李志华,黄玮玮,马 涛,王 毅,于湘友.miR-155通过促进炎症因子释放参与脓毒症肠道屏障功能障碍的发生发展[J].现代生物医学进展英文版,2023,(13):2412-2418. |
miR-155通过促进炎症因子释放参与脓毒症肠道屏障功能障碍的发生发展 |
MiR-155 is Involved In The Occurrence and Development of Intestinal Barrier Dysfunction In Sepsis by Prompting The Release of Inflammatory Cytokines |
Received:February 10, 2023 Revised:February 28, 2023 |
DOI:10.13241/j.cnki.pmb.2023.13.003 |
中文关键词: 脓毒症 肠道屏障功能障碍 miR-155 |
英文关键词: Sepsis Intestinal barrier dysfunction MiR-155 |
基金项目:国家自然科学基金项目(82160360);新疆维吾尔自治区科技支疆项目(2021E02064) |
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中文摘要: |
摘要 目的:探讨miR-155在脓毒症致肠道功能障碍中的表达及作用。方法:(1)临床实验:以2022年5月至2022年8月入住新疆医科大学第一附属医院重症医学科的脓毒症患者和同期健康体检者为研究对象,根据急性胃肠损伤分级(AGI)将脓毒症患者分为AGI组和非AGI组。根据28 d生存情况分为存活组和死亡组,采用实时荧光定量反转录-聚合酶链反应(qRT-PCR)法前瞻性观察各组外周血miR-155的变化。(2)体内实验:将20只雄性S/D大鼠按照随机数字表法分为假手术组(sham组)和盲肠结扎穿孔术组(CLP组),采用qRT-PCR及Western blot法检测miR-155、肠道紧密连接蛋白(ZO-1、claudin-1)的表达,ELISA法检测大鼠肠道组织中IL-1β、IL-6、IL-18的表达水平。(3)体外实验:培养人结直肠腺癌细胞(Caco-2),并分为正常对照组(完全培养基培养48 h)、脂多糖组(完全培养基培养24 h后加入10 μg/mL LPS处理24 h)。采用qRT-PCR检测miR-155的表达。在倒置荧光显微镜下观察细胞形态的变化。采用CCK-8检测细胞活力。采用免疫荧光观察Caco-2细胞中紧密连接蛋白ZO-1分布的变化。并行细胞旁通透性实验,观察两组细胞旁通透性的变化。结果:(1)脓毒症组和健康对照组相比,脓毒症患者外周血miR-155较健康对照组明显升高,差异具有统计学意义(P<0.05)。AGI组患者外周血miR-155表达较非AGI组明显升高,差异具有统计学意义(P<0.05)。脓毒症死亡患者外周血miR-155表达显著升高(P<0.05)。(2)应用CLP模型进行体内动物实验,CLP组大鼠肠道组织miR-155表达较假手术组(sham组)明显升高。CLP组大鼠肠道紧密连接蛋白(ZO-1、claudin-1)较sham组下降,差异具有统 计学意义(P<0.05)。ELISA结果提示,CLP大鼠肠道组织中IL-1β、IL-6、IL-18水平较sham组显著升高(P<0.05),且肠道组织中miR-155水平与IL-1β、IL-6、IL-18呈正相关(r=0.542,r=0.906,r=868,P<0.05)。(3)与正常对照组相比,经LPS处理后细胞形态破坏、细胞间紧密连接破坏、细胞活力减弱、细胞旁通透性增加。结论:脓毒症发生时伴有肠道屏障功能障碍,miR-155在脓毒症肠道屏障功能障碍中表达升高,并可能通过促进炎症因子的释放参与脓毒症肠道屏障功能障碍的发生发展。miR-155异常表达对脓毒症患者早期肠道功能障碍诊断及预后的评估具有重要价值,可作为脓毒症早期肠道损伤诊断及预后情况的重要指标。 |
英文摘要: |
ABSTRACT Objective: To investigate the mechanism of Mir-155 in intestinal tissue injury caused by sepsis. Methods: (1) Clinical experiment: Sepsis patients admitted to the Department of Critical Care Medicine of the First Affiliated Hospital of Xinjiang Medical University from May 2022 to August 2022 and healthy subjects during the same period were selected as the research objects. According to the grade of acute gastrointestinal injury (AGI), sepsis patients were divided into AGI group and non-AGI group. According to the 28-day survival, the patients were divided into survival group and death group. The changes of MiR-155 in peripheral blood of each group were prospectively observed by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). (2) In vivo experiment: Twenty male S/D rats were randomly divided into sham group (Sham group) and cecal ligation and perforation group (CLP group). The expressions of MiR-155 and intestinal tight junction proteins (ZO-1 and claudin-1) were detected by qRT-PCR and Western blot. The expression levels of IL-1β, IL-6 and IL-18 in rat intestinal tissues were determined by ELISA. (3) In vitro experiment: human colorectal adenocarcinoma cells (Caco-2) were cultured and divided into normal control group (complete culture medium for 48 h) and LPS group (complete culture medium for 24 h and then add 10 μg/mL LPS for 24 h). The expression of MiR-155 was detected by qRT-PCR. The changes of cell morphology were observed under an inverted fluorescence microscope. Cell viability was detected by CCK-8. The changes of intestinal tight junction protein (ZO-1) were observed by immunofluorescence. Transwell chamber was used for paracellular permeability test to observe the changes of paracellular permeability between the two groups. Results: (1) Compared with healthy control group, peripheral blood miR-155 in sepsis group was significantly higher than that in healthy control group, with statistical significance(P<0.05). The expression of miR-155 in peripheral blood of AGI group was significantly higher than that of non-AGI group, with statistical significance(P<0.05). The expression of miR-155 in peripheral blood of patients with sepsis death was significantly increased(P<0.05). (2) In vivo animal experiments using the CLP model showed that the expression of miR-155 in the intestinal tissues of rats in the CLP group was significantly higher than that in the sham group. Intestinal compact junction proteins (ZO-1 and claudin-1) in CLP group were decreased compared with those in sham group, and the difference was statistically significant(P<0.05). ELISA results indicated that the levels of IL-1β, IL-6 and IL-18 in the intestinal tissue of CLP rats were significantly higher than those of the sham group(P<0.05), and the level of miR-155 in the intestinal tissue was positively correlated with IL-1β, IL-6 and IL-18(r=0.542, r=0.906, r=868). P<0.05). (3) Compared with the control group, the cell morphology was damaged, the tight connections between cells were damaged, the cell viability was weakened, and the paracellular permeability was increased after LPS treatment. Conclusion: The occurrence of sepsis is accompanied by intestinal barrier dysfunction. The level of miR-155 in septic intestinal barrier dysfunction is increased, and may participate in the occurrence and development of septic intestinal barrier dysfunction by promoting the release of inflammatory factors. The abnormal expression of miR-155 is of great value for the diagnosis and prognosis of early intestinal dysfunction in patients with sepsis, and can be used as an important indicator for the diagnosis and prognosis of early intestinal damage in sepsis. |
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