Jia-feng Wang

Serum miR-146a and miR-223 as potential new biomarkers for sepsis.

OBJECTIVE Current biomarkers cannot completely distinguish sepsis from systemic inflammatory response syndrome (SIRS) caused by other non-infectious diseases. Circulating microRNAs (miRNAs) are promising biomarkers for several diseases, but their correlation with sepsis is not totally clarified. METHODS Seven miRNAs related to inflammation or infection were included in the present study. Serum miRNA expression was investigated in 50 patients diagnosed with sepsis, 30 patients with SIRS and 20 healthy controls to evaluate the diagnostic and prognostic value. Expression levels of serum miRNAs were determined by quantitative PCR using the Qiagen miScript system. Serum CRP and IL-6 levels were determined by enzyme linked immunosorbent assay. RESULTS Serum miR-146a and miR-223 were significantly reduced in septic patients compared with SIRS patients and healthy controls. The areas under the receiver operating characteristic curve of miR-146a, miR-223 and IL-6 were 0.858, 0.804 and 0.785, respectively. CONCLUSION Serum miR-146a and miR-223 might serve as new biomarkers for sepsis with high specificity and sensitivity. (ClinicalTrials.gov number, NCT00862290.).

Hydrogen-Rich Saline Protects Against Renal Ischemia/Reperfusion Injury in Rats

BACKGROUND Recently it has been demonstrated that hydrogen, as a novel antioxidant, can selectively reduce hydroxyl radicals (·OH) and peroxynitrite anion (ONOO-) in vitro and exert therapeutic antioxidant activity in many diseases. This study was designed to investigate the effect of hydrogen-rich saline on renal ischemia/reperfusion (I/R) injury in rats. METHODS A rat model of renal I/R injury was induced by 45-min occlusion of the bilateral renal pedicles and 24-h reperfusion. Physiologic saline, hydrogen-rich saline, or nitrogen-rich saline (8 mL/kg) were administered intraperitoneally at 5 min before reperfusion, respectively. RESULTS After I/R injury, serum blood urea nitrogen (BUN), creatinine (Cr), tissue malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OhdG), TNF-α, IL-1β, IL-6 levels, and myeloperoxidase (MPO) activity were all increased significantly, while tissue superoxide dismutase (SOD) and catalase (CAT) activities were all decreased significantly. Hydrogen-rich saline reversed these changes and relieved morphological renal injury and I/R-induced apoptosis, while no significant changes were observed in the nitrogen-rich saline-treated group compared with physiologic saline-treated group. CONCLUSIONS Hydrogen-rich saline is able to attenuate the renal I/R injury, which is possibly by reduction of oxidative stress and inflammation.

Screening plasma miRNAs as biomarkers for renal ischemia-reperfusion injury in rats

Background Acute kidney injury is a common clinical comorbidity and early diagnosis is crucial for improving prognosis, but there is still no ideal biomarker for early diagnosis. Material/Methods miRNA microarray was used for detecting miRNA in kidney subjected to renal ischemia-reperfusion injury 12 h after reperfusion. Real-time PCR was performed to validate the results of microarray. miRNAs in the ischemia group were twice as high as in the sham group. Kidney-enriched miR-10a, miR-192, and miR-194 were detected in rat plasma to screen potential biomarkers for renal ischemia-reperfusion injury. Aberrant expressed miRNA in plasma at 12 h were further detected at 1 h, 2 h, 6 h, 12 h, and 24 h to observe the changing trend of these miRNAs and were compared to blood urea nitrogen and serum creatinine. Results Thirty-six miRNAs were aberrantly expressed in kidney of rats with renal ischemia-reperfusion injury, among which 15 miRNAs had a 2-fold greater change. Results of real-time PCR were generally in accordance with microarray results. Levels of the 15 miRNAs differentially expressed in injured kidney were not significantly different from those in sham kidney. However, miR-10a, miR-192, and miR-194 were significantly increased in plasma of rats with renal ischemia-reperfusion injury, among which miR-10a was elevated within 1 h after reperfusion, whereas miR-192 and miR-194 were elevated at 6 h after injury. Blood urea nitrogen was increased at 12 h and serum creatinine was increased at 6 h after injury. Conclusions Plasma miR-10a, miR-192, and miR-194 were potential biomarkers for renal ischemia reperfusion injury in rats, and miR-10a might be the most promising plasma biomarker for renal injury because of its elevation within 1 h after renal injury, as well as renal specificity.

Up-regulation of programmed cell death 1 ligand 1 on neutrophils may be involved in sepsis-induced immunosuppression: an animal study and a prospective case-control study.

Background:Recent studies have shown that neutrophils may display an antigen-presenting function and inhibit lymphocyte proliferation by expressing programmed cell death 1 ligand 1 (PD-L1). The current study was performed to investigate the effect of neutrophils and their pathophysiological significance during sepsis. Methods:Neutrophil PD-L1 expression was determined in both septic mice (n = 6) and patients (n = 41). Neutrophils from septic mice were subtyped into PD-L1− and PD-L1+ populations to determine their phenotypes and functions. Septic neutrophils were cocultured with lymphocytes to observe the effect of septic neutrophils on lymphocyte apoptosis. Results:The PD-L1 level on neutrophils from septic mice was significantly up-regulated (21.41 ± 4.76%). This level increased with the progression of sepsis and the migration of neutrophils from the bone marrow to the blood and peritoneal cavity. The percentages of CD11a, CD62L, and C-C chemokine receptor type 2 were lower, whereas the percentages of CD16 and CD64 were higher on PD-L1+ neutrophils than on PD-L1− neutrophils. The migratory capacity of PD-L1+ neutrophils was compromised. Septic neutrophils induced lymphocyte apoptosis via a contact mechanism, and this process could be reversed by anti-PD-L1 antibody. PD-L1 was also up-regulated on neutrophils from patients with severe sepsis (14.6% [3.75%, 42.1%]). The levels were negatively correlated with the monocyte human leukocyte antigen-DR level and positively correlated with the severity of septic patients. Neutrophil PD-L1 was a predictor for the prognosis of severe sepsis, with an area of 0.74 under the receiver operating curve. Conclusions:PD-L1 is up-regulated on neutrophils during sepsis, which may be related to sepsis-induced immunosuppression.

Heat shock protein A12B protects against sepsis-induced impairment in vascular endothelial permeability

BACKGROUND As a common and life-threatening infectious syndrome, sepsis contributes significantly to morbidity and mortality in clinical settings. Vascular endothelial injury and hyperpermeability play an important role in the development of sepsis-induced organ dysfunction. Heat shock protein A12B (HSPA12B) is one of the HSP70 superfamily members and is mainly expressed in vascular endothelial cells. The present study was performed to investigate the role of HSPA12B in endothelial barrier dysfunction during sepsis. METHODS Human umbilical vein endothelial cells (HUVECs) were stimulated with 1 μg/mL of lipopolysaccharide (LPS) and harvested at 0, 3, 6, 9, 12, and 24 h. The messenger RNA and protein levels of HSPA12B were detected by Real Time-polymerase chain reaction and Western blot. Upregulation of HSPA12B was induced by transfection of pIRES2-EGFP plasmid carrying the HSPA12B complementary DNA. The in vitro effect of HSPA12B overexpression on endothelial permeability was manifested by the transendothelial electrical resistance value, expression of the adhesion molecules VE-cadherin, and the level of permeability-related kinase myosin light chain, SRC, and CDC42. Mice received cecal ligation and puncture surgery followed by nasal inhalation of nano-polymer-mediated siRNA. Lung endothelial permeability was assessed via intrajugular vein injection of Evans Blue 30 h after cecal ligation and puncture. RESULTS After LPS induction, the messenger RNA and protein level of HSPA12B in HUVECs increased and peaked at 12 h, whereas they returned to the baseline level at 24 h. Overexpression of HSPA12B can reduce the permeability of HUVEC stimulated by LPS in vitro, while increasing the expression of VE-Cadherin, myosin light chain, and CDC42. On the other hand, downregulating the expression of HSPA12B can significantly increase lung permeability in mice with sepsis-induced vascular injury. CONCLUSIONS HSPA12B plays a protective role in vascular endothelial barrier dysfunction by preserving the endothelial permeability during sepsis.

Remote ischemic preconditioning protects neurocognitive function of rats following cerebral hypoperfusion.

Summary Background Protection of remote ischemic preconditioning on neurocognitive function caused by bilateral common carotid artery occlusion has been investigated in rats. Material/Methods Thirty-six male Sprague-Dawley rats were divided into 3 groups – control group (Group C, n=12), bilateral carotid arteries occlusion group (Group B, n=12) and remote ischemic precondition group (Group P, n=12). In Group P, remote ischemic preconditioning (RIPC) was performed on the right femoral artery with 3 cycles (10 min) of occlusion/perfusion. After 3 cycles of preconditioning, bilateral carotid arteries were occluded immediately for 60 min. In Group B, ischemic insults were conducted without RIPC. Sham surgeries were performed in Group C. Evaluation of memory and learning capacity was performed on days 5–8 after surgery by Morris water maze testing of spatial learning capacity (n=6 for each group). Apoptosis of cells in the hippocampus region was determined by TUNEL tests and Bcl-2 at this region was determined by ELISA 24 h and 9 days after vessel occlusion (n=6 for each group). Results Neurocognitive tests showed that latency time was significantly longer in Group B than in Group P on day 7 (p=0.016) and day 8 (p=0.036). Moreover, frequency of platform crossings was significant less in group B than in the other 2 groups on day 9. Bcl-2 level was significantly increased in the hippocampal region of rats in Group P on days 1 and 9 after vessel occlusion. TUNEL test showed that apoptosis could be observed at 24 h after occlusion in Group B, but not in Group P and Group C. No apoptosis was observed on day 9. Conclusions Our results suggest that RIPC can protect neurocognitive function of rats after bilateral carotid occlusions, and that Bcl-2 may play an important role in this protective effect.

WS1 gene mutation analysis of Wolfram syndrome in a Chinese patient and a systematic review of literatures

Wolfram syndrome is a rare hereditary disease characterized by diabetes mellitus and optic atrophy. The outcome of this disease is always poor. WFS1 gene mutation is the main cause of this disease. A patient with diabetes mellitus, diabetes insipidus, renal tract disorder, psychiatric abnormality, and cataract was diagnosed with Wolfram syndrome. Mutations in open reading frame (ORF) of WFS1 gene was analyzed by sequencing. Mutations in WFS1 gene was also summarized by a systematic review in Pubmed and Chinese biological and medical database. Sequencing of WFS1 gene in this patient showed a new mutation, 1962G>A, and two other non-sense mutations, 2433A>G and 2565G>A. Systematic review included 219 patients in total and identified 172 WFS1 gene mutations, most of which were located in Exon 8. These mutations in WFS1 gene might be useful in prenatal diagnosis of Wolfram syndrome.

Plasma HSPA12B Is a Potential Predictor for Poor Outcome in Severe Sepsis

Introduction Endothelium-derived molecules may be predictive to organ injury. Heat shock protein (HSP) A12B is mainly located in endothelial cells, which can be detected in the plasma of septic patients. Whether it is correlated with prognosis of sepsis remains unclear. Methods Extracellular HSPA12B (eHSPA12B) was determined in plasma of septic mice at 6h, 12h, 24h and 48h after cecal ligation and puncture (CLP). It was also detected in plasma of patients with severe sepsis, sepsis, systemic inflammatory response syndrome and healthy volunteers. The predictive value for prognosis of severe sepsis was assessed by receiver operating curve (ROC) and Cox regression analyses. Results eHSPA12B was elevated in plasma of CLP mice at 6h and peaked at 24h after surgery. A total of 118 subjects were included in the clinical section, including 66 patients with severe sepsis, 21 patients with sepsis, 16 patients with SIRS and 15 volunteers. Plasma eHSPA12B was significantly higher in patients with severe sepsis than in patients with sepsis, SIRS and volunteers. The level of eHSPA12B was also higher in non-survivals than survivals with severe sepsis. The area under the curve (AUC) of eHSPA12B in predicting death among patients with severe sepsis was 0.782 (0.654–0.909) in ROC analysis, much higher than that of IL-6 and IL-10. Cox regression analysis showed that cardiovascular diseases, IL-6 and eHSPA12B were risk factors for mortality in patients with severe sepsis. Survival curve demonstrated a strikingly significant difference between 28-day survival rates of patients with an eHSPA12B lower or not lower than 1.466ng/ml. Conclusions Plasma eHSPA12B is elevated in both septic mice and patients. It may be a good predictor for poor outcome in patients with severe sepsis.

Methane-rich saline alleviates cerulein-induced acute pancreatitis by inhibiting inflammatory response, oxidative stress and pancreatic apoptosis in mice

Background Acute pancreatitis (AP) is a potentially life‐threatening gastrointestinal disease involving intracellular activation of digestive enzymes and pancreatic acinar cell injury. The present study was performed to investigate whether methane‐rich saline (MS) was involved in the regulation of AP. Methods MS (16 ml/kg) was administered at different dosing frequencies on mice with cerulein‐induced AP. Serum amylase, lipase and histopathological changes in the pancreas tissue were measured. Serum cytokine TNF&agr;, IL‐6, IFN&ggr; and IL‐10 were detected by ELISA. The mRNA levels of these inflammatory cytokines in the pancreas were detected by real time‐PCR. Myeloperoxidase (MPO) and superoxide dismutase (SOD) were determined using commercial kits. Apoptosis was assessed by immunohistochemistry and Western blot. Results MS treatment reversed the increased serum level of amylase and lipase, alleviated the pathological damage in the pancreas, and decreased the expression of TNF&agr;, IL‐6, IFN&ggr; and IL‐10 in cerulean‐induced AP mice. In addition, MPO was down‐regulated and SOD was up‐regulated in the MS treated pancreas, indicating that MS had an anti‐oxidant effect against AP. Furthermore, MS protected pancreatic cells against cerulean‐induced apoptosis and abolished cleaved caspase‐3. Conclusion MS exerted anti‐inflammatory, anti‐oxidant and anti‐apoptotic effects on cerulein‐induced AP in mice and may proved to be a promising therapeutic agent for the clinical treatment of pancreatitis. HighlightsMethane‐rich saline alleviated pancreas apoptosis in cerulein‐induced acute pancreatitis.Methane‐rich saline inhibits inflammation both in pancreas and systematically.Methane‐rich saline might be a promising treatment for acute pancreatitis in clinical practice.

Heat Shock Protein A12B Protects Vascular Endothelial Cells Against Sepsis-Induced Acute Lung Injury in Mice

Background: Pulmonary endothelial injury is a critical process in the pathogenesis of acute lung injury (ALI) during sepsis. Heat shock protein A12B (HSPA12B) is mainly expressed in endothelial cells and protects against several harmful factors. However, the effects of HSPA12B in sepsis-induced ALI and its potential mechanisms of action remain unclear. Methods: For in vivo experiments, C57BL/6 mice were randomly divided into four groups (n=15): a sham operation group, a cecal ligation and puncture (CLP) group, a HSPA12B siRNA-CLP group and a negative control (NC) siRNA-CLP group. The mice were treated by nasal inhalation of 2-OMe-modified HSPA12B siRNA or NC siRNA. Sepsis was induced by CLP. Samples were harvested 24 and 48 hours post-CLP surgery. Pathological changes and scoring of lung tissue samples were monitored using hematoxylin and eosin staining. Levels of pro-inflammatory cytokines (e.g., interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6) and myeloperoxidase activity in bronchoalveolar lavage fluid were analyzed by ELISA. Pulmonary edema was assessed using a wet-to-dry weight ratio. Neutrophils and alveolar macrophages were counted using flow cytometry. Pulmonary endothelial cell apoptosis was detected by TUNEL staining. Expression levels of MAPK family signaling molecules and caspase-3 were measured by Western blot analysis. In addition, 7-day survival was recorded. For in vitro experiments, human umbilical vein endothelial cells were pre-transfected with HSPA12B siRNA or pIRES2-EGFP-HSPA12B-Flag plasmid and treated with lipopolysaccharide; subsequently, the expression levels of MAPK family signaling molecules and caspase-3 were measured by Western blotting. Results: Nasal inhalation of nano-polymer-encapsulated HSPA12B siRNA specifically downregulated mRNA and protein expression levels of HSPA12B in lung tissues. The administration of HSPA12B siRNA aggravated lung pathological injury, upregulated pro-inflammatory cytokine (e.g., IL-1β, TNF-α, and IL-6) expression, and increased myeloperoxidase activity, neutrophil infiltration, pulmonary edema, and pulmonary endothelial cell apoptosis. Additionally, HSPA12B knockdown worsened survival after CLP surgery. The potential protective mechanisms of HSPA12B may involve the inhibition of ERK phosphorylation and caspase-3 activation in vivo and in vitro. Conclusion: HSPA12B protected against sepsis-induced ALI. The potential mechanism may be partly due to the inhibition of ERK phosphorylation and caspase-3 activation. These findings provide a potential therapeutic target for treating sepsis.