Animals and Ethics
The present study was approved by the Ethics Committee of Jinling Hospital, Nanjing University, and was performed in accordance with the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health. Adult male C57BL/6 mice weighing 25-32 g were purchased from the Animal Center of Jinling Hospital, Nanjing, China. Animals were housed in standard conditions and maintained in a 12/12-hour light-dark cycle with food and water ad libitum.
Polymicrobial sepsis was induced by CLP as previously described (12). Briefly, C57BL/6 mice were anesthetized with intraperitoneal injection of 2% sodium pentobarbital in saline (50 mg/kg; Sigma Chemical Co, St. Louis, MO). The cecum was isolated and then ligated with 4.0 silk below the ileocecal junction, approximately 1.2 cm from the distal end. The cecum was then punctured twice on the anti-mesenteric side with a sterile 22-gauge needle and was gently squeezed to extrude the fecal contents into the peritoneal cavity. Finally, the cecum was placed back into the abdomen and the incision was closed with sutures in two layers. For the mice served as controls, the operation was performed in the same manner as CLP except that the cecum was neither ligated nor punctured. All mice were then resuscitated with subcutaneous lactated Ringers 30 ml/kg immediately after surgery. The entire procedure was completed within 8 min.
Mice were randomly allocated into one of the following three groups (N=10 for each group). (1) Sham group: animals received an intraperitoneal injection of saline (10 ml/kg) at 1 hour before operation. (2) CLP group: animals received an intraperitoneal injection of saline (10 ml/kg) at 1 hour before CLP. (3) CLP + glibenclamide (CLP+GLB) group: animals received an intraperitoneal injection of 50 mg/kg glibenclamide (Glibenclamide was first dissolved in dimethylsulphoxide and then diluted with 0.9% saline) at 1 hour before CLP. The dose of glibenclamide was determined on the basis of a previous study (5) and our preliminary experiment demonstrating that this dose reduced inflammatory mediators in a polymicrobial sepsis mouse model. At the end of the 24-hour period, mice were euthanatized by an over dose of sodium pentobarbital of 80 mg/kg. Mice were then perfused with 10 ml of phosphate-buffered saline through the right ventricle to rinse the pulmonary circulation of blood. Thereafter, lung tissues were harvested for further tissue analyses.
Blood Glucose Measurement
Blood was obtained from eyes by puncture, and blood glucose levels were measured at 6, 12, 18, and 24 hours after sham operation or CLP (Life Scan, Inc, Inc. Milpitas, CA 95035 USA).
An isolated central lobe in the right lung was excised and immediately immersed into 4% formalin. The samples were sectioned and stained with hematoxylin and eosin for light microscopy. The severity of microscopic injury was graded from 0 (normal) to 4 (severe) based on the following categories: neutrophil infiltration, interstitial edema, hemorrhage, hyaline membrane. The sum of all scores was combined to calculate a composite score as described previously (12).
Enzyme-Llinked Immunosorbent Assay (ELISA)
Pulmonary levels of inflammatory cytokines were quantified using specific ELISA kits for rats according to the manufacturers' instructions [tumor necrosis factor (TNF)-a from Diaclone Research, Besanson Cedex, France; IL-1β and IL-18 from R&D Systems, Minneapolis, MN, USA; IL-6 from Biosource Europe SA, Nivelles].
Western Blotting Analysis
Western blotting was performed as previously described (12) using the following primary antibodies: The primary antibodies used to detect caspase-1, IL-1β, Toll-like receptor 4 (TLR4), nuclear factor (NF)-κB p65, and iNOS were from Santa Cruz Biotechnology. The antibody for β-actin was from Cell Signaling Technology. We used the NIH Image J software (National Institutes of Health, Bethesda, MD, USA) to quantitate protein band concentrations.
Myeloperoxidase (MPO) Activity and Nitric Oxide (NO) Concentration
Myeloperoxidase (MPO) activity, a marker for polymorphonuclear neutrophil infiltration into the lung, was determined using a MPO (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) assay kit according to the manufacturer's protocol. NO concentration was measured using a technique as previously described (12).
Malondialdehyde (MDA) and Superoxide Dismutase (SOD) Assay
MDA concentrations were determined by the thiobarbituric acid method and SOD activities were evaluated by the xanthine oxidase method as previously described (12). The absorbance was measured at 532 and 550 nm for MDA and SOD, respectively. MDA concentrations are expressed as nanomoles per milligram of protein and SOD activities are expressed as units per milligram of protein.
Wet-to-Dry Weight (W/D) Ratio
The left lower lobe of the lung was harvested, weighed, and dried for 48 hours in a heated stove. Lung wet to dry (W/D) weight ratio was calculated by dividing wet by the dry lung weight.
In additional three groups of animals: Sham group (N=12), CLP group (N=20), and glibenclamide + CLP group (N=20). Mice were then allowed to food and water ad libitum, animals were frequently observed by a researcher blinded to the group assignment to determine the 7-day survival rate.
Data are expressed as the mean±standard error of the mean (S.E.M.). Normal distribution for the continuous variable was tested using Kolmogorov-Smirnov test and all variables were normally distributed. Statistical significance was determined by one-way, two-way, or repeated-measures of analysis of variance (ANOVA) followed by a Bonferroni test, as appropriate. The survival rate was estimated by Kaplan-Meier method and compared by the log-rank test. A P value<0.05 was regarded as statistically significant difference.