[Agmatine alleviates sepsis-induced intestinal injury by modulating the nuclear factor-ΚB signaling pathway through immunoglobulin superfamily containing leucine rich repeat to suppress intestinal pyroptosis].

OBJECTIVE: To investigate the mechanism by which agmatine alleviates sepsis-induced intestinal injury through the inhibition of intestinal pyroptosis.

METHODS: 1) Bioinformatic analysis: Dataset GSE13904 was selected from the gene expression omnibus (GEO) database, gene ontology (GO), Kyoto encyclopedia of genes and genomes database (KEGG), and protein-protein interaction (PPI) analyses were performed to analyze transcriptomic changes in the blood of sepsis patients. 2) Animal experiments: Ten C57BL/6J mice were randomly divided into a sham group (laparotomy only without ligation) and a sepsis-induced intestinal injury model group [cecal ligation and puncture (CLP)], with 5 mice in each group. Mice were euthanized 48 hours after successful modeling, and ileal tissues were harvested. The expression of immunoglobulin superfamily containing leucine rich repeat (ISLR) in the intestinal tissues of mice was detected by quantitative polymerase chain reaction (qPCR) and Western blotting. 3) Cell experiments: ICE-6 cells were divided into the following groups: control group (cultured in complete medium), lipopolysaccharide (LPS) group (cultured in complete medium containing 10 mg/L lipopolysaccharide, LPS), LPS+agmatine group (treated with 120 μmol/L agmatine on the basis of the LPS group), and an LPS+agmatine+siRNA-ISLR group [transfected with small interfering RNA (siRNA)-ISLR on the basis of the LPS + agmatine group]. Furthermore, cells transfected with siRNA negative control (siRNA-NC) were used to verify the silencing effect of siRNA-ISLR on the ISLR gene. ICE-6 cells were divided into a control group, an LPS group, an LPS+agmatine group, and an LPS+agmatine+Aconine group [treated with 0.25 mmol/L Aconine, a nuclear factor-κB (NF-κB) signaling activator, on the basis of the LPS + agmatine group]. The expression levels of ISLR, NOD-like receptor protein 3 (NLRP3), interleukin-1β (IL-1β), and caspase-1 in cells from each group were detected by qPCR and Western blotting. The levels of IL-6, IL-1β, and tumor necrosis factor-α (TNF-α) in the cell supernatants were measured by enzyme-linked immunosorbent assay (ELISA).

RESULTS: 1) Bioinformatic analysis: GO, KEGG, and PPI analyses of dataset GSE13904 revealed abnormal expression of multiple molecules in sepsis patients, among which ISLR expression was significantly decreased. 2) Animal experiments: The expression levels of ISLR in the intestinal tissue of mice in the sepsis-induced intestinal injury model group were significantly lower than those in the sham group [ISLR mRNA (2-ΔΔCt): 0.52±0.12 vs. 1.02±0.21; ISLR/GAPDH: 0.55±0.01 vs. 1.00±0.01, both P < 0.05], which was consistent with the trend observed in the bioinformatic analysis; therefore, ISLR was selected as the target molecule for functional and mechanistic investigation. 3) Cell experiments: Compared with the control and siRNA-NC groups, ISLR expression was downregulated in the siRNA-ISLR group, confirming successful specific knockdown of the ISLR gene. Compared with the control group, ISLR expression was significantly downregulated in the LPS group, while the expression of NLRP3, IL-1β, and caspase-1 was significantly upregulated, and the levels of IL-6, IL-1β, and TNF-α in the supernatant were significantly increased (all P < 0.05), indicating successful establishment of the LPS-induced injury model. Compared with the LPS group, ISLR expression was significantly upregulated in the LPS+agmatine group [ISLR mRNA (2-ΔΔCt): 0.98±0.17 vs. 0.48±0.11, ISLR/GAPDH: 0.95±0.08 vs. 0.71±0.05], while the expression of NLRP3, IL-1β, and caspase-1 was significantly downregulated, and the levels of IL-6, IL-1β, and TNF-α in the supernatant were significantly decreased (all P < 0.05). Compared with the LPS+agmatine group, ISLR expression was significantly downregulated in both the LPS+agmatine+siRNA-ISLR group [ISLR mRNA (2-ΔΔCt): 0.62±0.20 vs. 0.98±0.17, ISLR/GAPDH: 0.68±0.03 vs. 0.95±0.08] and the LPS+agmatine+Aconine group [ISLR mRNA (2-ΔΔCt): 0.28±0.05 vs. 0.97±0.31, ISLR/GAPDH: 0.61±0.03 vs. 0.93±0.03], while the expression of NLRP3, IL-1β, and caspase-1 was significantly upregulated, and the levels of IL-6, IL-1β, and TNF-α in the supernatant were significantly increased (all P < 0.05), suggesting that the effects of agmatine were reversed by either siRNA-ISLR or the NF-κB signaling activator.

CONCLUSIONS: Agmatine may alleviate sepsis-induced intestinal cell injury by inhibiting intestinal cell pyroptosis, potentially through regulating NF-κB signaling via ISLR.