Specific pathogen-free (SPF) adult male C57BL/6 mice (6-8 wk) were bought from SLAC Laboratory Animal CO. (Shanghai, China). Before the experiments, the mice were kept in standard cages in a house controlled to a 12 hr light/dark cycle with temperature maintained at 22–24 °C, humidity at 50–60%) and SPF environment in Wenzhou Medical University. The mice were allowed free access to water and food. All animal procedures conformed to the Guide for the Care and Use of Laboratory Animals. This was approved by the Animal Studies Ethics Committee of Wenzhou Medical University.
Animal experimental groups
Mice were intraperitoneally injected with LPS (15 mg/kg, serotype 055: B5; Sigma, Saint Louis, MO, USA) for survival experiments and LPS (10 mg/kg) for other experiments. The effect of PCTR1 on LPS-induced sepsis model was determined by establishing four experimental groups: control group, LPS group, LPS+PCTR1 group, PCTR1 group. For the PCTR1 and control groups, the mice were intraperitoneally injected with PCTR1 100ng/mouse (Cayman Chemical, Ann Arbor, MI, USA) or an equal saline volume. Next, to determine the mechanism of PCTR1, four groups were established: control group, LPS group, LPS + PCTR1 group, LPS + PCTR1 + BOC-2 group, LPS + PCTR1 + EX527 group, and LPS + BAY11-7082 group. Mice were administered with BOC-2 (600 ng/kg，Biomol-Enzo Life Sciences，Farmingdale, NY), EX527 (10 mg/kg, MEC, Shanghai, China), and BAY11-7082 (20 mg/kg, Selleck, Houston, TX, USA) intraperitoneally following LPS administration. The mice were anesthetized with 1% pentobarbital and sacrificed 6hours later. We obtained blood samples via the ophthalmic artery from mice that survived in each group, and obtained lung specimens.
Cell culture and experimental groups
Human umbilical vein endothelial cells (HUVECs) were purchased from SGST (China). The cells were suspended in a DMEM medium containing 10% FBS and cultured in 25 cm2 flasks placed in an incubator controlled at 5% CO2 at 37°C. In all experiments, HUVECs were added to the wells of six-well plates at equal concentrations and were further sub-divided into six groups: control group, LPS group, LPS + PCTR1 group, LPS + PCTR1 + DMSO group (DMSO was used as the solvent for EX527), LPS + PCTR1 + EX527 group and LPS + BAY11-7082 group. DMSO, EX527 (1µM), and BAY11-7082 (1µM) were added to cells for 24 hours followed by LPS (1 µg/ml) and/or PCTR1 (100 nM) stimulation.
Invasive evaluation of respiratory mechanics
The lung function assay was conducted using a flexiVent system (Scireq, Montreal, QC, Canada), as described previously. In brief, mice were anesthetized with 90 mg/kg pentobarbital sodium injected intraperitoneally and then tracheotomized. Vecuronium bromide was administered into each mice via intravenous injection, followed by mechanical ventilation using a computer-moderated small-animal ventilator. A model of deep inflation was used to record the inspiratory capacity (IC). Cst (quasistatic compliance) was assessed based on the PV curves.
Left lungs were excised after anesthetization, and fixed overnight with 4% paraformaldehyde at room temperature. This was followed by staining of 5-μm sections using hematoxylin and eosin (HE) and microscopic examination. The lung injury score was determined based on the level of infiltration of inflammatory cells, hyperemia, and the thickness of the alveolar wall.
Lung vascular permeability assay
The evans blue dye (EBD) extravasation was utilized to assess pulmonary vascular permeability. Five and a half hours following LPS administration, EBD (20 mg/kg) was injected through the caudal vein. After the circulation of the dye for 30 minutes, perfusion of lungs was performed with saline (25 ml) under anaesthesia. Subsequently, the lungs were excised, blotted dry, weighed, and homogenized in formamide. After overnight extraction, the tissue fluid was centrifugated for 10 minutes at 12,000 ×g. A microplate reader was used to read the EBD concentration of the resultant supernatant at 620nm absorbance.
Wet-to-dry lung weight ratio
The index of pulmonary edema was determined from the wet-to-dry (W/D) lung weight ratio. Portions of the harvested wet left lungs were weighed and heated in an oven at 60°C for 48 hours. The W/D ratio was then determined after re-weighing the portions as dry weight.
Blood samples were obtained to determine level serum inflammatory cytokines, as well as the degradation products of glycocalyx in circulation. The blood was collected using the orbital sinus extraction method under anaesthesia. The serum was isolated from the blood samples for ELISA tests. Serum levels of cytokines including TNF-α, IL-1β, and IL-6 and glycocalyx-related proteins including HS, SDC-1, and HA were quantified using R&D systems and Boyun biotech ELISA kits following protocols provided by the manufacturer.
Lung tissues were homogenized with lysis buffer (RIPA: PMSF = 1:1) to obtain protein samples. The tissue homogenates were ultrasonicated and then centrifuged for 30min at 12,000 × g. The concentration of proteins in the supernatants was measured using the BCA kit. An equal amount of protein from each group was loaded onto 10% SDS-PAGE gels and resolved. The proteins were electro-transferred onto PVDF membranes. After being blocked with 10% milk for 2 hours, membranes were incubated with the primary antibodies: EXT-1 (1:2000, GeneTex, Irvine, CA, USA), HPA (1:1000, Abcam, Cambridge, MA, USA), p65 (1:1000, Abcam, Cambridge, MA, USA), p-p65 (1:1000, Abcam, Cambridge, MA, US), SIRT1 (1:1000, Abcam, Cambridge, MA, USA), and β-actin (1:1000, BOYUN, Shanghai, China) overnight at 4 ℃. Next, the membranes were washed thrice and incubated with secondary antibody (1:3000, BOYUN, Shanghai, China) at room temperature for 1 hour followed by another three-times wash. The protein bands were analyzed by Image Quant LAS 4000 mini (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The band intensity was analyzed with ImageJ.
Immunofluorescence was carried out using lung tissues and HUVECs. Lung sections were prepared for immunofluorescence after deparaffinized, dehydrated and antigen retrieval. This was followed by fixation of the HUVECs in 4% paraformaldehyde. Subsequently, the fixed cells and tissue samples were mounted on the cover glass, blocked with donkey serum (Solarbio, Beijing, China) and probed with HSPG2-antibody (1:200). After rinsing thrice with PBS, the fixed cells and tissue sections were incubated with the secondary antibody (1:200) at 37 ℃ for 1 hour and further incubated with DAPI (Abcam) for 5 min. Eventually, both fixed cells and sectioned tissues were covered with an antifade mounting medium (Solarbio, Beijing, China) for fluorescence microscopy (Leica).
The mean values are presented as the mean ± SD and were analyzed with GraphPad Prism 7.0 software. Mean values of groups were compared one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. Kaplan-Meier analysis was conducted to evaluate survival and a log-rank (Mantel-Cox) test was employed to determine statistical significance. P < .05 was considered statistically significant.