论文部分内容阅读
目的:探讨高迁移率族蛋白B1（HMGB1）在内毒素脂多糖（LPS）诱导脓毒症大鼠肠黏膜损伤中的作用及机制。方法:用腹腔注射LPS构建脓毒症大鼠模型；用HMGB1抑制剂EP溶液40 mg/kg干预来观察HMGB1在脓毒症中的作用，同时设磷酸盐缓冲液（PBS）对照组。制模后72 h后取各组大鼠腹主动脉血，用酶联免疫吸附试验（ELISA）检测黏膜屏障通透性血浆标志物D-乳酸和二胺氧化酶（DAO）水平；光镜下观察小肠组织黏膜病理学改变并进行肠黏膜损伤评分（Chiu评分），透射电镜下观察小肠上皮超微结构改变；用实时定量反转录-聚合酶链反应（RT-qPCR）和蛋白质免疫印迹试验（Western Blot）分别检测小肠组织中紧密连接蛋白封闭蛋白（Occludin）、炎性因子HMGB1及其下游信号分子核转录因子-κB p65（NF-κB p65）的mRNA与蛋白表达水平。结果:小肠组织病理学及超微结构观察结果显示，LPS组小肠组织黏膜明显水肿，部分腺体不完整，白细胞浸润增多，微绒毛缺失，排列紊乱，紧密连接数量较PBS对照组明显减少；LPS组黏膜屏障通透性血浆标志物D-乳酸和DAO水平、炎性因子HMGB1及其下游信号分子NF-κB p65的mRNA与蛋白表达水平均较PBS对照组明显升高，小肠组织中Occludin的mRNA和蛋白表达水平较PBS对照组明显降低，提示脓毒症大鼠小肠组织肠黏膜屏障损伤，通透性增加，且结构受损。而给予HMGB1抑制剂EP干预后，LPS诱导的小肠组织肠黏膜屏障损伤得到明显改善，表现为：EP干预组小肠组织Chiu评分及血浆D-乳酸和DAO水平均较LPS组明显降低〔Chiu评分（分）：1.60±0.48比3.40±0.48，D-乳酸（mmol/L）：3.30±0.22比5.30±0.16，DAO（U/L）：23.66±0.97比30.47±1.11，均n P<0.05〕，且小肠组织中Occludin的mRNA和蛋白表达水平较LPS组明显升高〔Occludin mRNA（2n -ΔΔCt）：0.82±0.05比0.37±0.08，Occludin蛋白（Occludin/β-actin）：1.04±0.09比0.75±0.11，均n P<0.05〕，而炎性因子HMGB1及其下游信号分子NF-κB p65的mRNA和蛋白表达水平较LPS组明显降低〔HMGB1 mRNA（2n -ΔΔCt）：1.63±0.10比3.57±0.10，HMGB1蛋白（HMGB1/β-actin）：1.40±0.07比1.87±0.07；NF-κB p65 mRNA（2n -ΔΔCt）：1.47±0.09比2.62±0.13，NF-κB p65蛋白（NF-κB p65/β-actin）：1.24±0.14比1.60±0.13，均n P<0.05〕。n 结论:脓毒症大鼠肠黏膜屏障损伤，通透性增加，且结构受损，其机制可能是炎性因子HMGB1表达上调并促进其下游信号分子NF-κB激活，从而介导了炎症级联反应，导致肠黏膜损伤。“,”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, alln P < 0.05]. Occludin mRNA and protein expression levels were significantly higher than those in the LPS group [Occludin mRNA (2 n -ΔΔCt): 0.82±0.05 vs. 0.37±0.08, Occludin protein (Occludin/β-actin): 1.04±0.09 vs. 0.75±0.11, bothn 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 n -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 (2n -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, alln P < 0.05].n 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.