Necroptosis is a key mediator of enterocytes loss in intestinal ischaemia/reperfusion injury

Abstract

Cell death is an important biological process that is believed to have a central role in intestinal ischaemia/reperfusion (I/R) injury. While the apoptosis inhibition is pivotal in preventing intestinal I/R, how necrotic cell death is regulated remains unknown. Necroptosis represents a newly discovered form of programmed cell death that combines the features of both apoptosis and necrosis, and it has been implicated in the development of a range of inflammatory diseases. Here, we show that receptor-interacting protein 1/3 (RIP1/3) kinase and mixed lineage kinase domain-like protein recruitment mediates necroptosis in a rat model of ischaemic intestinal injury in vivo. Furthermore, necroptosis was specifically blocked by the RIP1 kinase inhibitor necrostatin-1. In addition, the combined treatment of necrostatin-1 and the pan-caspase inhibitor Z-VAD acted synergistically to protect against intestinal I/R injury, and these two pathways can be converted to one another when one is inhibited. In vitro, necrostatin-1 pre-treatment reduced the necroptotic death of oxygen-glucose deprivation challenged intestinal epithelial cell-6 cells, which in turn dampened the production of pro-inflammatory cytokines (tumour necrosis factor-α and interleukin-1β), and suppressed high-mobility group box-1 (HMGB1) translocation from the nucleus to the cytoplasm and the subsequent release of HMGB1 into the supernatant, thus decreasing the activation of Toll-like receptor 4 and the receptor for advanced glycation end products. Collectively, our study reveals a robust RIP1/RIP3-dependent necroptosis pathway in intestinal I/R-induced intestinal injury in vivo and in vitro and suggests that the HMGB1 signalling is highly involved in this process, making it a novel therapeutic target for acute ischaemic intestinal injury.

Keywords: high-mobility group box-1; intestine; ischaemia/reperfusion injury; programmed necrosis.

Figure 1

Intestinal protection by necrostatin‐1 versus intestinal I/R injury in vivo. (A) Histopathologic changes in the intestinal mucosa. Haematoxylin and eosin stained small intestine after 1 hr ischaemia followed by 6 hrs/24 hrs of reperfusion. Magnification is ×200, bar denotes 100 mm. Black arrows indicate denuded, fused villi and haemorrhage. Black asterisks indicate Gruenhagen's space. (B) Injury scores of the intestinal mucosa morphology. (C) Intestinal cellular injury evaluated by serum DAO activity. (D) Serum HMGB1 level were analysed by ELISA. The images are representative for each group. The data are shown as the means ± S.D. (n = 8 per group). *P < 0.05, **P < 0.01 compared with sham group, ^# P < 0.05, ^## P < 0.05 compared with I/R group.

Figure 2

Necroptosis is an essential contributor to intestinal I/R injury in vivo. (A) Serum concentration of tumour necrosis factor‐α (TNF‐α). (B) Expression of tumour necrosis factor receptor 1 (TNFR1) (green fluorescence). Nuclei were counterstained with Hoechst (blue). (C) Fluorescent intensity of TNFR1 in the intestine. (D) TUNEL and cleaved caspase‐3 dual immunofluorescent labelling in the intestine. TUNEL (green) and cleaved caspase‐3 (red) staining were performed after 6 and 24 hrs of reperfusion. Nuclei were stained with Hoechst (blue). Both TUNEL‐ and cleaved caspase‐3‐positive cells were apoptotic, while TUNEL‐positive but cleaved caspase‐3‐negative cells were necroptic. (E) The number of TUNEL(+)/cleaved caspase‐3(‐) cells per ×20 field in the intestine. Bar denotes 40 μm; inset, magnified photographs. The data are shown as the means ± S.D. (n = 8 per group). *P < 0.05, **P < 0.01 compared with sham group; ^# P < 0.05, ^## P < 0.01 compared with I/R group.

Figure 3

Necrostatin‐1 inhibits necroptosis‐related protein expressions and protects the intestine independent of apoptosis in vivo. (A and B) Western blot and quantification showed increased RIP1 and RIP3 protein expression levels after intestinal I/R. (C and D) Western blot and quantification showed increased MLKL protein expression levels after intestinal I/R. (E and F) MLKL recruitment to RIP1 was significantly decreased after Nec‐1 treatment. (G and H) Pre‐treatment with Nec‐1 did not affect caspase‐3 cleavage. The data are shown as the means ± S.D. (n = 8 per group). *P < 0.05, **P < 0.01 compared with sham group; ^# P < 0.05, ^## P < 0.01 compared with I/R group.

Figure 4

Increased protection from intestinal I/R injury by the combined blockade of necroptosis and apoptosis after 1 hr of ischaemia/24 hrs of reperfusion in vivo. (A) Histopathologic changes of the intestinal mucosa. Haematoxylin and eosin stained small intestine. Magnification is ×200, bar denotes 100 mm. (B) Injury scores of the intestinal mucosa morphology. (C) Intestinal cellular injury was evaluated by serum DAO activity. (D and E) Z‐VAD with/without Nec‐1 treatment decreased the caspase‐3 cleavage. (F and G) Treatment with Z‐VAD alone had no effect on RIP3 up‐regulation. Caspase inhibition shifted intestinal I/R‐induced epithelial cell death from apoptosis to necroptosis. The images are representative for each group. The data are shown as the means ± S.D. (n = 8 per group). *P < 0.05, **P < 0.01 compared with sham group, ^## P < 0.01 compared with I/R group and DMSO group, ^δ P < 0.05 compared with Nec‐1 group and Z‐VAD group.

Figure 5

Necrostatin‐1 decreases IEC‐6 cell death and pro‐inflammatory cytokine gene expression after OGD in vitro. Cultured IEC‐6 cell injury was induced by depriving culture media of oxygen and glucose (OGD). (A) Viability after different time courses of OGD. (B) Immunofluorescence for TUNEL staining (green, bar denotes 20 μm) and the quantification of TUNEL‐positive cells per ×20 field in IEC‐6 cells (C). (D and E) Western blot and quantification show that RIP3 proteins were expressed at a higher level in the OGD, DMSO and Z‐VAD groups but were attenuated after Nec‐1 treatment. (F and G) Q‐PCR for TNF‐α and IL‐1β mRNA levels in OGD‐challenged IEC‐6 cells after Z‐VAD and Nec‐1 treatment. The data are shown as the means ± S.D. (n = 6 per group). *P < 0.05, **P < 0.01 compared with control group, ^# P < 0.05, ^## P < 0.01 compared with OGD group and DMSO group, ^δ P < 0.05 compared with Z‐VAD group and Nec‐1 group.

Figure 6

Necrostatin‐1 inhibits OGD‐induced HMGB1 translocation from the nucleus to the cytoplasm and HMGB1 signalling activation. (A) Translocation of HMGB1 (green) was detected in cells after OGD challenge. The nuclei were stained with Hoechst (blue, bar denotes 20 μm). White arrows indicate HMGB1 translocation from the nucleus to the cytoplasm. (B) IEC‐6 cytoplasmic and total extracts were analysed for HMGB1 expression by Western blot. (C) Supernatants HMGB1 levels were analysed by ELISA. (D and E) Western blot and quantification show the down‐regulated expression of RAGE and TLR4 after Nec‐1 pre‐treatment. The data are shown as the means ± S.D. (n = 6 per group). *P < 0.05, **P < 0.01 compared with control group, ^## P < 0.01 compared with OGD group and DMSO group.

Figure 7

Putative mechanism of necroptosis in the development of intestinal injury following intestinal I/R. In the proposed model, intestinal I/R stimulates the endogenous TNF‐α production and TNFR1 combination, and activation of the downstream RIP1/RIP3‐MLKL signalling pathway, which triggers necroptotic intestinal epithelium death and release of HMGB1 from the nucleus to the cytoplasm, further contributing to the additional loss of functional epithelium and subsequent mucosal barrier dysfunction. Nec‐1 administration reduced the number of necroptotic cells and extracellular HMGB1 level, which decreases the inflammatory drive that both increases TLR4 and RAGE expression and activates the detrimental effect of HMGB1 signalling in intestinal ischaemia.