[Interferon regulatory factor 7 participates in hepatic ischemia-reperfusion injury via modulating non-classical...

To explore the role and mechanism of interferon regulatory factor 7 (IRF7) in regulating caspase-11-mediated non-canonical pyroptosis in mice hepatic ischemia-reperfusion injury (HIRI).

(1) Animal experiments: Thirty male 4- to 6-week-old C57BL/6 mice were randomly divided into a sham operation (Sham) group and reperfusion 2, 6, 12, and 24 hours groups, with six mice in each group. The reperfusion 2, 6, 12, and 24 h groups underwent a 70% hepatic ischemia-reperfusion (IR) model, which involved clamping the middle and left hepatic lobes with an arterial clamp for 1.5 hours, followed by blood flow reperfusion at 2, 6, 12, and 24 hours time points after releasing the clamp. The Sham group mice underwent laparotomy without vessel clamping. Liver tissue and serum were collected at 2, 6, 12, and 24 hours of reperfusion, and the mice were sacrificed. Transcriptomic sequencing and data analysis of liver tissue from the Sham group and reperfusion 6 hours group were performed using the Illumina sequencing platform. Immunohistochemical staining was followed by light microscopy to observe the positive expression of IRF7 in each group. Transmission electron microscopy was used to observe the ultrastructural changes in liver tissue cells in each group. Western blotting was used to detect the protein expression of IRF7, caspase-11, gasdermin D (GSDMD), GSDMD-N terminal domain (GSDMD-N), and interleukin-1β (IL-1β) in liver tissue from each group. (2) Cell experiments: The mouse AML-12 hepatocyte cell line was transfected with IRF7 overexpression plasmid, knockdown IRF7 plasmid (shIRF7), and corresponding empty vector (NC), respectively. The cells were then subjected to hypoxia/reoxygenation (H/R) treatment, and divided into the following groups: shIRF7 NC+H/R group, shIRF7+H/R group, IRF7 overexpression NC+H/R group, and IRF7 overexpression+H/R group. Western blotting was used to detect the protein expression of IRF7, caspase-11, GSDMD, GSDMD-N, and IL-1β in each group of cells. Immunofluorescence double staining was followed by inverted fluorescence microscopy to observe the co-expression of caspase-11 and GSDMD in each group of cells. Inverted microscopy was used to observe the survival of cells in each group; the content of IL-1β in the supernatant of each group of cells was measured.

(1) Animal experiments: Preliminary findings from transcriptomics sequencing revealed that the expression of IRF7 mRNA in the 6-hour reperfusion group was significantly upregulated compared to the Sham group. Light microscopy showed that IRF7 was mainly localized in the cytoplasm and nucleus; at various time points during reperfusion, the expression of IRF7 in mouse liver tissue cells was upregulated compared to the Sham group, with the highest expression at 6 hours of reperfusion [IRF7 (A value): 0.59±0.04 vs. 0.04±0.01, P<0.05]. Transmission electron microscopy revealed that at various time points during reperfusion, mouse liver cells exhibited varying degrees of nuclear membrane damage and rupture, mitochondrial swelling, and cytoplasmic vacuolization, with the most pronounced changes observed at 6 hours of reperfusion. Compared to the Sham group, the protein expression of IRF7, caspase-11, GSDMD, GSDMD-N, and IL-1β in mouse liver tissue was upregulated after reperfusion in all groups. Among these, the upregulation of IRF7, caspase-11, GSDMD, and GSDMD-N proteins was most pronounced in the 6 h reperfusion group [IRF7 protein (IRF7/GAPDH): 0.59±0.06 vs. 0.38±0.02, caspase-11 protein (caspase-11/GAPDH): 0.42±0.01 vs. 0.15±0.01, GSDMD protein (GSDMD/GAPDH): 0.60±0.02 vs. 0.11±0.01, GSDMD-N protein (GSDMD-N/GAPDH): 0.66±0.03 vs. 0.35±0.03], while IL-1β protein expression was most markedly upregulated in the 12 h reperfusion group (IL-1β/GAPDH: 0.64±0.01 vs. 0.15±0.01), all P<0.05. (2) Cell experiments: Compared to the IRF7 overexpression NC+H/R group, the protein expression of caspase-11, GSDMD, GSDMD-N, and IL-1β was upregulated in the IRF7 overexpression+H/R group (all P<0.05). Compared with the shIRF7 NC+H/R group, the shIRF7+H/R group exhibited downregulation in the protein expression of pyroptosis-related proteins caspase-11, GSDMD, GSDMD-N, and IL-1β [caspase-11 protein (caspase-11/GAPDH): 0.59±0.05 vs. 1.02±0.06, GSDMD protein (GSDMD/GAPDH): 0.87±0.04 vs. 1.09±0.05, GSDMD-N protein (GSDMD-N/GAPDH): 0.38±0.05 vs. 0.80±0.04, IL-1β protein (IL-1β/GAPDH): 0.81±0.03 vs. 1.02±0.03]. Additionally, the number of caspase-11/GSDMD double-positive cells decreased (average fluorescence intensity: 45.50±6.59 vs. 61.83±8.93), while the number of viable cells increased (15 549±1 962 vs. 9 675±3 460), and the IL-1β content in the cell supernatant decreased (ng/L: 147.60±19.36 vs. 253.70±23.96). All differences were statistically significant (all P<0.05).

IRF7 aggravates HIRI in mice, which may be related to the non-classical pyroptosis promotion mediated by caspase-11.