Animals
Wild type (WT) male C57BL/6J mice (8 weeks old) and MLKL KO male mice (8 weeks old) were housed at GemPharmatech Co, Ltd (China). All mice were maintained on a 12-hour light/dark cycle with ad libitum access to standard water and chow and supplements under specific pathogen-free conditions. All animals received humane care according to a protocol approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (number NMU08-092), and procedures for all animals complied with relevant legal and ethical requirements.
Mouse Hepatic Ir Model
Model of mouse partial hepatic warm IR injury was established as previously described.(20) Briefly, after successful anesthesia, an atraumatic clip was used to interrupt the arterial and portal venous blood supply to the cephalic lobes of the liver for 90 min and then the clip was removed to initiate hepatic reperfusion. Sham controls underwent the same procedure but without vascular occlusion. The mice were euthanized 6 h after reperfusion, and their blood samples and liver tissues were collected.
Serum Biochemical Measurements And Liver Histopathology
Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using an automatic chemical analyzer (Olympus, Japan). Portions of the liver specimens were fixed in 10% buffered formalin and embedded in paraffin. Sections of the paraffin-embedded liver tissues were stained with H&E. Severity of the hepatic IR was graded blindly from 0 to 4, according to the Suzuki criteria. No necrosis and no hyperemia/centrilobular globular changes were scored as 0, whereas severe hyperemia and > 60% lobular necrosis were scored as 4.
Isolation Of Liver Cells And Hypoxia/reoxygenation (H/r) Model
As previously reported,(21) livers were perfused in situ via the portal vein followed by 0.5% collagenase IV (Sigma, USA) and teased using 70 µm nylon mesh cell strainers (Biosharp, China). The liver cells were resuspended and then divided into non-parenchymal cells and hepatocytes. The non-parenchymal cells were replated for 30 min and the attached cells were considered to be macrophages.
Primary hepatocyte culture hypoxia/reoxygenation (H/R) patterns were cultured in a hypoxic incubator for 1 h (28887166).(22) The culture conditions were then adjusted to normal for 6 h. The cells and supernatants were used in the subsequent experiments.
Detection Of Cell Survival And Death
Hepatocyte viability was quantified using the Cell Counting Kit-8 assay (Yeasen Biotechnology, China). Moreover, hepatocyte cytotoxicity was determined by measuring lactate dehydrogenase activity (Promega, USA) according to the manufacturer's protocol.
Pten-induced Kinase1(Pink1) Knockdown
For knockdown of PINK1 in vivo, adeno-associated virus (AAV) vectors carrying PINK1 short hairpin RNA (shRNA) or control shRNA (GeneChem, China) were administered intravenously at a dose of 4 × 1011 PFU per mouse into the tail vein of 8-week-old male WT or MLKL KO mice. After two weeks, all the mice were established as liver ischemia-reperfusion models.
In vitro, primary hepatocytes were prepared and transiently transfected with PINK1 small interfering RNA (siRNA) or control siRNA (GeneChem, China) using Lipofectamine 3000 (Thermo Fisher Scientific, USA).
Sting And 8-hydroxy-2deoxyguanosine (8-ohdg) Signaling Intervention
STING and 8-hydroxy-2deoxyguanosine (8-OHdG) signaling intervention
DMXAA (MedChemExpress, USA), a STING-specific agonist, was injected intraperitoneally at a dose of 10 mg/kg per mouse, 3 h before the onset of liver ischemia.
To study the effects of 8-OHdG inhibition on hepatic IR, 8-week-old WT or MLKL KO male mice were intraperitoneally injected with mouse monoclonal anti-8-OHdG antibody (GeneTex, USA) at a dose of 10 mg/kg per mouse, 3 h before the onset of liver ischemia.
Immunohistochemical Staining
For tissue immunofluorescence analysis, the liver tissue sections of the mice (4 µm thick) were stained with the indicated primary antibodies MLKL (Proteintech, China), mouse EGF-like module-containing mucin-like hormone receptor-like1 (F4/80) (Cell Signaling Technology, USA), STING (Cell Signaling Technology, USA), and 8-OHdG (Santa Cruz Biotechnology, USA) and incubated overnight at 4°C. The sections were then washed twice and incubated with the secondary Cy3-conjugated goat IgG (Sigma, USA) and FITC-conjugated IgG (Sigma, USA) according to the manufacturer's instructions for 30 min at 37°C. In addition, 4′,6-diamidino‐2‐phenylindole (Beyotime Biotechnology, China) was used for nuclear staining. Representative images were captured and viewed using a confocal microscope (Zeiss).
Western Blotting
Proteins of the liver tissue or cells were extracted prepared. Antibodies against GAPDH, MLKL, cGAS, TANK-binding kinase1 (TBK1), phosphor-TANK-binding kinase1 (P-TBK1) (Cell Signaling Technology, USA), PINK1, P62, LC3B (Proteintech, China), and TOM20 (Santa Cruz Biotechnology, USA) were used.
Quantitative Real-time-pcr
Total RNA was extracted from the liver tissues and cells using an RNA extraction kit (Invitrogen, USA). Reverse transcription of the RNA into complementary DNA was performed using an RR047A PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Japan). Quantitative real-time PCR was performed and repeated thrice. The expression of target genes was normalized to that of GAPDH.
Dihydroethidium (Dhe) Staining
Fluorescent probes of DHE were used to detect intracellular reactive oxygen species (ROS) in the liver tissues. Cryosections of the liver tissues were incubated with 10 µM DHE for 30 min at 37°C. Fluorescence was detected at 488 nm excitation and 525 nm emission wavelengths using a confocal microscope.
Measurement Of Malondialdehyde (Mda), Superoxide Dismutase (Sod), And Ros Levels
The levels of MDA, SOD, and ROS in the liver tissues were estimated using commercial kits. The liver tissues were washed with PBS, homogenized in lysis buffer, and sonicated. After sonication, the lysed tissues were centrifuged (10,000 × g, 10 min) to remove debris and to retain the supernatant. The contents of MDA, SOD, and ROS in the supernatant were determined using a microplate reader. In addition, MDA, SOD, and ROS levels were normalized to protein concentration.
Measurement Of Mitochondrial Membrane Potential (Mmp)
MMP was detected by flow cytometry and immunofluorescence staining using a JC-10 assay kit (Yeasen Biotechnology, China). As a lipophilic dye, it is concentrated in healthy mitochondria to form reversible red fluorescent aggregates and is released into the cytoplasm to form green fluorescent monomers when mitochondria are damaged, resulting in a decrease or disappearance of membrane potential. A reduction in the red signal indicates a decrease in the MMP.
Detection Of Mitophagy
Primary hepatocytes from the mouse liver tissues were transduced with adenovirus-coated-COX8-mt-mkeima (ObiO Technology Corp., Ltd, China) for 24 h to assess the mitophagy levels. Moreover, mt-keima can bind to the mitochondrial matrix, and its fluorescence varies in different pH environments. An increase in red signal indicates a higher level of mitophagy flux. The levels of mitophagy were measured by the pixel area in the red channel (acidic) and normalized to the signal in the green channel (neutral).
Statistical And Analysis
Results are shown as the mean ± SD and represent at least two independent experiments. Statistical analysis was performed using Student's test between two groups and one-way analysis of variance between multiple groups followed by Bonferroni's post hoc test. All analyses were performed with Graphpad8.0. p-value < 0.05 (two‐tailed) was considered statistically significant.