Protective effect of NSA on intestinal epithelial cells in a necroptosis model

Abstract

Objective: This study aimed to investigate the protective effect of the necroptosis inhibitor necrosulfonamide (NSA) on intestinal epithelial cells using a novel in vitro necroptosis model that mimics inflammatory bowel disease (IBD).

Methods: 2,4,6-trinitrobenzenesulfonic acid (TNBS) was perfused into the rectum of BALB/c mice to established a colitis model. Pathologic injury and cell death were evaluated. A novel in vitro model of necroptosis was established in Caco-2 cells using TNF-α and Z-VAD-fmk, and the cells were treated with or without NSA. Morphologic changes, manner of cell death and the levels of phosphorylation of receptor-interacting protein kinase 3 (p-RIPK3) and mixed-lineage kinase domain-like (p-MLKL) were detected.

Results: In the TNBS-induced colitis in mice, TUNEL-positive and caspase-3-negative cells were observed in the intestinal mucosa, and p-RIPK3 was found to be elevated. Under the stimulation of TNF-α and Z-VAD-fmk, the morphologic damage in the Caco-2 cells was aggravated, the proportion of necrosis was increased, and the level of p-RIPK3 and p-MLKL were increased, confirming that the regulated cell death was necroptosis. NSA reversed the morphological abnormalities and reduced necrotic cell death induced by TNF-α and Z-VAD-fmk.

Conclusion: NSA can inhibit necroptosis in intestinal epithelial cells in vitro and might confer a potential protective effect against IBD.

Keywords: colitis; inflammatory bowel disease; intestinal epithelial cells; necroptosis; necrosulfonamide.

Figure 1. Histopathologic changes and MPO activity in TNBS-induced colitis

(A) Histopathologic changes in the colonic tissues upon H&E staining (100× magnification). Arrows denote necrotic materials, and arrowheads denote intestinal infiltration by inflammatory cells. (B) Pathologic scores were elevated in TNBS-induced colitis. (C) MPO activity was increased in TNBS-induced colitis. The results are the mean ± standard deviation. *P<0.05 compared with the control group. TNBS, 2,4,6-trinitrobenzenesulfonic acid; MPO, Myeloperoxidase.

Figure 2. Necroptosis in TNBS-induced colitis

(A) Immunofluorescence of colonic tissues stained with cleaved caspase-3 (red), TUNEL (green) and DAPI (blue). The enlarged image shows green fluorescence representing TUNEL-positive and caspase-3-negative necroptotic cells and yellow fluorescence representing TUNEL-positive and caspase-3-positive apoptotic cells. (B) Western blot analysis of p-RIPK3. p-RIPK3 is increased in TNBS-induced colitis. *P<0.05 compared with the control group. TNBS, 2,4,6-trinitrobenzenesulfonic acid; p-RIPK3, Phosphorylated receptor-interacting protein kinase 3.

Figure 3. TNF-α mRNA and protein in colonic tissues

TNF-α mRNA and protein levels were both up-regulated in TNBS-induced colitis. *P<0.05 compared with the control group.

Figure 4. Morphologic changes in Caco-2 cells treated with TNF-α (T) and Z-VAD-fmk (V) with or without necrosulfonamide (NSA)

(A) Cells under different conditions were observed with a light microscope (LM) for morphologic change, a fluorescence microscope for Hoechst-dye fluorescence, and an electron microscope (EM) for ultrastructural change. Cells became elongated and/or enlarged, and some cells were detached. Hoechst dye showed increasing irregular cell nuclei, nuclear condensation and fragmentation. Cellular swelling, organelle swelling and plasma membrane rupture were observed under the electron microscope. (B) Quantification of Cao-2 cells number in each group. (C) Quantification of cell death which was indicated by Hoechst 33258 Stains. *P<0.05 compared with the control group; # P<0.05 compared with the T+V group.

Figure 5. Regulated necrosis and early apoptosis of Caco-2 cells

Caco-2 cells were treated with TNF-α (T) and Z-VAD-fmk (V) with or without necrosulfonamide (NSA). (A) Typical flow cytometric graph of necrotic cells under different conditions, and histogram showing proportions of propidium iodide (PI)-positive cells or Annexin V-positive PI negative cells. Data in the histogram represent four experiments. (B) Cell metabolic activity was measured by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and expressed as the percentage of control. Data in the histogram represent four experiments. The results are the mean ± standard deviation. *P<0.05 compared with the control group; # P<0.05 compared with the T+V group.

Figure 6. Western blot analysis of p-RIPK3 and p-MLKL in Caco-2 cells treated with TNF-α (T) and Z-VAD-fmk (V) with or without necrosulfonamide (NSA)

*P<0.05 compared with the control group. p-RIPK3, phosphorylated receptor-interacting protein kinase 3; p-MLKL, phosphorylated mixed-lineage kinase domain-like.