Ulinastatin protects intestinal mucosal barrier by inhibiting the activation of intestinal NLRP3 inflammasomes in septic rats

Abstract

To explore the damage of the intestinal mucosal barrier of septic rats by the activation of NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasomes and the role of Ulinastatin (UTI) on the expression of intestinal nuclear factor-κB (NF-κB)/NLRP3 inflammasome signaling pathway in septic rats. According to the random number table method, 64 male Wistar rats were divided into sham operation group (Sham group), cecal ligation and puncture (CLP) group, UTI treatment group (100 kU/kg UTI was intraperitoneally injected 1, 6, 12 and 18 hours after CLP), and UTI pretreatment group (100 kU/kg UTI was given 1 hour before CLP), with 16 rats in each group. The survival of rats was observed after 24 hours, and the blood was collected from abdominal aorta at 24 hours after modeling, then rats were killed and their ileum tissues were taken. Hematoxylin-eosin (HE) staining was used to observe histopathological changes and Chiu score. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and intestinal fatty acid binding protein (I-FABP) in serum were detected by enzyme linked immunosorbent assay (ELISA). The protein expression of NF-κB p65 in intestinal tissue was detected by Western blotting. The expression of intestinal tight junction proteins Claudin-1, Occludin and the inflammasome NLRP3, apoptosis-associated speck-like protein containing CARD (ASC) and caspase-1 were detected by immunohistochemistry. Compared with Sham group, the 24-hour survival rate of CLP group was significantly reduced. Histopathological results showed that the CLP group had severe edema of mucosa and submucosal stroma with obvious infiltration of inflammatory cells and disordered villi arrangement. Some glands were incomplete, and the villus structure was severely damaged. The Chiu score was significantly increased. The levels of TNF-α, IL-1β, I-FABP in serum and the protein expression of NF-κB p65 in intestinal tissue were significantly increased. The positive expressions of NLRP3, caspase-1 and ASC were also significantly increased. However, the positive expression of tight junction protein in small intestine tissue such as Occludin and Claudin-1 were significantly reduced. It suggested that when sepsis occurs, small intestinal mucosal barrier dysfunction happens, and mucosal permeability increases, while tight junction protein expression decreases, NLRP3 inflammasome and its upstream molecule NF-κB p65 were activated. After UTI treatment and UTI pretreatment intervention, although there was no significant difference in 24-hour survival compared with CLP group (62.5%, 68.8% vs. 43.8%, both P > 0.05), the intestinal tissue damage of septic rats was significantly improved. Specifically: Chiu score and the levels of TNF-α, IL-1β, I-FABP in serum were significantly decreased [Chiu score: 3.37±0.25, 3.23±0.16 vs. 4.08±0.13, TNF-α (ng/L): 147.62±20.74, 140.71±24.81 vs. 222.82±16.84, IL-1β (ng/L): 80.64±5.68, 78.11±4.75 vs. 133.73±3.92, I-FABP (μg/L): 38.29±3.60, 35.88±4.52 vs. 59.81±4.66, all P < 0.05]; the protein expression of NF-κB p65 was significantly decreased (NF-κB p65/β-actin: 0.65±0.10, 0.69±0.11 vs. 0.99±0.10, both P < 0.05), the positive expressions of Claudin-1 and Occludin in the small intestine tissue were increased [Claudin-1 positive expression area: (19.43±3.08)%, (23.99±6.27)% vs. (7.77±2.03)%; Occludin positive expression area: (19.58±4.75)%, (23.28±3.68)% vs. (11.69±4.30)%, all P < 0.05], while the positive expressions of NLRP3, caspase-1, ASC were decreased [NLRP3 positive expression area: (7.80±3.14)%, (6.86±2.63)% vs. (14.44±3.68)%; caspase-1 positive expression area: (10.62±3.52)%, (9.49±3.09)% vs. (26.69±8.05)%; ASC positive expression area: (9.95±2.81)%, (10.53±3.61)% vs. (24.16±5.48)%, all P < 0.05]. However, there was no significant difference in the improvement effect between UTI treatment group and UTI pretreatment group. Intestinal barrier dysfunction in sepsis may be related to the activation of NLRP3 inflammasomes in the intestinal mucosa. The protective effect of UTI in the intestinal mucosa may be related to inhibiting the activation of NLRP3 inflammasomes in the intestinal mucosa, but UTI pretreatment has no obvious advantage compared with UTI treatment.