Activation of NOD-like receptor protein 3 inflammasome mediates inflammatory response and apoptosis in septic intestinal injury model / 中华危重病急救医学

ABSTRACT

Objective:To investigate the expression of NOD-like receptor protein 3 (NLRP3) inflammasome in intestinal injury models with different severity of sepsis and the inflammatory response and apoptosis mediated by NLRP3 inflammasome.Methods:Human colorectal adenocarcinoma cells (Caco-2) were cultured in vitro. The logarithmic growth phase cells were divided into blank control group (normal culture in complete medium) and lipopolysaccharide (LPS) 1, 2 and 4 mg/L groups (complete medium containing 1, 2 and 4 mg/L LPS, respectively). The supernatant were collected at 6, 12 and 24 hours, and the levels of tumor necrosis factor-α (TNF-α), interleukins (IL-6, IL-1β, IL-18) were detected by enzyme linked immunosorbent assay (ELISA). The apoptotic level of cells was detected by flow cytometry. The cells were harvested, and the real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the mRNA expressions of NLRP3 and silent information regulator 1 (SIRT1). Western blotting was used to detect the protein expressions of NLRP3, SIRT1, caspase-1 and apoptosis-associated speck-like protein (ASC). Results:ELISA results showed that the levels of IL-6, TNF-α, IL-1β, and IL-18 in cell supernatant of LPS groups increased in a dose-dependent and time-dependent manner as compared with the blank control group during the same intervention period. The increase was most significant in LPS 4 mg/L group at 24 hours [IL-6 (ng/L) 3.55±0.06 vs. 0.67±0.09, TNF-α (ng/L) 15.37±0.19 vs. 5.04±0.14, IL-1β (ng/L) 2.26±0.10 vs. 0.56±0.09, IL-18 (ng/L) 433.92±22.55 vs. 93.55±21.13, all P < 0.05]. The results of the apoptotic test showed that, compared with the blank control group, the apoptotic rate of LPS groups increased in a dose-dependent and time-dependent manner, and the apoptotic rate of LPS 4 mg/L group increased most significantly at 24 hours [(14.83±3.73)% vs. (5.87±1.17)%, P < 0.05]. RT-qPCR results showed that the expression level of NLRP3 mRNA was increased, while the expression level of SIRT1 mRNA was decreased with the increase of LPS intervention dose and the prolonging of intervention time. At 24 hours, there were significant differences between LPS 4 mg/L group and blank control group [NLRP3 mRNA (2 -&Delta;&Delta;Ct) 8.20±2.82 vs. 1.00±0.36, SIRT1 mRNA (2 -&Delta;&Delta;Ct) 0.58±0.01 vs. 1.03±0.06, both P < 0.05]. Western blotting showed that compared with the blank control group, the protein expression levels of NLRP3, caspase-1 and ASC in LPS groups were significantly increased, while the protein expression levels of SIRT1 were significantly decreased. During each intervention period, with the increase of LPS dose, the expressions of NLRP3, caspase-1 and ASC protein increased gradually, while the expression of SIRT1 protein decreased gradually. At 24 hours, the difference between the LPS 4 mg/L group and the blank control group was significant [NLRP3 protein (NLRP3/β-actin) 1.48±0.03 vs. 0.90±0.12, caspase-1 protein (caspase-1/β-actin) 1.18±0.11 vs. 0.72±0.09, ASC protein (ASC/β-actin) 1.09±0.01 vs. 0.82±0.03, SIRT1 protein (SIRT1/β-actin) 0.48±0.03 vs. 0.76±0.05, all P < 0.05]. Conclusion:In vitro, in the sepsis induced intestinal inflammation model, with the increase of LPS intervention dose and the prolongation of intervention time, intestinal inflammatory response and cell apoptosis showed an increasing trend, which may be related to the up-regulation of NLRP3 inflammasome and its downstream products ASC and caspase-1, and to the down-regulation of SIRT1 expression.