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Abstract：
BACKGROUND: Cardiac arrest (CA), a common disease with a high mortality rate, is a leading cause of ischemia/reperfusion (I/R)-induced dysfunction of the intestinal barrier. Long non-coding RNAs (lncRNAs) play crucial roles in multiple pathological processes. However, the effect of the lncRNA maternally expressed 3 (MEG3) on intestinal I/R injury and the intestinal barrier has not been fully determined. Therefore, this study aimed to investigate the function of MEG3 in CA-induced intestinal barrier dysfunction.
METHODS: The oxygen and glucose deprivation (OGD) model in the human colorectal adenocarcinoma Caco-2 cells and in vivo cardiac arrest-induced intestinal barrier dysfunction model in Sprague-Dawley (SD) rats were established. The effect and underlying mechanism of MEG3 on the intestinal barrier from cardiac arrest-induced ischemia/reperfusion injury were analyzed by methyl thiazolyl tetrazolium (MTT) assays, Annexin V-FITC/PI apoptosis detection kit, Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) staining, quantitative polymerase chain reaction (qPCR) assays, Western blot analysis, luciferase reporter gene assays, transepithelial electrical resistance (TEER) measurements, immunofluorescence analysis, and enzyme-linked immunosorbent assay (ELISA) assays.
RESULTS: Interestingly, we found that MEG3 could protect Caco-2 cells from oxygen-glucose deprivation (OGD)/reoxygenation-induced I/R injury by modulating cell proliferation and apoptosis. Moreover, MEG3 relieved OGD-induced intestinal barrier dysfunction in vitro, as demonstrated by its significant rescue effect on transepithelial electrical resistance and the expression of tight junction proteins such as occludin and claudin-1 (CLDN1), which were impaired in OGD-treated Caco-2 cells. Mechanistically, MEG3 inhibited the expression of inflammatory factors including interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interferon-gamma (IFN)-γ, inflammatory factors including interleukin (IL)-10, and transforming growth factor beta (TGFb)-1, as well as nuclear factor-kappa B (NF-κB) signaling. In response to OGD treatment in vitro, MEG3 also activated the expression of sirtuin 1 (SIRT1) by Caco-2 cells via sponging miR-34a-3p. Furthermore, MEG3 relieved CA-induced intestinal barrier dysfunction through NF-κB signaling in vivo.
CONCLUSIONS: LncRNA MEG3 can protect the intestinal barrier from cardiac arrest-induced I/R injury via miR-34a-3p/SIRT1/NF-κB signaling. This finding provides new insight into the mechanism by which MEG3 restores intestinal barrier function following I/R injury, presenting it as a potential therapeutic candidate or strategy in intestinal injury.
METHODS: The oxygen and glucose deprivation (OGD) model in the human colorectal adenocarcinoma Caco-2 cells and in vivo cardiac arrest-induced intestinal barrier dysfunction model in Sprague-Dawley (SD) rats were established. The effect and underlying mechanism of MEG3 on the intestinal barrier from cardiac arrest-induced ischemia/reperfusion injury were analyzed by methyl thiazolyl tetrazolium (MTT) assays, Annexin V-FITC/PI apoptosis detection kit, Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) staining, quantitative polymerase chain reaction (qPCR) assays, Western blot analysis, luciferase reporter gene assays, transepithelial electrical resistance (TEER) measurements, immunofluorescence analysis, and enzyme-linked immunosorbent assay (ELISA) assays.
RESULTS: Interestingly, we found that MEG3 could protect Caco-2 cells from oxygen-glucose deprivation (OGD)/reoxygenation-induced I/R injury by modulating cell proliferation and apoptosis. Moreover, MEG3 relieved OGD-induced intestinal barrier dysfunction in vitro, as demonstrated by its significant rescue effect on transepithelial electrical resistance and the expression of tight junction proteins such as occludin and claudin-1 (CLDN1), which were impaired in OGD-treated Caco-2 cells. Mechanistically, MEG3 inhibited the expression of inflammatory factors including interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interferon-gamma (IFN)-γ, inflammatory factors including interleukin (IL)-10, and transforming growth factor beta (TGFb)-1, as well as nuclear factor-kappa B (NF-κB) signaling. In response to OGD treatment in vitro, MEG3 also activated the expression of sirtuin 1 (SIRT1) by Caco-2 cells via sponging miR-34a-3p. Furthermore, MEG3 relieved CA-induced intestinal barrier dysfunction through NF-κB signaling in vivo.
CONCLUSIONS: LncRNA MEG3 can protect the intestinal barrier from cardiac arrest-induced I/R injury via miR-34a-3p/SIRT1/NF-κB signaling. This finding provides new insight into the mechanism by which MEG3 restores intestinal barrier function following I/R injury, presenting it as a potential therapeutic candidate or strategy in intestinal injury.
摘要：
背景：心脏骤停（CA），一种高死亡率的常见疾病，是缺血/再灌注(I/R)诱导的肠屏障功能障碍的主要原因。长链非编码RNA(lncRNAs)在多种病理过程中起着至关重要的作用。然而,母体表达的lncRNA3（MEG3）对肠I/R损伤和肠屏障的影响尚未完全确定。因此,本研究旨在探讨MEG3在CA诱导的肠屏障功能障碍中的作用。
方法：建立人结直肠腺癌Caco-2细胞氧糖剥夺（OGD）模型和SD大鼠心脏骤停诱导的肠屏障功能障碍模型。通过甲基噻唑基四唑（MTT）分析MEG3对心脏骤停引起的缺血/再灌注损伤肠屏障的影响和潜在机制。膜联蛋白V-FITC/PI凋亡检测试剂盒,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）染色，定量聚合酶链反应（qPCR）测定，蛋白质印迹分析,荧光素酶报告基因测定，跨上皮电阻（TEER）测量，免疫荧光分析，和酶联免疫吸附测定(ELISA)测定。
结果：有趣的是，我们发现MEG3可以通过调节细胞增殖和凋亡来保护Caco-2细胞免受氧糖剥夺（OGD）/复氧诱导的I/R损伤。此外，MEG3在体外减轻OGD诱导的肠屏障功能障碍，如其对跨上皮电阻和紧密连接蛋白如occludin和claudin-1（CLDN1）的表达的显着挽救作用所证明的，在OGD处理的Caco-2细胞中受损。机械上,MEG3抑制炎症因子白细胞介素(IL)-1β的表达,肿瘤坏死因子（TNF）-α，干扰素-γ（IFN）-γ，炎症因子包括白细胞介素（IL）-10和转化生长因子β（TGFb）-1，以及核因子-κB（NF-κB）信号。为了响应体外OGD处理，MEG3还通过海绵作用miR-34a-3p激活Caco-2细胞的沉默调节蛋白1(SIRT1)的表达。此外，MEG3通过体内NF-κB信号缓解CA诱导的肠屏障功能障碍。
结论：LncRNAMEG3可通过miR-34a-3p/SIRT1/NF-κB信号通路保护肠屏障免受心脏骤停诱导的I/R损伤。这一发现为MEG3在I/R损伤后恢复肠屏障功能的机制提供了新的见解。将其作为肠道损伤的潜在治疗候选物或策略。
方法：建立人结直肠腺癌Caco-2细胞氧糖剥夺（OGD）模型和SD大鼠心脏骤停诱导的肠屏障功能障碍模型。通过甲基噻唑基四唑（MTT）分析MEG3对心脏骤停引起的缺血/再灌注损伤肠屏障的影响和潜在机制。膜联蛋白V-FITC/PI凋亡检测试剂盒,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）染色，定量聚合酶链反应（qPCR）测定，蛋白质印迹分析,荧光素酶报告基因测定，跨上皮电阻（TEER）测量，免疫荧光分析，和酶联免疫吸附测定(ELISA)测定。
结果：有趣的是，我们发现MEG3可以通过调节细胞增殖和凋亡来保护Caco-2细胞免受氧糖剥夺（OGD）/复氧诱导的I/R损伤。此外，MEG3在体外减轻OGD诱导的肠屏障功能障碍，如其对跨上皮电阻和紧密连接蛋白如occludin和claudin-1（CLDN1）的表达的显着挽救作用所证明的，在OGD处理的Caco-2细胞中受损。机械上,MEG3抑制炎症因子白细胞介素(IL)-1β的表达,肿瘤坏死因子（TNF）-α，干扰素-γ（IFN）-γ，炎症因子包括白细胞介素（IL）-10和转化生长因子β（TGFb）-1，以及核因子-κB（NF-κB）信号。为了响应体外OGD处理，MEG3还通过海绵作用miR-34a-3p激活Caco-2细胞的沉默调节蛋白1(SIRT1)的表达。此外，MEG3通过体内NF-κB信号缓解CA诱导的肠屏障功能障碍。
结论：LncRNAMEG3可通过miR-34a-3p/SIRT1/NF-κB信号通路保护肠屏障免受心脏骤停诱导的I/R损伤。这一发现为MEG3在I/R损伤后恢复肠屏障功能的机制提供了新的见解。将其作为肠道损伤的潜在治疗候选物或策略。
