Chen Wendong, Ma Li, Shao Jianlin, Bi Chun, Li Junjie, Yang Wei
Department of Anesthesiology, The First Affiliated Hospital of Kunming Medical University, Kunming 650032, Yunnan Province, China.
Heliyon. 2023 Jul 16;9(7):e18325. doi: 10.1016/j.heliyon.2023.e18325. eCollection 2023 Jul.
Intestinal ischemia-reperfusion (II/R) is a common pathological injury in clinic, and the systemic inflammatory response it causes will lead to multiple organ damage and functional failure. miR-185-5p has been reported to be a regulator of inflammatory response and autophagy, but whether it participates in the regulation of autophagy in II/R is still unclear. Therefore, we aimed to explore the mechanism of miR-185-5p regulating intestinal barrier injury in (II/R).
Caco-2 cells was induced by oxygen-glucose deprivation/reoxygenation (OGD/R) to establish II/R model. The superior mesenteric artery of C57BL/6 mice was clamped for 45 min and then subjected to reperfusion for 4 h for the establishment of II/R mice model. miR-185-5p mimic, miR-185-5p inhibitor, pcDNA-autophagy-related 101 (ATG101) were respectively transfected into Caco-2 cells. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to assess miR-185-5p expression. Western blot detected the level of ATG101 and tight junction-associated proteins ZO1, Occludin, E-cadherin, β-catenin, as well as autophagy markers ATG5, ATG12, LC3Ⅰ/Ⅱ, Beclin1 and SQSTM1. Transepithelial electrical resistance (TEER) values was detected by a resistance meter. FITC-Dextran was performed to measure cell permeability. 5-ethynyl-2'-deoxyuridine (EDU) staining measured cell proliferation. Transmission electron microscope was conducted to observe autophagosomes. Hematoxylin & eosin (H&E) staining observed the damage of mice intestinal. Immunohistochemistry (IHC) measured the percentage of ki67 positive cells. TdT-mediated dUTP nick-end labeling (TUNEL) assay assessed cell apoptosis in intestinal tissues of II/R. Dual-luciferase assay verified the targeting relationship between miR-185-5p and ATG101.Results miR-185-5p was overexpressed in OGD/R-induced Caco-2 cells and intestinal tissues of II/R mice. Knocking down miR-185-5p markedly promoted autophagy and TEER values, reduced cell permeability, and alleviated intestinal barrier damage. ATG101 was a target of miR-185-5p, and overexpression of ATG101 promoted autophagy and dampened OGD/R-induced intestinal barrier damage. Overexpression of miR-185-5p reversed the effect of overexpressed ATG101 on OGD/R-induced Caco-2 cells.
Knockdown of miR-185-5p enhanced autophagy and alleviated II/R intestinal barrier damage by targeting ATG101.
肠缺血再灌注（II/R）是临床上常见的病理损伤，其引发的全身炎症反应会导致多器官损伤和功能衰竭。据报道，miR-185-5p是炎症反应和自噬的调节因子，但它是否参与II/R中自噬的调节仍不清楚。因此，我们旨在探讨miR-185-5p调节II/R中肠屏障损伤的机制。
通过氧糖剥夺/复氧（OGD/R）诱导Caco-2细胞建立II/R模型。夹闭C57BL/6小鼠的肠系膜上动脉45分钟，然后再灌注4小时以建立II/R小鼠模型。将miR-185-5p模拟物、miR-185-5p抑制剂、pcDNA-自噬相关101（ATG101）分别转染到Caco-2细胞中。采用实时定量聚合酶链反应（RT-qPCR）评估miR-185-5p的表达。蛋白质免疫印迹法检测ATG101和紧密连接相关蛋白ZO1、闭合蛋白、E-钙黏蛋白、β-连环蛋白的水平，以及自噬标志物ATG5、ATG12、LC3Ⅰ/Ⅱ、Beclin1和SQSTM1。用电阻仪检测跨上皮电阻（TEER）值。采用异硫氰酸荧光素-葡聚糖（FITC-Dextran）检测细胞通透性。用5-乙炔基-2'-脱氧尿苷（EDU）染色检测细胞增殖。通过透射电子显微镜观察自噬体。采用苏木精-伊红（H&E）染色观察小鼠肠道损伤情况。免疫组织化学（IHC）检测ki67阳性细胞的百分比。采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）法评估II/R小鼠肠道组织中的细胞凋亡。双荧光素酶报告基因检测验证miR-185-5p与ATG101之间的靶向关系。结果miR-185-5p在OGD/R诱导的Caco-2细胞和II/R小鼠的肠道组织中过表达。敲低miR-185-5p可显著促进自噬和提高TEER值，降低细胞通透性，并减轻肠屏障损伤。ATG101是miR-185-5p的靶标，ATG101的过表达促进自噬并减轻OGD/R诱导的肠屏障损伤。miR-185-5p的过表达逆转了过表达ATG101对OGD/R诱导的Caco-2细胞的影响。
敲低miR-185-5p通过靶向ATG101增强自噬并减轻II/R肠屏障损伤。
