YQFM was obtained from Tasly Pharmaceutical Co., Ltd. (China), which is composed of the ethanol extract (78°C) of three herbals (Panax ginseng: Ophiopogon japonicas: Schisandra chinensis = 1: 3: 1.5). After extraction, filtration and lyophilization, aseptic packaging and strict quality control were done before appearing on the market. Dexamethasone sodium phosphate (lyophilized) was purchased from Biosharp biotechnology company (China).
Animal model of CLP and treatment of drugs
C57BL/6J male mice (18-22g) were obtained from the Experimental Animal Center of Yangzhou University (Yangzhou, China). All mice were housed for 3-5 days to adapt to the environment with controlled temperature (23±1°C), humidity (30%−40%), light (12 h light-dark cycle) and a commercial standard solid rodent chow and water ad libitum. All experiments were in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all protocols were approved by the Animal Ethics Committee of China Pharmaceutical University.
Animals were randomly divided into six groups: Sham group, Model group, Dexamethasone group (Dex) and three YQFM groups of different dosages. Mice were injected with 5 mg/kg dexamethasone or multi-doses of YQFM (1, 2, 4 g/kg) from tail vein before surgery. Sepsis in mice was induced by the CLP surgery as described previously within 1 min after drug administration. The mice were anesthetized by intraperitoneal injection of 4% chloral hydrate (400 mg/kg) and then fixed on the operating table in supine position. After shaved the abdominal hair of mice, a 1 cm incision was made along the ventral white line. The cecum was isolated through blunt separation and ligated with a 5-0 nylon monofilament suture. The length from the free end of cecum to the ligated position accounts for 75% of the total length of cecum. The cecum was punctured in twice with a 16-gauge needle from the mesenteric side without blood vessels to the non-mesenteral side, avoiding damaging the cecum vessels. A small amount of feces was extruded from the two ends of the pinhole. Then the cecum was put back into the abdominal cavity. The incision of mice was sutured layer by layer and iodine was used to prevent infection. Mice in the sham group were injected with same volume of saline and performed with the same surgical procedures without ligation and perforation of the cecum. The mice were sacrificed and relevant examinations were carried out at 18 h after surgery.
Determination of wet weight to dry weight (W/D) ratio of lung tissues
The mice were sacrificed at 18 h after CLP. The lungs were taken out and the wet weight of tissue was accurately measured. After dried in an oven at 120°C for 48 h, the lung tissues were weighed again, recording as dry weight. The wet weight to dry weight ratio of lung tissues were calculated to determine the degree of lung edema.
Lung hematoxylin-eosin (H&E) staining
Histomorphological analysis was measured by H&E staining. At 18 h after CLP, the mice were euthanized. The lungs were rapidly taken out and dipped in 4% paraformaldehyde. The examination was finished in the pathology department of Jiangsu Center for Safety Evaluation of drugs (Jiangsu, China).
Determination of cell contents in bronchoalveolar lavage fluid (BALF)
The contents of white blood cells, neutrophil and lymphocyte in BALF were measured as previous. At 18 h after CLP, the mice were sacrificed and the trachea was peeled off. The alveoli were lavaged with 500 μL phosphate buffer saline (PBS) for 3 times. The BALF were collected and centrifuged (1500 rpm, 5 min, 4°C). The precipitation was resuspended in 800 μL PBS and the cell contents determinations were finished in the Center for New Drug Safety Evaluation and Research in China Pharmaceutical University (Jiangsu, China).
Determination of myeloperoxidase (MPO) content in lung, BALF and plasma
The content of MPO in lung, BALF and plasma was determined by Myeloperoxidase kit (Nanjing Jiancheng Biotechnology Research Institute, China). At 18 h after CLP, the blood, BALF and lung tissues of mice were collected. The determination of MPO was performed according to the manufacturer’s instructions. The absorbance at 450 nm was measured spectrophotometrically using an Infinite M200 Pro plate reader (Tecan, NC, USA). The MPO content in lung homogenate was expressed as Unit per gram of wet lung tissue, while that in BALF or plasma was expressed as Unit per litre.
Measurement of evans blue (EB) leakage into the lung tissue
EB extravasation was used to determine pulmonary microvascular permeability. At 16 h after CLP, EB dye (50 mg/kg, Sigma, USA) was injected via the tail vein. The mice were anesthetized at 2 h after injection of EB and then perfused with saline. The lungs were rapidly taken out and imaged. The lung tissues were weighed and homogenized in formamide (1 mL/100 mg tissue) and centrifuged (5000 g, 30 min). The supernatants were collected to determine the quantity of EB, the absorbance at 620 nm was measured spectrophotometrically using an Infinite M200 Pro plate reader (Tecan, NC, USA). EB leakage was assessed with a standard curve and expressed as micrograms per gram of wet lung tissue.
Western blotting analysis
Western blotting analysis was performed as previously described. The lung tissues and were lysed and centrifuged. The protein concentration was calculated using a bicinchoninic acid protein assay kit (Beyotime Biotechnology, China). Equal amounts of proteins were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, USA) using electrophoresis. The membranes were blocked with 5% bovine serum albumin (BSA) for 2 h and incubated overnight at 4°C with primary antibodies against VE-cadherin (1:1000, Santa Cruz, USA), p120-catenin (1:1000, Abcam, USA), phospho-Src (Y416) (1:1000, Life, USA), Src (1:1000, Life, USA), TLR4 (1:1000, Santa Cruz, USA) and β-actin (1:5000, Bioworld, USA), followed by incubation with the HRP-conjugated secondary antibodies (1:8000, Bioworld, USA) and visualization using enhanced chemiluminescence (ECL, Vazyme Biotech, P.R. China). The bolts were quantified by the Image Lab™ software (version 5.2, Bio-Rad, USA) and the relative values were expressed relative to GAPDH signals.
Immunofluorescence (IF) analysis
Immunofluorescence analysis were performed as previously described. The lung tissues were sectioned at 8 µm thicknesses to adhesive slides. The specimens were treated with blocking buffer (5% BSA, 0.1%Triton-100) at 4°C for 1 h and then incubated overnight at 4°C with primary antibody against CD31 (1:100 dilution, R&D Systems, USA), VE-cadherin (1:200 dilution, Abcam, UK), p120-catenin (1:100 dilution, Santa Cruz Biotechnology, USA), followed by incubation with an Alexa Fluors 488-conjugated donkey anti-rabbit IgG (HþL) antibody (Invitrogen, USA) and 4′,6-Diamidino-2-phenylindole (DAPI, Beyotime Biotechnology, P.R.China). Fluorescent images were observed with confocal laser scanning microscope (LSM700, Zeiss, Germany) and processed using the ZEN imaging software.
All analyses were performed using GraphPad Prism Version 5.01 (GraphPad Software Inc., USA). Results are expressed as the means ± SEM. Statistical analyses were performed using Student's two-tailed t test for comparison between two groups and one-way analysis of variance (ANOVA) followed by Dunnett's test when the data involved three or more groups. The tests were considered statistically significant at P < 0.05.