Drugs and reagents
Cangrelor (98% pure) was obtained from Vcare PharmaTech Co., Ltd (Shanghai, China). Myeloperoxidase (MPO) kit were obtained from Nanjing Jiancheng (Jiangsu, China). BCA Protein Assay was obtained from Thermo Fisher (MA, USA). anti-CD40L (#15094, 1:1000) and β-actin (#4970, 1:1000) were obtained from Cell Signalling Technology (MA, USA). Anti-GPR17 (ab75553, 1:500) was obtained from Abcam (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits were obtained from RapidBio (CA, USA). TRIzol reagent was obtained from Life Technology (CA, USA). PrimeScript® RT Master Mix and SYBR® Premix Ex Taq™ were purchased from Takara (Dalian, China). All other reagents were obtained from Sigma-Aldrich (Shanghai, China).
Animal Treatment
C57BL/6 mice (20 ± 2 g) were purchased from Shanghai Laboratory Animal Center (Shanghai, China) and fed according to guidelines approved by the Experimental Animal Ethical Committee of Pudong New Area Gongli Hospital. They were raised in a constant temperature and humidity room (22 ± 1 °C, 65 ± 5% humidity) with standard diet and water in Pudong New Area Gongli Hospital. For the survival study, 58 mice were randomly separated into four groups: (1) sham (n=8), (2) cecal ligation and double puncture (CLP) (n=18), (3) CLP + cangrelor (5 mg/kg) (n=18), and (4) CLP + cangrelor (20 mg/kg) (n=18). Another 32 mice were randomly separated into four groups: (1) sham(n=8), (2) cecal ligation and double puncture (CLP) (n=8), (3) CLP + cangrelor (5 mg/kg) (n=8), and (4) CLP + cangrelor (20 mg/kg) (n=8). The CLP was performed on isoflurane-anesthetized mice as previously described [21, 22]. Sham control animals underwent a laparotomy without ligation or double puncture. Mice were orally administrated with cangrelor (5 or 20 mg/kg) 2 h before surgery. After surgery but prior to emergence, mice were fluid-resuscitated (1mL/mouse of sterile saline, subcutaneously). For the mechanism study, 32 mice were randomly separated into four groups: (1) sham (n=8), (2) cecal ligation and double puncture (CLP) (n=8), (3) CLP + cangrelor (5 mg/kg) (n=8), and (4) CLP + cangrelor (20 mg/kg) (n=8). Mice were sacrificed 24 h after induction of CLP, plasma and lung were saved.
Masson staining of the lung
Paraffin sections of lung tissues were analyzed after several methods containing dewaxing rehydration, Ponceau S Fuchs in acid solution staining for 300 seconds, by washing in 1% phosphomolybdic acid differentiation solution for 5 min, aniline blue solution counterstaining for 300 seconds, with 1% glacial acetic acid for 1 min, alcohol gradient dehydration, transparency production using dimethylbenzene and xylene, and mounting.
Flow cytometry
For detect of surface CD40L expression on platelets, plasma were placed for 10 min at 36.5°C that blocked FcγIII/II receptors in order to decrease nonspecific labeling, next incubated with fluorescein isothiocyanate-conjugated anti-CD41. Immobilization of cells with 1% formaldehyde; erythrocytes were lysed with fluorescence-activated cell sorting lysing solution (BD Biosciences, Shanghai, China), and neutrophils and platelets were then recovered by centrifugation at 300 × g for 10 min. Platelet activation was analyzed by using PE‐conjugated JON/A antibody (Emfret Analytics, Wuerzburg, Germany), which bound to the high affinity conformation of mouse αffini.
RNA isolation and quantitative real-time PCR (qRT-PCR)
mRNA was obtained using TRIzol, respectively. cDNA was synthesised using M-MLV reverse transcriptase and Sangon reverse transcription primers. Relative transcript abundance was determined by the 2−ΔΔCt method and mRNA expression was normalised against β-actin. All amplification analyses were completed on QuantStudio™ 6 Flex Real-Time PCR System.
Protein extraction and western blot analysis
Lung tissues were lysed by RIPA buffer and protein concentrations were assessed by the BCA protein assay. About 20 μg of protein from each example was divided by a 10% SDS-polyacrylamide gel and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 10% skim milk in Tris Buffered Saline with Tween-20 (TBST) and incubated with primary antibodies overnight at 4°C. Membranes were incubated with the corresponding secondary antibody for 1 h at room temperature and washed in TBST. Protein signals were detected using the Super ECL Plus Detection Reagent.
Enzyme-linked immunosorbent assay (ELISA)
Concentrations of the inflammatory cytokines in mice serum were measured with an ELISA kit following to manufacture protocols.
Statistical Analysis
The effects of Cang on the survival of mice were analyzed using the Kaplan–Meier curve and log-rank test. All other results were analyzed using one-way ANOVA analysis by SPSS statistical software for Windows, version 22.0 (SPSS, Chicago, IL, USA). The measurement data are expressed as the mean ± SD. A value of p < 0.05 was deemed statistically significant.