2.1 Human subjects
Our study included four patient populations: 23 control subjects (group 1), 35 patients with sepsis without fungal infection (group 2), 35 patients with severe sepsis without fungal infection (group 3), and 23 patients with severe sepsis with C. albicans infection (group 4). All patients were selected from the Intensive Care Unit (ICU) and the Department of Hepatobiliary Surgery, the Second Affiliated Hospital of Chongqing Medical University, China between May 2015 and May 2016. Patient selection was based on diagnostic criteria from the International Guidelines for Management of Severe Sepsis and Septic Shock 2012 . Patients with C. albicans sepsis were accorded with diagnostic criteria for severe sepsis, and C. albicans cultures from peripheral bloods were positive at least once. Healthy participants were randomly selected from outpatients of the Second Affiliated Hospital of Chongqing Medical University, as controls. This study was conducted in accordance with the Declaration of Helsinki, and was approved by the Human Research Ethics Committee of Chongqing Medical University (CHICTR-OCC-13003185). Informed consent was obtained from participants, which were given a full explanation of the study.
2.2 Blood sample collection and assays
Peripheral blood samples were collected after an overnight fast, when the sepsis diagnosis was confirmed. Samples were collected in pyrogen-free tubes containing heparin (Bioendo, Xiamen, China). Approximately 250 µl peripheral blood was immediately added to 750 µl lysate solution (Bioteke, Beijing, China), and stored at -70 °C. The remaining blood was centrifuged at 3500 rpm for 15 minutes, after which the serum was divided into five separate 1.5 ml tubes and stored at -70 °C until assaying.
Clinical indices, including blood routine, liver and kidney functions, coagulation metrics, C-reactive protein(CRP), procalcitonin(PCT) and 1,3-β-D-Glucan(BDG) were measured by the Department of Laboratory Medicine, the Second Affiliated Hospital of Chongqing Medical University.
2.3 Animal care
Male BALB/c mice (body weight: 20–25 g and 6–8 weeks old) were obtained from the Experimental Animal Center of Chongqing Medical University. Mice were housed in a pathogen-free environment and given ad libitum access to food and water. Indoor temperature were maintained at 22 ± 2 °C with an artificial light/dark period of 12 hours. All animal experimental operations were in accordance with Chongqing Management Approach of Laboratory Animal (chongqing government order NO.195 ).
2.4 Establishment of an immunosuppressive mouse model
Forty BALB/c male healthy mice were intraperitoneally injected with 100 mg/kg/d cyclophosphamide (Sigma, USA) for three days. Mouse blood was taken by tail-breaking at baseline (one day before cyclophosphamide injection) and on the third day of cyclophosphamide injection. Blood routines were measured by the Department of Laboratory Medicine, the Second Affiliated Hospital of Chongqing Medical University.
2.5 Selection of optimum C. albicans cell densities
C. albicans (ATCC 14053) was supplied by the microbiology laboratory at the Second Affiliated Hospital of Chongqing Medical University. Cells were cultured for 48 h in Sabao weak medium (Microbial reagent company, Hangzhou, China) at 37 °C and serially passaged three times prior to infection. Different C. albicans inoculum densities were prepared using sterilized saline.
On the fourth day after immunosuppression, mice in each group (n = 8) were assigned different C. albicans cell density inoculums (1 × 105, 2 × 105, 5 × 105, 1 × 106, 1 × 107 cells per ml). Mice were injected with 200 µl of these densities via the lateral tail vein . Mortality in each group was observed for 10 days after infection. Based on these mortality rates, the C. albicans inoculum 2 × 105 was selected for subsequent experiments.
2.6 Establishment of an invasive C. albicans infection model in mice
Mice in the invasive C. albicans infection group (n = 12) were injected with 200 µl of the 2 × 105 inoculum via the tail vein on the fourth day after immunosuppression. Mice in the immunosuppressive group (n = 3) were injected with 200 µl saline. Infected mice on days 1, 3, 5, and 7 after infection (n = 3 mice/time point) and mice in the immunosuppressive group (n = 3) were then euthanized and the lungs, liver and kidneys were aseptically removed. Part of the liver, lungs and kidney were weighed, homogenized in phosphate buffered solution (PBS) (8.06 mM sodium phosphate, 1.94 mM potassium phosphate, 2.7 mM KCl, and 137 mM NaCl) (pH 7.4), serially diluted, and plated in duplicate on Sabao weak medium and CHROMagar Candida medium (Microbial reagent company, Hangzhou, China). Plates were incubated at 37 °C and C. albicans growth was assessed at 48 h. Part of the liver, lung and kidney were fixed in 4% paraformaldehyde for 24 h, after which tissues were dehydrated by conventional alcohol gradient processing, embedded in paraffin, serial sectioned and stained with hematoxylin-eosin (H&E) or periodic acid-Schiff (PAS).
2.7 EP treatment in mice infected with invasive C. albicans
EP (28 mM) (Sigma, St. Louis, MO) was prepared in solution with sodium (130 mM), potassium (4 mM), calcium (2.7 mM) and chloride (139 mM) (pH 7.0).
Eighty mice were randomly divided into four groups (n = 20 for each group): 1) the control group, 2) the immunosuppressive group, 3) the invasive C. albicans infection group, and 4) the EP group. Mice in the EP and the invasive C. albicans infection groups were intraperitoneally injected with 100 mg/kg/d cyclophosphamide for three days. On the fourth day, both groups were injected with 200 µl of the 2 × 105 inoculum via the tail vein.
Mice in the EP group were intraperitoneally injected with 60 mg/kg EP four hours after infection, twice a day for three days. Mice in the invasive C. albicans infection group were injected with the same volume of saline. Three days after intraperitoneal injection of cyclophosphamide, the immunosuppressive group were given the same volume of saline. The control group were injected with the same volume of saline. In addition, the mortality in each group (n = 15) was observed during all procedures.
Clinical symptoms (e.g., appetite, activities and weight changes, etc.) were observed and recorded at 8:00 a.m. everyday. Mice were euthanized on days 1, 3, 5, and 7 after infection (n = 5 mice/time point). Blood was aseptically drawn from the orbital venous plexus and collected in pyrogen-free tubes containing heparin. Approximately 250 µl of peripheral blood was immediately added to 750 µl lysate solution (Bioteke, Beijing, China) and stored at -70 °C. The remaining blood was centrifuged at 3500 rpm for 15 minutes at 4 °C. The supernatant was collected in a clean micro-tube, which was stored at -70 °C until assaying.
After euthanasia, the lungs, liver and kidneys were aseptically removed. Some sections were frozen in liquid nitrogen and other sections were fixed in 10% formalin and embedded in paraffin. Tissue sections were stained with H&E or placed on poly- L-lysine-coated glass slides for immunohistochemistry.
2.8 Quantitative real-time polymerase chain reaction (qRT-PCR) analysis
Total RNA was extracted using the total RNA isolation kit (Bioteke, Beijing, China), according to manufacturers’ instructions. The RNA was reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara, Dalian, China). Quantitative real-time PCR analysis was performed using the SYBR Premix Ex TaqTM II kit (Takara, Dalian, China). HMGB1 primers were designed using Primier4.1 software and GADPH was used as a reference gene. Primers were purchased from Huada, Dalian, China.
Human HMGB1 primers were: 5’-GCGGACAAGGCCCGTTA-3’ (sense) and 5’-AGAGGAAGAAGGCCGAAGGAA − 3’ (antisense), and GADPH primers were 5’-TGCCAAATATGATGACATCAAGAA-3’ (sense) and 5’-GGAGTGGGTGTCGCTGTTG-3’ (antisense).
Mouse HMGB1 primers were: 5’-ACCCGGATGCTTCTGTCAACTTCT-3’ (sense) and 5’-GCCTTGTCAGCCTTTGCCATATCT-3’ (antisense); and mouse GADPH primers were 5’-CATGGCCTTCCGTGTTCCTA-3’ (sense) and 5’-GCGGCACGTCAGATCCA-3’ (antisense). The mRNA expression levels of target genes were normalized to GADPH using the 2−∆∆Ct method, where ∆Ct = target gene Ct - GAPDH Ct and ∆∆Ct = ∆Ct treatment-∆Ct control. Data values of 2−∆∆Ct > 2, were considered statistically significant. Three independent experiments were performed in triplicate.
2.9 Enzyme-linked immunosorbent assay (ELISA) analysis
The recombinant HMGB1 (rHMGB1, Novoprotein, Shanghai, China) series concentrations, ranging from 15.625 ng/ml to 1 µg/ml, and serum diluted with PBS (1∶8) from patients or mice, were incubated overnight in 96-well plates at 4 °C. Plates were then washed five times in PBS Tween-20 buffer (PBST; 0.05% Tween-20 in PBS). This step was performed after each incubation period. The plates were blocked for two hours at 37 °C using 3% bovine serum albumin (BSA) in PBST. After this, rabbit anti-HMGB1 polyclonal antibody (1:1,500, Abcam, U.S.A), diluted in PBS buffer containing 2% skimmed milk, was added to all wells and incubated for two hours at 37 °C. The plates were then incubated for one hour at the same temperature, with anti-IgG rabbit antibody conjugated to horseradish peroxidase (1:5,000, Golden Bridge Biotech Co Ltd, Beijing, China). After washing, tetramethylbenzidine (Abcam, U.S.A) was added to each well and plates were incubated in the dark for 10–20 minutes at 37 °C. Reactions were monitored by measuring the absorbance at 460 nm (Thermo, U.S.A). All assays were run in duplicate on each test plate.
TNF-α and IL-6 levels were assessed using commercial ELISA kits (4A Biotech Co., Ltd, Beijing, China).
2.10 Western blot analysis
Tissue total proteins were extracted using the total protein isolation kit (Solarbio, Beijing, China), according to manufacturers’ instructions. Proteins were quantified using the Nanodrop 1000 (Thermo Scientific, Wilmington, Del.). Samples were denatured at 100 °C for five minutes. Equal protein concentrations were loaded onto 12% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (Millipore Corporation, Billerica, Mass.). Membranes containing proteins were blocked in 5% BSA and incubated with rabbit anti-HMGB1 polyclonal antibody (1:1,000, Abcam, U.S.A), followed by incubation with horseradish peroxidase-linked secondary antibodies (1:5,000; Golden Bridge Biotech Co. Ltd, Beijing, China). For standardization and expression comparisons, the membranes were also hybridized with a primary anti-β-actin antibody (1:1,000; Wuhan Boster Biological Technology, Wuhan, China). The bands appearing on film were analyzed with GeneTools software (Syngene, Frederick, Md).
Tissue sections on poly-L-lysine-coated glass slides were deparaffinized in xylene and rehydrated in a graded alcohol series. Then the slides were blocked by 3% hydrogen peroxide for 10 minutes at room temperature (RT), and antigen retrieval for 15 minutes at 100 °C. The slides were then incubated in 5% BSA for one hour to block nonspecific protein binding sites. After washing in PBS, the slides were incubated with an anti-HMGB1 polyclonal antibody (1:1,000; Abcam, U.S.A) overnight at 4 °C. PBS, without primary antibody, was used as a negative control. The slides were washed three times in PBS, then incubated with horseradish peroxidase-linked secondary antibodies (1:5,000; Golden Bridge Biotech Co. Ltd) for 15–20 minutes at RT. Slides were colored with diaminobenzidine and hydrogen peroxide (Golden Bridge Biotech Co Ltd), counterstained with hematoxylin, dehydrated and mounted for viewing (Nikon, Japan). Two different sections from each organ, per time point, were assessed per experiment.
2.12 Statistical analysis
We used SPSS version 20.0 software for statistical analysis. Data were displayed as the mean ± SD. Categorical variables were analyzed using χ2 or Fisher exact tests. Bonferroni tests were applied when normality and homogeneity of variance were satisfied, otherwise Tamhane’s T2 tests were applied. Differences in survival rates among groups were compared by the Log-Rank test. A P-value < 0.05 was considered statistically significant.