Mingshi Yang

HSF1 regulates expression of G-CSF through the binding element for NF-IL6/CCAAT enhancer binding protein beta

Heat shock factor 1 (HSF1) is the major heat shock transcription factor and plays an essential role in mediating the cellular response to physiological and environmental stress. We found that LPS-induced expression of the granulocyte-colony stimulating factor (G-CSF) gene was upregulated in HSF1 knock-out (HSF1−/−) mice using a gene array. In order to determine whether and how HSF1 regulates the induced expression of G-CSF, mRNA, and protein levels of G-CSF were detected by Northern blotting and ELISA, the promoter of G-CSF was analyzed with an online transcription element search system and the transcriptional activity of the G-CSF promoter was analyzed by EMSA and a reporter gene assay. The results showed that transcription and protein secretion of G-CSF induced by LPS are both inhibited by HSF1. Three high affinity binding sites for NF-IL6/CCAAT enhancer binding protein beta, but no heat shock element, were identified in the core promoter of G-CSF. The DNA-binding capability of NF-IL6 to the G-CSF promoter was reinforced by LPS but not influenced by heat shock or HSF1. However, HSF1 was observed to bind to the binding sites of NF-IL6 in the G-CSF promoter. The transcriptional activity of the G-CSF promoter was enhanced by LPS or NF-IL6 and inhibited by HSF1 in a dose dependent manner. We conclude that HSF1 regulates expression of G-CSF through binding to the NF-IL6-binding element.

Protective effect of pioglitazone on sepsis-induced intestinal injury in a rodent model

BACKGROUND Pathogenesis and treatment of inflammatory gut barrier failure is an important problem in critical care. In this study, we examined the role of pioglitazone, an agonist of peroxisome proliferator-activated receptor gamma, in gut barrier failure during experimental peritonitis in rats. MATERIALS AND METHODS Male rats were randomly divided into three groups as follows: sham, sepsis, and sepsis + pioglitazone. Sepsis was achieved by means of the cecal ligation and puncture (CLP). Pioglitazone was administered intraperitoneally (10 mg/kg/d) for 7 d before the experiment. Animals were killed at 24 h or followed 72 h for survival. The tissue level of tumor necrosis factor-α, interleukin-6, superoxide dismutase, malondialdehyde, and myeloperoxidase was measured. Intestinal mucosa injury was assessed histologically. The plasma fluorescein isothiocyanate-dextran, D-lactic acid, and intestinal diamine oxidase were determined to evaluate the permeability and integrity of intestinal mucosal epithelium. Vena cava blood and tissue samples were used to monitor bacterial translocation. RESULTS Intestinal inflammation, oxidize stress, neutrophil infiltration, morphology injury, and impaired permeability of the small intestine in the CLP group were found more severe than those in the sham group. Application of pioglitazone not only minimized all the indicators of intestinal injury and barrier failure but also improved the survival of septic rats induced by CLP. CONCLUSIONS Our novel findings suggest that pioglitazone could protect against intestinal injury and maintain intestinal barrier integrity and might be a useful strategy to ameliorate intestinal failure in polymicrobial sepsis.

ANGIOGENESIS INHIBITOR ENDOSTATIN PROTECTS MICE WITH SEPSIS FROM MULTIPLE ORGAN DYSFUNCTION SYNDROME.

ABSTRACT Endostatin is an endogenous inhibitor of vascular endothelium. It can inhibit endothelial cell migration, proliferation, and vascular angiogenesis and is mainly used for anticancer therapy. We have previously found that endostatin is an important node protein in the pathogenesis of sepsis. However, its impacts on sepsis have not yet been reported. We established a septic mouse model using cecal ligation and puncture (CLP) and gave the mice either endostatin or placebo (saline). The effects of endostatin on serum enzyme, Evans blue leakage, lung wet-to-dry weight ratio, and cytokine (tumor necrosis factor &agr;, interleukin 1&bgr; [IL-1&bgr;], and IL-6) production were assessed. Survival rates were observed for up to 3 days. In addition, we examined the effects of endostatin on serum vascular endothelial growth factor A (VEGF-A), VEGF-C, and pathological changes and scores of lung tissues as well as the phosphorylation of JNK, p38, and ERKl/2 proteins in lung tissues of mice with sepsis. We found that endostatin can increase the survival of septic mice in a time- and dose-dependent manner probably by reducing multiorgan dysfunctions shown by serum indicators, morphologic changes, Evans blue leakage, wet-to-dry weight ratio, and inflammation of lung tissues. In addition, endostatin could reduce serum tumor necrosis factor &agr;, IL-1&bgr;, IL-6, and VEGF-C levels in septic mice as well as inhibit phosphorylation of p38 and ERK1/2 in lung tissues of septic mice. This is the first study demonstrating the protective effect of endostatin on sepsis and its possible underlying mechanisms from the aspects of inhibiting inflammatory responses, blocking VEGF receptor, attenuating VEGF-C expression, and reducing vascular permeability. Overall, the study revealed the potential protect role for endostatin in the treatment of sepsis.

Identification of potential biomarkers by serum proteomics analysis in rats with sepsis.

ABSTRACT This study was aimed to find new biomarkers for diagnosis and prediction of prognosis of sepsis. Serum samples from nonsurvivor, survivor, and control groups were obtained at 12 h after the induction of sepsis and labeled with isobaric tags (iTRAQ) and then analyzed by two-dimensional liquid chromatography and tandem mass spectrometry. Protein identification and quantification were obtained using mass spectrometry and the ProteinPilot software. Bioinformatics annotation was performed by searching against the PANTHER database. Enzyme-linked immunosorbent assays were used to further confirm the protein identification and differential expression. A logistic regression was then used to screen the index set for diagnosis and prognosis of sepsis. We found that 47 proteins were preferentially elevated in septic rats (both nonsurvivors and survivors) compared with the control rats, and 28 proteins were preferentially elevated in the NS rats as compared with the S group. Several biomarkers, such as multimerin 1, ficolin 1, carboxypeptidase N (CPN2), serine protease 1, and platelet factor 4, were tightly correlated with the diagnosis of sepsis. Logistic regression analyses established multimerin 1, pro–platelet basic protein, fibrinogen-&agr;, and fibrinogen-&bgr; for prognosis of sepsis.

Interleukin-1 Receptor 2: A New Biomarker for Sepsis Diagnosis and Gram-negative/gram-positive Bacterial Differentiation.

ABSTRACT This study was undertaken to explore the value of interleukin-1 receptor 2 (IL1R2) as a novel potential biomarker for diagnosis of sepsis and discrimination of gram-negative (G−)/gram-positive (G+) bacterial sepsis. The study was performed in Kunming mice and septic patients. Inactive Escherichia coli or Staphylococcus aureus were used to stimulate Kunming mice (109 CFU/kg). In clinical study, septic patients with different pathogen infection were studied, and healthy volunteers and patients with systemic inflammatory response syndrome without definite infection were enrolled as control. IL1R2 transcriptions of human subjects’ peripheral leukocytes were measured by real-time quantitative polymerase chain reaction assay. IL1R2 serum concentrations of mice and human subjects were measured by enzyme-linked immunosorbent assay. The value of IL1R2 as a biomarker was compared with procalcitonin (PCT), C-reactive protein (CRP), and Acute Physiology and Chronic Health Evaluation II (APACHE II). The results showed that IL1R2 expression was upregulated in mice treated with inactive Escherichia coli and septic patients. The elevation of serum IL1R2 was more significant in septic patients infected by Escherichia coli or G− bacteria than in those infected by Staphylococcus aureus or G+ bacteria. For sepsis diagnosis and G−/G+ bacterial sepsis discrimination, serum IL1R2 was more sensitive and specific than the traditional biomarkers such as PCT, CRP, and APACHE II as shown by the receiver operating characteristic curves. It was suggested that IL1R2 was a potential biomarker for diagnosis and G−/G+ bacterial differentiation in sepsis.

Severe multiple organ injury in HSF1 knockout mice induced by lipopolysaccharide is associated with an increase in neutrophil infiltration and surface expression of adhesion molecules

We have reported previously that HSF1 is essential in protection against the lethal systemic inflammation induced by LPS. However, the mechanism by which HSF1 protects against LPS‐induced systemic inflammation remains unknown. In this study, HSF1−/− mice were subjected to endotoxemia by a bolus injection of LPS (10 mg/kg, i.p.). The serum levels of LDH, BUN, and transaminase (ALT and AST) were measured. PMN infiltration in lung, liver, and kidney tissues after endotoxemia was observed with immunohistochemistry. Comparing with the WT control, LPS administration induced more severe multiple organ dysfunction and lower survival rates in the HSF1−/− mice. Moreover, PMN infiltration into lungs, liver, and kidneys in HSF1−/− mice was more than that in the WT mice. The augmented tissue PMN infiltration in HSF1−/− mice was associated with their enhanced adhesive properties to endothelium in vivo. In addition, HSF1−/− caused greater surface expression of PSGL‐1 and CD11b on the PMN surface after LPS treatment. These findings suggested that HSF1 alleviated LPS‐induced multiple organ injury in mice by suppressing the surface expression of adhesion molecules on PMNs and subsequent infiltration of PMNs in tissues.

Naringin attenuates MLC phosphorylation and NF-κB activation to protect sepsis-induced intestinal injury via RhoA/ROCK pathway

BACKGROUND Sepsis is commonly associated with excessive stimulation of host immune system and result in multi-organ failure dysfunction. Naringin has been reported to exhibit a variety of biological effects. The present study aimed to investigate the protective effect of naringin on sepsis-induced injury of intestinal barrier function in vivo and in vitro. METHODS Mice were randomly divided into 4 groups named sham (n = 20), CLP + vehicle (n = 20), CLP + NG (30 mg/kg) (n = 20) and CLP + NG (60 mg/kg) (n = 20) groups. Sepsis was induced by cecal ligation and puncture (CLP). H&E staining and transmission electron microscopy (TEM) were performed to observe intestinal mucosal morphology. ELISA was used to determine the intestinal permeability and inflammatory response in vivo and in vitro. Western blot and RhoA activity assay were performed to determine the levels of tight junction proteins and the activation of indicated signaling pathways. MTT assay was used to determine cell viability. RESULTS Naringin improved survival rate of CLP mice and alleviated sepsis-induced intestinal mucosal injury. Furthermore, naringin improved impaired intestinal permeability and inhibited the release of TNF-α and IL-6, while increased IL-10 level in CLP mice and lipopolysaccharide (LPS)-stimulated MODE-K cells in a dose-dependent manner. Naringin increased the expression of tight junction proteins ZO-1 and claudin-1 via RhoA/ROCK/NF-κB/MLCK/MLC signaling pathway in vivo and in vitro. CONCLUSION Naringin improved sepsis-induced intestinal injury via RhoA/ROCK/NF-κB/MLCK/MLC signaling pathway in vivo and in vitro.

Intraintestinal administration of ulinastatin protects against sepsis by relieving intestinal damage

BACKGROUND Intravenous administration of ulinastatin (UTI), a broad spectral protease inhibitor, has been used on an experimental basis with severe sepsis patients in Asia. However, the effects of intraintestinal administration of UTI on intestinal and multiple organ damage in sepsis have not been reported. MATERIALS AND METHODS In this study, we established a sepsis model in rats using cecal ligation and puncture and compared the effects of intraintestinal administration of UTI through an artificial fistula of duodenum and intraperitoneal administration of UTI on the pathophysiological changes of sepsis. RESULTS It was found that intraintestinal administration of UTI (1) significantly improved the survival of septic rats, (2) significantly reduced the serum levels of tumor necrosis factor-α, interleukin-1β, interleukin-6 as well as intestinal injury biomarkers diamine oxidase, D-lactic acid, and fluorescein isothiocyanate-dextran 4, and (3) significantly reduced intestinal microscopic and ultrastructural damage of septic rats. In addition, the protective effects of intraintestinal administration of UTI were significantly better than those of intraperitoneal administration of UTI. CONCLUSIONS Overall, the present study for the first time revealed that intraintestinal administration of protease inhibitor UTI could reduce systemic inflammatory responses and multiple organ dysfunction in rats with sepsis by inhibiting autodigestion of intestinal wall due to proteases and provided new research ideas and experimental evidences for treatment of sepsis by intraintestinal administration of UTI.

Expression of microRNA-23b in patients with sepsis and its effect on leukocytes and the expression of E-selectin and ICAM-1

The expression of microRNA-23b in peripheral blood leukocytes of patients with sepsis was investigated to assess the correlations with leukocyte, E-selectin, ICAM-1 and disease severity. The expression of microRNA-23b in peripheral blood leukocytes from 87 patients with sepsis, 50 patients with systemic inflammatory response syndrome (SIRS) and 50 normal controls were measured by reverse transcription-quantitative PCR (RT-qPCR), and stability of microRNA-23b was evaluated. Enzyme-linked immunosorbent assay (ELISA) was used to detect E-selectin and ICAM-1. Sequential organ failure assessment (SOFA) scoring system was used to assess the severity of sepsis patients. Correlation analysis was performed between microRNA-23b and the total number of white blood cells (WBC), E-selectin, ICAM-1, and SOFA scores. Compared with the normal control group, the expression level of microRNA-23b in the sepsis group was significantly decreased (P<0.05), and WBC and E-selectin were significantly increased (P<0.05). ICAM-1 level in the sepsis and the SIRS groups was significantly higher than that in the control group (P<0.05), and it is also higher in the sepsis group than in the SIRS group. In the sepsis group, the expression level of microRNA-23b in the death group was significantly lower than that in the survivor group (P<0.05), while the level of E-selectin, ICAM-1, and SOFA scores were significantly higher in the death group than in the survivor group (P<0.05), while there was no significant difference in WBC between the groups (P>0.05). The expression level of microRNA-23b in patients with sepsis was significantly negatively correlated with SOFA scores, E-selectin, and ICAM-1 (r=−0.633, −0.585, and −0.439, respectively, P<0.05). The expression of microRNA-23b in peripheral blood of patients with sepsis is related to the manifestation of the inflammatory state, and can be used to judge the severity and prognosis of patients with this disease.

The CD40 rs1883832 Polymorphism Affects Sepsis Susceptibility and sCD40L Levels

Sepsis is a severe and progressive disease characterized by systemic inflammatory response syndrome (SIRS). CD40 serves as a vital link between immune response and inflammation. This study was designed to investigate the potential association between a functional single-nucleotide polymorphism (SNP) of CD40 (rs1883832) and susceptibility to sepsis. We first performed a case-control study to explore the relationship between the CD40 rs1883832 polymorphism and sepsis. CD40 mRNA expression and protein expression were determined by real-time PCR and western blotting, respectively, in peripheral blood mononuclear cells (PBMCs) from sepsis patients and healthy controls. The plasma sCD40L levels in the two groups were measured by ELISA. The results showed that the frequencies of the TT genotype and the CD40 rs1883832 T allele were significantly higher in sepsis patients than in healthy controls. Plasma sCD40L levels were also significantly increased in sepsis patients. In addition, TT genotype carriers among sepsis patients displayed the highest CD40 expression at both the mRNA and protein levels, accompanied by the highest plasma sCD40L concentrations. In conclusion, the CD40 rs1883832 T allele acts as a risk factor for increased susceptibility to sepsis and may be involved in the process of sepsis through regulation of CD40 expression and plasma sCD40L levels.