NOD1 induces pyroptotic cell death to aggravate liver ischemia-reperfusion injury in mice

Abstract

Nucleotide-binding oligomerization domain 1 (NOD1) can direct the release of inflammatory factors and influence autophagy and apoptosis in hepatic ischemia-reperfusion injury (IRI) in mice. As pyroptosis is involved in a number of inflammatory reactions, in this report, we investigated the potential for NOD1 to affect pyroptosis. We found that an increased expression of NOD1 during IRI was related to activation of the pyroptotic signaling pathway. With NOD1 activation, cleavage fragments of Caspase-1, gasdermin D (GSDMD), and interleukin (IL)-1β were all increased. Moreover, downregulation of NOD1 expression in AML12 cells exerted an opposite effect. Expression levels of cleaved-Caspase-1 and cleaved-GSDMD decreased after exposure to IRI and the number of cell membrane pores and apoptotic or pyroptotic cells decreased, along with the contents of inflammatory factors and lactate dehydrogenase in the supernatants of AML12 cells. Based on these findings, we conclude that NOD1 aggravates the pyroptotic cell death associated with hepatic ischemia-reperfusion injury in a mouse model via the Caspase-1/GSDMD axis. These findings help to alleviate pyroptotic cell death during liver transplantation or resection, providing new insights into novel protective therapies for liver IRI.

Keywords: NOD1; hepatocytes; ischemia‐reperfusion; pyroptosis.

FIGURE 1

Serology, pathology, and morphology in a mouse hepatic IRI model. (A) Pathological changes in an IR liver (×200, black arrow). (B) Suzuki's score. (C) IR increased serum ALT and AST, with levels reaching a peak at approximately 12 h. (D) Serum contents of IL‐1β and IL‐18 in the IR group were increased as compared with those in the sham group. (E and F) Cell apoptosis or pyroptosis as measured using TUNEL and analysis of TUNEL‐positive cells (apoptotic nuclei were stained red and normal nuclei blue). (G) TEM images of mouse hepatocytes after IR. Compared with sham group: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

FIGURE 2

NOD1 and pyroptosis indicators in a mouse hepatic IRI model. (A) Western blot showing expressions of NOD1, Caspase‐1, cleaved‐Caspase‐1, and GSDMD cleaved‐GSDMD. IL‐13, cleaved‐IL‐1B, and IL‐18 protein levels were increased after IR treatment, achieving peak values at 6 or 12 h and then gradually decreasing (B–G and I and J). Statistical analyses of NOD1 and pyroptosis indicators in the different groups. (H) At each reperfusion time point in mouse liver samples subjected to IR, Caspase‐1, and GSDMD, mRNA were determined using qRT‐PCR analysis. (K) Expressions of NOD1 and GSDMD at each of the reperfusion time points as detected with use of immunohistochemistry. Compared with sham group: *p < 0.05, **p < 0.01, ***p < 0.001, **p < 0.0001

FIGURE 3

Activation of NOD1 by iE‐DAP on liver pyroptosis in a mouse hepatic IRI model. (A) HE staining was used to assess pathological changes in the liver of the IR and iE‐DAP + IR groups (×200, black arrow). (B) TEM images from the iE‐DAP + IR and IR groups. Compared with IR group. (C) mRNA levels of Caspase‐1 and GSDMD in mouse liver samples as measured by qRT‐PCR in the iE‐DAP + IR and IR groups. (D) Immunohistochemistry results displaying the expressions of NOD1 and GSDMD in mouse liver samples in the two groups. (E and J) Cell apoptosis or pyroptosis as detected using TUNEL, with representative images for the two groups. The apoptotic or pyroptotic nuclei were dyed green and normal nuclei blue. Rate of TUNEL‐positive cells in mouse liver samples were analyzed using lmageJ software. (F and I) Western blots demonstrating that expressions of NOD1, Caspase‐1, cleaved‐Caspase‐1, cleaved GSDMD, IL‐1B cleaved‐IL‐1β, and IL‐18 were increased in the iE‐DAP + IR versus the IR group. Statistical analysis of NODi and pyroptosis protein indicators in the iE DAP + IR and IR groups. (G and H) Serum levels of ALT and AST IL‐1β and IL‐18 in the iE‐DAP + IR group were increased as compared with the IR group: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

FIGURE 4

NOD1 knockout on pyroptosis in AML12 cells. (A and F) Hoechst/PI staining and positive rate of apoptosis or pyroptosis in AML12 cells in the NOD1 SiRNA and NC SiRNA groups. (B) Immunofluorescence of AML12 cells displayed a decrease in the intensity of the fluorescent signal within the GSDMD as compared with NC SiRNA group. (C) TEM images from the NC SiRNA and the NOD1 SiRNA groups. Compared with NC SIRNA. (D and H) Hepatocellular apoptosis or pyroptosis rates within the NC SiRNA and the NOD1 SiRNA groups. (E) Statistical analysis of NOD1 and pyroptosis protein indicators. (G) Relative release rate of LDH in AML12 cells. (I) Levels of IL‐18 and IL‐1β in the supernatant of AML12 cells in two groups. (J)Western blot analysis of NOD1, Caspase‐1, cleaved‐Caspase‐1, GSDMD cleaved‐GSDMD, IL‐1β, and IL‐18 in AML12 cells treated or not with NOD1 SiRNA: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001