ABSTRACT
Objective:To investigate the role and mechanism of high mobility group box 1(HMGB1) in the injury of Caco-2 intestinal epithelial barrier induced by lipopolysaccharide (LPS).Methods:The Caco-2 cellular monolayer barrier was established with Transwell chamber. After the Caco-2 monolayer model was established, the transepithelial electrical resistance (TEER) values were measured. When the TEER value reached 500 Ω·cm 2, the cells were divided into 3 groups: control group, LPS treatment group, and LPS+ ethyl pyruvate (EP) treatment group. The concentration of LPS and EP were 100 μg/mL, 50 μg/mL, separately. Then TEER values were measured at 12, 24, 48 and 72 h, and FITC-dextran permeability was detected at 24 h. The cells were seeded on 6-well plates. After cell density reached 80%, treatments were given as the above. The real-time polymerase chain reaction (RT-PCR) and Western blot were used to measure the changes in the protein and mRNA expressions of Occludin, HMGB1, and nuclear factor-κB (NF-κB). Results:Compared with the control group, the TEER values (Ω·cm 2) reduced at 12, 24, 48 and 72 h in the LPS treatment group [(514.22±12.59) vs (304.96±9.69), (521.65±13.35) vs (276.21±7.82), (523.99±8.18) vs (206.64±15.85), (491.21±6.72) vs (156.33±10.83), all P<0.05]. The FITC-dextran permeability increased significantly at 24 h [(2.58±0.07) vs (1.04±0.06), P<0.05]. The expression levels of Occludin protein and mRNA were decreased (all P<0.05), while the expression levels of HMGB1 and NF-κB protein and mRNA were significantly increased (all P<0.05). Compared with the LPS treatment group, the TEER values (Ω·cm 2) increased significantly at 12, 24, 48 and 72 h in the EP treatment group [(519.00±5.66) vs (304.96±9.69), (504.69±8.57) vs (276.21±7.82), (453.65±10.74) vs (206.64±15.85), (385.28±7.57) vs (156.33±10.83), all P<0.05]. The FITC-dextran permeability decreased at 24 h [(1.23±0.11) vs (2.58±0.07), P<0.05]. The expression level of Occludin protein and mRNA were increased ( P<0.05), while the expression levels of HMGB1 and NF-κB protein and mRNA were significantly decreased (all P<0.05). Conclusions:LPS can injure intestinal barrier directly in vitro and reduces the expression of tight junction proteins between cells. The mechanism may be related to the increased expression of HMGB1 and NF-κB protein.
ABSTRACT
Objective:To investigate the role and mechanism of the high mobility group box 1 (HMGB1) in intestinal mucosal barrier injury in rat with sepsis induced by endotoxin lipopolysaccharide (LPS).Methods:The rats were given intraperitoneal injection of LPS to reproduce a model of sepsis. The effect of HMGB1 inhibitor EP solution (40 mg/kg) on sepsis was observed, and phosphate buffer (PBS) control group was set up. Seventy-two hours after modeling, abdominal aortic blood was obtained, and enzyme-linked immunosorbent assay (ELISA) was used to measure the plasma levels of D-lactic acid and diamine oxidase (DAO) of mucosal barrier permeability. The pathological changes of the intestinal mucosal were observed with light microscope and the Chiu score was recorded. The intestinal mucosal ultrastructural changes were observed with electron microscopy. Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and Western Blot were used to measure the mRNA and protein expressions of Occludin, inflammatory factor HMGB1 and its downstream signal molecule nuclear transcription factor-κB p65 (NF-κB p65) in the rat small intestine.Results:The results of histopathology and ultrastructure of the small intestine showed that in the LPS group, the intestinal mucosa tissue swelled obviously, part of the glands were incomplete, the infiltration of neutrophils increased, themicrovillus cells were absent, arranged indisorder, and the number of tight connections significantly reduced compared with the PBS control group. The levels of D-lactic acid and DAO indicating mucosal barrier permeability, the levels of inflammatory factor HMGB1 and its downstream signaling molecule NF-κB p65 mRNA and protein expressions in the LPS group were significantly higher than those in the PBS control group, and the mRNA and protein expression of Occludin in the small intestine was significantly lower than that in the PBS control group, suggesting that the intestinal mucosal barrier function in septic rats was damaged, permeability increased, and the structure was damaged. After the administration of the HMGB1 inhibitor EP, the intestinal mucosal barrier damage was significantly improved. The performance was as follows: the Chiu score of the small intestine tissue and the plasma D-lactic acid and DAO levels in the EP intervene group were significantly lower than those in the LPS group [Chiu score: 1.60±0.48 vs. 3.40±0.48, D-lactic acid (mmol/L): 3.30±0.22 vs. 5.30±0.16, DAO (U/L): 23.66±0.97 vs. 30.47±1.11, all P < 0.05]. Occludin mRNA and protein expression levels were significantly higher than those in the LPS group [Occludin mRNA (2 -ΔΔCt): 0.82±0.05 vs. 0.37±0.08, Occludin protein (Occludin/β-actin): 1.04±0.09 vs. 0.75±0.11, both P < 0.05], while the mRNA and protein expression levels of HMGB1 and NF-κB p65 were significantly lower than those in the LPS group [HMGB1 mRNA (2 -ΔΔCt): 1.63±0.10 vs. 3.57±0.10, HMGB1 protein (HMGB1/β-actin): 1.40±0.07 vs. 1.87±0.07; NF-κB p65 mRNA (2 -ΔΔCt): 1.47±0.09 vs. 2.62±0.13, NF-κB p65 protein (NF-κB p65/β-actin): 1.24±0.14 vs. 1.60±0.13, all P < 0.05]. Conclusions:Intestinal mucosal barrier function of septic rats was damaged, permeability increased, and structure was damaged. The mechanism may be that the expression of inflammatory factor HMGB1 was up-regulated and promoted the activation of its downstream signaling molecule NF-κB, thereby mediated the inflammatory cascade reaction and caused damage to the intestinal mucosa.