Patient samples

Liver specimens from patients undergoing hepatectomy at Affiliated Yancheng School of Clinical Medicine of Nanjing Medical University were collected for this study. All patients donating samples for this project signed informed consents, and all experiments were approved by the Medical Ethics Committee of Yancheng Third People’s Hospital.

Mouse liver partial warm ischemia models

6–8 weeks wild-type C57BL/6 male mice were fasted for at least 8 h before surgery and intraperitoneally injected with SYK inhibitors: GS-9973 (MCE, HY15968) (20 mg/kg), R406 (MCE, HY12067) (10 mg/kg), and Piceatannol (MCE, HY13518) (20 mg/kg) 4 h before surgery. The mice were induced with anesthesia using 2% isoflurane, followed by midline laparotomy. Heparin was injected, and non-traumatic vascular clamps were applied to occlude the arteries and portal veins of cephalic lobe, inducing 70% partial hepatic ischemia. After 90 min of partial hepatic ischemia, reperfusion was performed for 3 to 24 h. The sham-operated group underwent the same procedures without vascular occlusion.

In vivo/in vitro SYK knockdown

In vivo: SYK siRNA (20 mg/kg, Santa Cruze, California, USA) was coupled with a mannose-conjugated polymer (polyplus transfection, Illkich, France), injected into the tail vein of mice 4 h before surgery and delivered to liver CD206hi macrophages.

In vitro: Bone marrow-derived macrophages were collected from C57BL/6 mice. The shSYK lentivirus (Genepharm, Shanghai, China) was synthesized to transfect BMDMs collected from C57BL/6 mice. 3 days after the induction of BMDMs, the cells were infected with 100 MOI shSYK-LV or NC-LV. After 24 h, stale medium were replaced by fresh medium with murine M-CSF (20 ng/mL). After 7 days, BMDMs were collected and used for subsequent experiments.

Neutrophil isolation

Preoperative and postoperative peripheral blood was collected from patients or mice with hepatic IR and isolated with the EasySep Direct Human Neutrophil Isolation Kit (STEMCELL Technology, #19666) according to the manufacturer’s protocol. Mouse neutrophils were isolated using EasySep Mouse Neutrophil Enrichment Kit (STEMCELL Technology,#19762A). In some experiments, we treated neutrophils with SYK inhibitors (5µM).

Isolation of neutrophils from Mouse liver

Preheated PBS was used to perfuse the liver (5 ml/min). After the liver became completely white, the liver was perfused with type IV collagenase (2 ml/min) dissolved in HBSS. After removing the gallbladder, the liver was harvested, and the digested cells were filtered with a 70 μm cell strainer to prepare a single-cell suspension. Cells were collected by centrifugation at 427 g for 10 min. 3 ml of 70% Percoll was added to the centrifuge tube, cells were resuspended on top of 8 ml of 40% Percoll added to 70% Percoll, and centrifuged at 872 g for 30 min. Cells on the 40%/70% Percoll boundary surface were washed twice for neutrophil separation.

Extraction and cultivation of bone marrow-derived macrophages (BMDMs)

Bone marrow cells were isolated from the femurs and tibias of mice, filtered through a 70 μm cell strainer, followed by removal of erythrocytes with Red Blood Cell Lysis Buffer. Cells were cultured in DMEM medium supplemented with 10% FBS and 20% cell-free L929 medium. After one week, the medium was replaced for subsequent experiments.

Flow cytometry

Isolated cells were preincubated with Fc receptor blockers to reduce nonspecific staining. Cells were fixed with Fixation/Permeabilization solution (BD Bioscience, 554722) at 4 ℃ for 20 min. After washing, cells were resuspended in BD Perm/Wash™ (BD Bioscience, 554723) buffer and incubated in the dark with the corresponding antibodies for 30 min: F4/80 (invitrogen, 2300247), CD11b (Biolegend, 101218), Ly6G (Biolegend, 127627), CD4 (Biolegend, 100443), FOXP3 (Biolegend, 126407). Flow cytometric analysis was performed using a flow cytometer (Beckman, California, CA, USA), and the data were analyzed using FlowJo (version 10.8.1).

Co-immunoprecipitation

The isolated peripheral blood neutrophils were lysed with NP40 for 30 min, centrifuged at 12,000 g for 15 min to collect the supernatant. The cell lysates were immunoprecipitated using primary antibodies and Protein A + G Agarose. Total lysates or immunoprecipitated complexes were subsequently subjected to SDS-PAGE electrophoresis, followed by immunoblot using corresponding antibodies.

Western blot

Liver tissues or cells were lysed using RIPA, separated by SDS-PAGE gel electrophoresis and then transferred to PVDF membranes. The proteins were then immunoblotted with corresponding primary antibody: anti-P-SYK (Tyr525/526) (Cell Signaling Technology, 2710), anti-SYK (Cell Signaling Technology, 2712), anti-PKM2 (Cell Signaling Technology, 4053), anti-P-STAT3 (Cell Signaling Technology, 9145), anti-β-actin (Cell Signaling Technology, 4970), anti-MPO (ab208670, Abcam), anti-CitH3 (ab281584, Abcam), anti-NE (bs-6982R, Bioss), anti-β-actin (Cell Signaling Technology, 4970), using chemiluminescence-based method for color development.

qPCR

RNA was extracted from tissues or cells and reverse transcribed into cDNA. Real-time fluorescence quantitative polymerase chain reaction was conduct using SYBR green master mix. all expression levels of target genes were normalized to β-actin expression.

Determination of serum ALT, AST

The blood samples collected from patients or mice were stored at 4 °C overnight and then centrifuged at 300 rpm for 15 min. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the serum were determined using corresponding kits (Servicebio, GM1102, GM1103). The results were measured using an automatic biochemical analyzer (Rayto Life and Analytical Sciences Co., Ltd., Chemray 240).

Histopathology

The liver tissue specimens were fixed in 4% formaldehyde, embedded in paraffin, and sectioned into 4 μm thick slices for hematoxylin-eosin (H&E) staining. The severity of liver injury was graded based on the Suzuki scoring system.

Immunohistochemistry and Immunofluorescence

For Immunofluorescence, the fixed tissue sections were placed in a repair kit (Servicebio, China) filled with EDTA antigen retrieval buffer (PH = 9.0), and then boiled in a microwave for repair. BSA (3%) solution was added for 30 min to block nonspecific binding, and then the corresponding primary antibodies were added: anti-P-SYK (Bioss, bs-3434R), anti-PKM2 (Cell Signaling Technology, 4053), anti-P-STAT3 (Cell Signaling Technology, 9145), anti-MPO (Abcam, ab208670), anti-CitH3 (Abcam, ab281584).

TUNEL assay

Freshly frozen liver tissues were collected and stained using a TUNEL kit (Servicebio, G1504-50T), and images were collected using a fluorescence microscope to calculate the rate of positively stained cells.

ELISA assay

Murine serum and cell culture supernatant was collected, and the levels of the cytokine were measured using a mouse IL-1β ELISA kit (Abcam, ab197742).

Statistical analysis

The data are expressed as mean ± SD, and the statistical difference between subgroups is determined by GraphPad Prism. Statistical significance was analyzed using the Student’s unpaired t test. Linear regression is used to assess the strength of the linear relationship between variables. Two-tailed P-value less than 0.05 is considered statistically significant.