of interleukins (IL-4, IL-10),tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in serum were detected by enzyme linked immunosorbent assay (ELISA). Real-time polymerase chain reaction (PCR) was employed to detect the levels of IL-10 mRNA and TNF-αmRNA in abdominal macrophages. This experiment was meant to evaluate immune state in mice with chronic SJ infestation. Experiment 2: lipopolysaccharide (LPS) was intraperitoneally injected to reproduce sepsis model. Thirty mice were randomly grouped into LPS group (n=15) and SJ-LPS group (n=15). The levels of cytokines were determined by ELISA at 0, 24, 48 and 72 hours after LPS injection. This experiment was meant to detect the effect of chronic SJ infestation in mice during the septic process. Experiment 3: two types of sepsis model were reproduced by cecal ligation and puncture (CLP) and LPS injection, respectively. The survival rate of mice with chronic SJ infestation in 72 hours in either type of sepsis was evaluated. RESULTS: Experiment 1: compared with normal group [IL-4 (56.32±8.66) ng/L, IL-10 (48.17±7.23) ng/L], chronic SJ infestation showed an increase in serum IL-4 [(151.35±12.24) ng/L] and IL-10 [(133.22±11.09) ng/L, both P<0.05]. Chronic SJ infestation also resulted in an increase in IL-10 mRNA expression (SJ group 4.46±1.82, normal group 1.52±0.60) and inhibited TNF-α mRNA expression (SJ group 1.61±0.93, normal group 2.32±1.03) in abdominal macrophages (both P<0.05), indicating that macrophages could be differentiated into alternative activated macrophages. Experiments 2 and 3 showed that the levels of serum IL-4 and IL-10 were increased at 0 hour after LPS injection, and then gradually decreased in SJ-LPS group, but the levels were still higher than those in LPS group at 72 hours [IL-4 (ng/L): 92.2±7.6 vs. 41.5±4.5; IL-10 (ng/L): 92.1±7.8 vs. 35.6±4.0, both P<0.05]; the levels of TNF-α and IFN-γ were increased at 24 hours, and then decreased in SJ-LPS group, and the levels were lower than those in LPS group at 72 hours [TNF-α (ng/L): 82.9±5.6 vs. 91.5±5.2; IFN-γ (ng/L): 44.1±4.8 vs. 52.6±4.0, both P<0.05]. Therefore, chronic SJ infestation could improve the survival rate of mice with sepsis induced by CLP or LPS (CLP: 80% vs. 20%, LPS: 70% vs. 30%, both P<0.05). CONCLUSION: Chronic SJ infestation could elevate anti-inflammatory factors in septic mice, thus ameliorating the survival rate, so it has protective effect on mice with sepsis.

[The significance of the changes in expressions of heat shock protein 70 and heme oxygenase-1 in the myocardium in patients died suddenly due to acute myocardial infarction].

OBJECTIVE: To investigate the changes and its clinical significance of the expressions of the mRNA and protein of heat shock protein 70 (HSP70) and heme oxygenase-1 (HO-1) genes in the myocardium of acute myocardial infarction (AMI) areas in patients who died suddenly due to AMI. METHODS: Specimens of myocardial tissue at infarct site was obtained during autopsy from 18 patients who died suddenly due to AMI, and they were enlisted as study group, and that of myocardial tissue from 17 normal hearts of patients died rapidly after accidents were as control group. The levels of mRNA expression of HSP70 and HO-1 genes were measured in all the specimens by reverse transcription-polymerase chain reaction (RT-PCR) using cDNA samples, and the levels and locations of HSP70 and HO-1 protein expressions in myocardial cells of all specimens were examined by immunohistochemistry (IHC), and the pathological changes in the myocardial tissue were observed. RESULTS: The expressions of HSP70 mRNA (0.841+/-0.058), HO-1 mRNA (0.918+/-0.161) and HSP70 protein (3 556.68+/-574.19), HO-1 protein (4 336.68+/-865.30) in the myocardium of the AMI areas in the study group were significantly higher than those in control group (the expressions of HSP70 mRNA: 0.105+/-0.034, HO-1 mRNA: 0.086+/-0.053, HSP70 protein: 289.21+/-68.51, HO-1 protein: 1 556.78+/-506.26, all P<0.01), and a weak expression of HSP70 and HO-1 protein was found in the cardiocytes of the AMI area in study group. In the control group, HSP70 protein expression was negative in the cardiocytes, and there was a weak expression of HO-1 protein in the cardiocytes. There was a significant positive correlation between the expressions of mRNA and protein of HSP70 and HO-1 genes at the cardiocytes of patients died suddenly of AMI in study group (r(1)=0.865, r(2)=0.816, both P<0.01). CONCLUSION: HSP70 and HO-1 probably were both involved in the pathological and physiological processes of AMI, while HO-1 may express in cardiocytes, but it is more abundant in the cardiocytes in the AMI area.

[Polymorphism and structural analysis of suppressor of cytokine signaling-1 of rat].

OBJECTIVE: To identify the suppressor of cytokine signaling-1 (SOCS-1) of rat from the amplified gene with the help of bioinformatics to predict the deduced protein's structure and function in order to lay the foundation for further theoretical study. METHODS: The full-length rat SOCS-1 gene was amplified and identified from the GeneBank Nucleotide database, and the corresponding structure and function of its deduced protein was predicted by the bioinformatics analyzing tools online and the complicated bioinformatics software package Vector NTI suite 8.0, meanwhile the molecular cladogram was reconstructed. RESULTS: Two sequences were obtained by polymerase chain reaction (PCR) amplification of different SOCS-1 gene. The gene was comprised of 639 base pairs in the length, deduced 212 amino acids (aa), contained a SOCS box (aa172-aa208), a SH2 domain (aa80-aa155) and a nuclear localization sequence (aa160-aa174). The primary structure contained two linear B cell epitopes, all of them were on the surface of the protein and far away from the spatial structure. CONCLUSION: SOCS-1 gene has a polymorphism, its conservative SH2 region and SOCS box are related to its inhibitory effect on the signal transduction pathway. The nucleic localization sequence may affect other nuclear transduction factors. The B cell linear epitopes may be a candidate of immunodiagnosis with promising prospects.

[Study on the relationship between the expression of SOCS-1/3 in the peripheral blood mononuclear cells in patients with multiple organ dysfunction syndrome and their prognosis].

OBJECTIVE: To observe the mRNA and protein expression of suppressor of cytokines signaling protein 1/3 (SOCS-1/3) in the peripheral blood mononuclear cells (PBMCs) in patients with multiple organ dysfunction syndrome (MODS), and to explore the relationship between the SOCS expression and the patients' prognosis. METHODS: Peripheral blood specimens of 32 patients with MODS and 24 healthy volunteers (controls) were collected and the PBMCs were isolated by centrifugation and Ficoll-Hypaque sedimentation. The mRNA of SOCS-1/3 was determined by reverse transcription-polymerase chain reaction (RT-PCR). And the protein content of SOCS-1/3 was determined by Western blotting. The relationship between the SOCS expression and the patients' prognosis was analyzed. RESULTS: There was no significant difference in the SOCS-1 mRNA expression between control group and MODS group (0.526+/-0.044 vs. 0.466+/-0.047, P>0.05). The SOCS-1 expression of MODS group was significantly higher than that of control group (0.814+/-0.045 vs. 0.479+/-0.021, P<0.05). In the MODS group, the SOCS-1 mRNA expression and protein content of dead patients were significantly lower than those of survived patients (mRNA 0.487+/-0.032 vs. 0.532+/-0.028, protein 0.787+/-0. 029 vs. 0.838+/-0.040, both P<0.05). There was significant negative correlation between the SOCS-1 mRNA expression and the MODS score (r1=-0.731,P<0.01). There was also significant negative correlation between the SOCS-1 content and the MODS score (r2=-0.526,P<0.01). The SOCS-3 mRNA expression of MODS group was higher than that of control group (0.993+/-0.415 vs. 0.461+/-0.046, P<0.05). The SOCS-3 content in the PBMCs of MODS groups was significant higher than that of control group (0.458+/-0.033 vs. 0.403+/-0. 024, P<0.05). In the MODS group, the SOCS-3 mRNA expression and protein content of dead patients were significant higher than those of survived patients (mRNA 1. 245+/-0.408 vs. 0.805+/-0.326, protein 0.486+/-0.021 vs. 0.425+/-0.016, both P<0.05). There was positive correlation between the SOCS-3 mRNA expression and the MODS score but the correlation was not significant (r=0. 468, P>0.05). And there was significant positive correlation between the SOCS-3 content and the MODS score (r=0.783, P<0.01). CONCLUSION: SOCS-1 may protect the tissue from further damage while SOCS-3 may cause tissue damage indirectly. The detection of SOCS-1/3 may help to predict the prognosis of MODS.

[Role of nuclear factor-KappaB in endothelial injury in acute myocardial infarction].

OBJECTIVE: To examine the change in nuclear factor-KappaB (NF-KappaB) activity, tumor necrosis factor-alpha (TNF-alpha) and soluble thrombomodulin (sTM) levels at different time following reperfusion in acute myocardial infarction (AMI), and to identify the role of ischemia/reperfusion after ischemia in injury to endothelial cells and its relevant mechanism. METHODS: AMI group included 8 randomly selected patients with AMI, and a normal control group (n=8) composing individuals who underwent health check. NF-KappaB activity in monocytes was determined by electrophoretic mobility shift assays (EMSA). The level of TNF-alpha was measured by radio-immunity and sTM was measured by enzyme linked immunosorbent assay (ELISA). RESULTS: The NF-KappaB activity, TNF-alpha and sTM levels raised dramatically at 0.5 hour after reperfusion, reaching peak at 1 hour and declined gradually at 3, 12 and 24 hours. The levels of all the determined parameters at every time point were significantly higher than that of normal control group, and their levels at 1 hour were significantly higher than that at 24 hours (all P<0.05). There was a positive correlation between the NF-KappaB activity and the levels of TNF-alpha and sTM (all P<0.05). CONCLUSION: These results indicate that NF-KappaB is activated and the levels of TNF-alpha and sTM rise significantly after reperfusion in AMI. The activation of NF-KappaB maybe one of the most important pathogenic mechanism of endothelial injury.

[Study of the effect of p53 and Bcl-2 protein expression in myocardial apoptosis in septic rats].

OBJECTIVE: To explore the significance of p53 and Bcl-2 gene in myocardial apoptosis in septic rats. METHODS: Adult male Sprague-Dawley (SD) rats were randomly divided into normal control group (n=6), sham operation group (n=6) and cecal ligation and puncture (CLP) group (n=24). The sepsis model was reproduced by CLP. Rats in CLP group were divided into four subgroups according to the time points of 3, 9, 12, and 24 hours after operation, with 6 rats in each subgroup, and their hearts were examined according to the experimental protocol. Myocardial apoptosis was detected with electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique. Bcl-2 and p53 protein expression was measured by immunohistochemistry method. RESULTS: Myocardial apoptosis index (AI) in septic rats were increased, higher than that in normal control group [(1.500+/- 0.141)%] and sham operation group [(1.567+/-0.258)%, all P<0.05]. The myocardial AI peaked at 12 hours [(55.633+/-2.073)%], and lowered at 24 hours [(33.683+/-2.070)å]. The expression level of p53 protein in CLP group was lowest at 3 hours [(13.817+/-0.964)%], peaked at 12 hours [(80.567+/-5.055)%], and higher than that in normal control group [(0.617+/-0.232)%] and sham operation group [(0.600+/-0.297)%, all P<0.05 ]. The trend was parallel with that of the results of TUNEL. However, the expression level of Bcl-2 protein peaked at 3 hours [(31.650+/-1.799)%], and was lowest at 12 hours [(0.650+/-0.308)%], and lower than that in normal control group [(47.017+/-0.691)%] and sham operation group [(46.817+/-0.567)%, all P<0.05]. The trend was opposite with that of the results of TUNEL. CONCLUSION: Myocardial apoptosis may be a mechanism of the pathogenesis of myocardial injury in sepsis. The change in the modulating genes p53 and Bcl-2 of apoptosis may serve as indicators of myocardial injury. p53 and Bcl-2 gene may be used as an interventional means in sepsis to change its outcome.

[The expression and clinical implication of advanced oxidized protein products in patients with multiple organ dysfunction syndrome].

OBJECTIVE: To investigate the expression and clinical implication of advanced oxidized protein products (AOPP) in patients with multiple organ dysfunction syndrome (MODS). METHODS: Serum concentrations of C-reactive protein (CRP) and AOPP were determined in 180 patients with systemic inflammatory response syndrome (SIRS) or MODS (90 patients, respectively). The acute physiology and chronic health evaluation III (APACHE III) scoring system was applied to assess severity of patients' condition. The contents of serum CRP and AOPP in MODS group, SIRS group and normal control group, and also in survivor and dead patients in MODS group were determined and compared. The correlation between CRP and AOPP levels and the correlation between AOPP levels and severity of MODS were also observed. Ninety healthy volunteers who matched with study subjects in age and gender comprised the normal control group. RESULTS: The CRP [(22.22+/-4.32) mg/L] and AOPP [(130.66+/-18.08) micromol/L] levels in patients with MODS were significantly higher than those in normal control group [(2.38+/-0.89) mg/L and (33.20+/-5.32) micromol/L, respectively] and SIRS group [(5.32+/-1.22) mg/L and (48.58+/-6.03) micromol/L, respectively, all P < 0.05], and were positively correlated with APACHE III scores [(98.66+/-20.87) scores] of the patient (r1 = 0.469, r2 = 0.528, both P < 0.01). However, there was no significant difference between SIRS group and normal control group. The CRP and AOPP levels were found to be significantly higher in the patients who eventually died (47 cases) as compared to those in the patients who survived (43 cases, both P < 0.05). Positive correlations were noted between AOPP and CRP level (r = 0.448, P < 0.01). The serum concentrations of CRP and AOPP levels were elevated with the increase of the number of failed organs in MODS patients(all P < 0.05). CONCLUSION: The data show that AOPP might participate in the process of pathogenesis of MODS. The serum AOPP level may be taken as a diagnostic and prognostic indicator for MODS.

[E1A gene transfection of human undifferentiated thyroid cancer cell line HTC/3 by nanoparticles].

OBJECTIVE: To prepare nanoparticles containing E1A gene and observe the efficiency and feasibility of transfecting E1A gene into human undifferentiated thyroid cancer cell line HTC/3. To examine the sensitivity of transgene cells to X-ray and X-ray-induced apoptosis in those cells. METHODS: Nanoparticle-DNA complex was prepared with PLGA coating adenoviral early expression gene E1A, and the package efficiency, release progress in vitro, and size of the complex were determined. The nanoparticle-DNA was transfected into the HTC/3 cells. Lipofectamine was used to transfect E1A gene as a control. RT-PCR was used to examine E1A gene mRNA expression in the transfected cells. The survival ratio of HTC/3-E1A and control cells, and the growth inhibition ratio induced by different doses of X-ray in HTC/3-E1A cells were examined by MTT assay. The apoptosis in HTC/3-E1A cells induced by 2 Gy X-ray iradiation was examined by flow cytometry and DNA electrophoresis. RESULTS: The package efficiency, release progress in vitro, and size of the nanoparticle-DNA complex were 0.78%, 18 days, and 150-280 nm, respectively when transfected the plasmid at the same level, the nanoparticle group got more positive transgene cell clones than that in lipofectamine group, with a statistically significant difference (P < 0.05). RT-PCR showed that transgenic cells from both nanoparticle-DNA and lipofectamine groups had E1A gene mRNA expression. The HTC/3-E1A cells grew slowly, and their doubling time was prolongated (1.44 times in comparison with that in parental cells). According to IC50, the sensitivity of HTC/3-E1A cells to X-ray was improved 2.9 and 2.8 times, respectively, in comparison with that in HTC/3-Vect and HTC/3 cells. The ratio of subG0/G1 phase of HTC/3-E1A cells was significantly higher than that in HTC/3-Vect and HTC/3 cells (P < 0.01). The ratio of S phase of HTC/3-E1A cells was significantly lower than that in HTC/3-Vect and HTC/3 cells (P < 0.01). A typical DNA ladder pattern of apoptosis in HTC/3-E1A cells was observed by electrophoresis, but not found in HTC/3-Vect and HTC/3 cells. CONCLUSION: A nanoparticle-DNA complex has been successfully prepared, and it may carry a foreign gene into cells. The sensitivity of HTC/3-E1A cells to X-ray is significantly improved. Moreover, apoptosis is induced by x-ray in the E1A gene-transfected cells.

[Study on the expression of suppressor of cytokine signaling-1 in liver and spleen of septic mice].

OBJECTIVE: To investigate the change in suppressor of cytokine signaling-1 (SOCS1) gene in the liver and the spleen of septic mouse, and to find out its probable mechanisms of action in sepsis. METHODS: Cecal ligation and puncture (CLP) was adopted to reproduce sepsis model. The liver and the spleen tissues were harvested and RNA and protein were respectively extracted. The contents of the regulatory genes SOCS1 mRNA were determined by reverse transcription-polymerase chain reaction (RT-PCR) and regulatory content of protein was detected by Western blotting. The SPSS statistics software was adopted to calculate the correlation. RESULTS: The expressions of SOCS1 on gene and protein in the liver were markedly upregulated at 6 th hour. The gene expression peaked at the 24 th hour (P<0.05).The expression of protein was persistently high. However, the expression of SOCS1 was only detected in the spleen, and it obviously rose in strength with the passage of time, and it remained in a high level. By statistical analysis, positive correlations were found between the gene and protein expressions of SOCS1 (y=0.110+/-5.765 x 10(-3) x, r=0.837, F=93.309, P<0.01). CONCLUSION: CLP induced sepsis can induce the up-regulation of the expressions of SOCS1, indicating that SOCS1 play important role in the change in immune system in sepsis. They may be used to intervene sepsis so as to improve the outcome of sepsis.

Downregulated SOCS1 expression activates the JAK1/STAT1 pathway and promotes polarization of macrophages into M1 type.

Macrophage polarization is flexible, and involves in different signaling pathways and various transcription factors. Suppressor of cytokine signaling (SOCS) is an important inhibitor of cytokine signaling pathways and also a key physiological regulator for natural and acquired immunity systems. Following transfection of SOCS1 short hairpin (sh)RNA into mouse macrophage cells, reverse transcription­quantitative polymerase chain reaction demonstrated that the mRNA levels of Janus kinase (JAK)1 and signal transducer and activator of transcription (STAT)1 increased significantly. In addition, western blotting indicated that JAK1, STAT1 and p­STAT1 expression was significantly enhanced. Fludarabine can inhibit phosphorylation of STAT1 and SOCS1 expression. When fludarabine was added and SOCS1 shRNA was transfected, the inhibition of fludarabine was weakened, and p­STAT1 expression was upregulated. Flow cytometry detection indicated that, following the downregulation of SOCS1 expression, M1­type cells significantly increased, but the proportion of M2­type cells did not change significantly. Fludarabine can reduce the effect of SOCS1 shRNA on promoting M1­type cell polarization, and macrophages can polarize into both M1 and M2 phenotypes. Further ELISA results presented that, when downregulating SOCS1 expression, interleukin (IL)­4 and IL­10 expression was both downregulated, and tumor necrosis factor (TNF)­α and interferon (IFN)­Î³ expression was significantly upregulated. When adding fludarabine or injecting with the traditional Chinese medicine Xuebijing, IL­4 and IL­10 expression was both significantly upregulated, and TNF­α and IFN­Î³ expression was significantly downregulated. When adding fludarabine and downregulating SOCS1, IL­4, IL­10, TNF­α and IFN­Î³ expression presented no significant changes. The above results indicated that, when SOCS1 expression is downregulated, it will activate the JAK1/STAT1 pathway, and thereby promote the polarization of macrophages into M1 type. The findings are of great importance for understanding occurrence, development and treatment of various immune­related diseases.

A meta-analysis of cardiopulmonary resuscitation with and without the administration of thrombolytic agents.

OBJECTIVE: To pool data on the role of thrombolytic agents in cardiopulmonary resuscitation (CPR) and evaluate the efficacy and safety of thrombolysis. MATERIALS AND METHODS: The clinical studies in MEDLINE database from 1966 to August 2004 that studied the efficacy and safety in CPR with and without treatment with thrombolytic agents were assessed by a meta-analysis performed to evaluate the effect of the treatment. RESULTS: A total of eight papers evaluating the effect of thrombolysis in CPR were identified. This meta-analysis showed that thrombolytic agents significantly improved the rate of return of spontaneous circulation, 24 h survival rate, survival to discharge and long-term neurological function in patients treated with CPR (p < 0.01). However, the patients receiving thrombolysis had a risk of severe bleeding (p < 0.01). CONCLUSION: Thrombolytic agents during CPR can improve the survival rate to discharge and neurological function.

[Preparation and identification of polyclonal antiserum against heat shock protein 72].

OBJECTIVE: To express fusion protein of histamine (His) and human heat shock protein 72 (hHSP72) in Escherichia coli (E. coli), and to prepare hHSP72 antiserum in rabbit. METHODS: hHSP72 gene was inserted into pPROEX-1. The recombinant vector was identified by restriction endonuclease digestion analysis and sequence. Fusion protein His-hHSP72 was expressed in E. coli under isopropyl-beta-D-thiogalactoside (IPTG) induction. The rabbit antibody against His-hHSP72 was prepared by using purified His-hHSP72 protein as immunogen, and the specificity and sensitivity of polyclonal antibody were identified by Western blot. RESULTS: The restriction endonuclease digestion analysis of recombinant plasmid and sequence demonstrated that the hHSP72 gene had been exactly inserted into pPROEX-1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the relative molecular mass of the fusion protein was about 73 ku. Western blot result proved that the rabbit polyclonal antiserum could fuse with over 20 ng hHSP72 protein when diluted to 1:100,000. CONCLUSION: The polyclonal antibody against hHSP72 can be prepared in E. coli, it is a new reagent with high specificity and sensitivity for the research of hHSP72.

[Study of apoptosis of peripheral blood mononuclear cells in patients with multiple organ dysfunction syndrome].

OBJECTIVE: To study apoptosis of peripheral blood mononuclear cells (PBMCs) in patients with multiple organ dysfunction syndrome (MODS) and its associated gene Bcl-2 and p53 expression. METHODS: Twenty-five patients with MODS and 18 healthy volunteers were enrolled for the study. Flow cytometry assay, electron microscopy, acridine orange-ethidium bromide staining and fluorescence microscopy, DNA agarose gel electrophoresis were used to identify and quantify apoptosis of PBMCs. Reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify Bcl-2 mRNA and p53 mRNA expression. RESULTS: Typical morphological features of apoptotic PBMCs were observed in the patients. The apoptosis rate in MODS group was (25.4+/-9.2)%, and it was higher than that of control group (15.9+/-6.8)% (P<0.01). The number of apoptotic cells was (1.040+/-0.096)/high power field in MODS group, and it was higher than that in control group (0.235+/-0.028)/high power field (P<0.05). Bcl-2 mRNA expression of PBMCs in patients was significantly lower than that of healthy volunteers (0.11+/-0.09 vs. 0.19+/-0.06, P<0.05), while p53 mRNA expression was higher in patients than that of healthy volunteers (0.45+/-0.09 vs. 0.25+/-0.12, P<0.05). CONCLUSION: PBMCs apoptosis in patients with MODS is increased abnormally. The expression of Bcl-2 mRNA in patients is decreased while p53 mRNA expression is increased. The results suggest that abnormal apoptosis of monocytes as well as abnormal expression of apoptosis associated genes occur in patients with MODS.