Twelve-week-old Sprague-Dawley rats purchased from Liaoning Changsheng Biotech Biotech Co., Ltd. (Benxi, China) were used for the HS model. The rats were allowed free access to food and water under a condition of 22 ± 1°C and 12-h light/dark cycle for a one-week acclimation period. The femoral artery was cannulated with a catheter for withdrawing blood until mean arterial pressure (MAP) was decreased to 40–50 mmHg. MAP was maintained at this level for 60 min. After 1 h of shock, the rats were resuscitated with Lactated Ringer’s solution for blood pressure restoration. Three hours after resuscitation, all rats were euthanized for tissue collection. The sham-operated rats underwent the same catheter implantation procedure but without bloodletting or resuscitation. The body temperature of the rats was maintained at 37°C during the surgery. For knocking down PTPRO in the rats, 48 h before hemorrhage by a catheter inserted into the femoral artery, the rat was infected with 1×108 TU lentiviral encoding PTPRO short hairpin RNA (shRNA) or its negative control shRNA (shNC) by intratracheal instillation through the mouth (Figure 1b). The rats were categorized into four groups (n=6 per group). This study was performed following the Institution Animal Care and Use Committee of Wuxi 9th Affiliated Hospital of Soochow University. After euthanasia, the blood, bronchoalveolar lavage fluid (BALF), and lung tissues were collected and stored at -70°C or fixed with 4% paraformaldehyde.
A clinical study was conducted in 7 patients with severe HS after polytrauma from Wuxi 9th Affiliated Hospital of Soochow University. Thirty healthy participants served as the controls. The blood samples were collected from all subjects after obtaining written informed consent. The study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Wuxi 9th Affiliated Hospital of Soochow University (KT2020029).
Evans blue (EB) dye extravasation was performed to assess capillary leakage in the lungs. Twenty minutes before euthanasia, the rats were subjected to the tail vein injection with EB dye (50 ml/kg; Aladdin, Shanghai, China). After being euthanized, the heart was perfused with saline to remove the redundant dye in the vessels. EB was extracted from the lung tissues by incubation in formamide (4 m/g tissue; Aladdin, Shanghai, China) at 60°C for 48 h. The supernatant was detected with a microplate reader (800TS; BioTek, Winooski, VT, USA) at 620 nm after centrifugation. EB content was calculated according to a standard curve of EB in formamide and used to represent extravasation.
Quantitative real-time PCR
Using the Total RNA Isolation Kit (Tiangen Biotech Co. Ltd., Beijing, China) and BeyoRT II M-MLV reverse transcriptase (Beyotime, Shanghai, China), total RNA was isolated from the lungs and reverse transcribed into the first cDNA. A quantitative PCR assay was carried out by ExicyclerTM 96 Real-time PCR System (Bioneer Corporation, Daejeon, Korea) using SYBR Green (Solarbio, Beijing, China). The level of mRNA was normalized to GAPDH. The primers were synthesized by Genscript Biotechnology Co., Ltd. (Nanjing, China), and the sequences were shown in Table 1.
Western blot analysis
The lung tissues separated from the rats were lysed using RIPA buffer (Solarbio, Beijing, China) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF; Solarbio, Beijing, China) to isolate total proteins. Nuclear proteins were extracted using Nuclear Protein Extraction Kit (Solarbio, Beijing, China) according to the manufacturer's instructions. Protein quantified by bicinchoninic acid (BCA) Kit (Solarbio, Beijing, China) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins were transferred onto a polyvinylidene difluoride membrane (PVDF; Millipore, Billerica, MA, USA). The membrane was blocked with 5% skim milk (Sangon Biotech, Shanghai, China) for 1 h. Afterward, the membrane was incubated with the primary antibody including PTPRO antibody (1:500 dilution; Affinity, Cincinnati, OH, USA), p-IKKα/β antibody (1:2000 dilution; Affinity, Cincinnati, OH, USA), IKKα/β antibody (1:1000 dilution; Affinity, Cincinnati, OH, USA), p-P65 (S536) antibody (1:1000 dilution; ABclonal, Shanghai, China), NF-κB (P65) antibody (1:500 dilution; ABclonal, Shanghai, China), GAPDH antibody (1:10000 dilution; Proteintech, Rosemont, IL, USA), and Histone H3 antibody (1:5000 dilution; GeneTex, Alton PkwyIrvine, CA, USA) overnight at 4°C, followed by incubation with the secondary anti-rabbit or anti-mouse horseradish peroxidase-linked antibodies (1:300 dilution) at 37°C for 1 h. Protein bands were visualized using electrochemiluminescence (ECL) reagent (Solarbio, Beijing, China). Band intensities were analyzed with Gel-Pro-Analyzer software and normalized to band intensity of GAPDH or Histone H3.
Enzyme-linked immunosorbent assay (ELISA)
The levels of tumor necrosis factor α (TNF-α) and interleukin (IL)-6 in BALF were detected with respective specific ELISA kits (LIANKE Biotech, Hangzhou, China) according to the manufacturer's instructions.
Histological changes in the lungs were detected with hematoxylin and eosin (H&E) staining. The lung tissues fixed with 4% paraformaldehyde were embedded in paraffin and cut into 5-μm sections. After dewaxing and rehydration, the slides were stained with hematoxylin (Solarbio, Beijing, China) and eosin (Sangon Biotech, Shanghai, China). The images were captured under a microscope (BX53, Olympus, Tokyo, Japan) at 200 × magnification.
The lung tissue slides embedded in paraffin were exposed to citrate solution for antigen retrieval after dewaxing and rehydration. The slides were blocked with goat serum (Solarbio, Beijing, China) at room temperature for 15 min and incubated with MPO antibody (1:100 dilution; ABclonal, Shanghai, China) or P65 antibody (1:100 dilution; ABclonal, Shanghai, China) overnight at 4°C. Then the tissues were incubated in the dark with a Cy3-conjugated goat secondary antibody (1:200 dilution; Beyotime, Shanghai, China) at room temperature for 1 h. The tissues were re-stained with 4',6-diamidino-2-phenylindole (DAPI; Beyotime, Shanghai, China), and the images were captured by a fluorescence microscope (BX53, Olympus, Tokyo, Japan).
Myeloperoxidase (MPO) activity
Infiltration of neutrophils into the lung tissue was assessed by MPO activity. Myeloperoxidase assay kit (Jiancheng Bioengineering Institute, Nanjing, China) was used for the detection of MPO activity in the lungs according to the manufacturer's instructions.
The data were analyzed using GraphPad Prism 7.0 and expressed as mean ± standard deviations (SD). The values of different groups were compared with one-way analysis of variance (ANOVA) followed by Tukey’s test. Significance was considered when P < 0.05 between different groups.