Abstract：
BACKGROUND: Endothelial barrier dysfunction is critical for the pathogenesis of sepsis-induced acute lung injury (ALI). Lipopolysaccharide (LPS)-stimulated human pulmonary microvascular endothelial cells (HPMECs) are widely used as the cell model of sepsis-associated ALI for exploration of endothelial barrier dysfunction. Dickkopf (DKK) family proteins were reported to mediate endothelial functions in various diseases. The present study explored the effect of Dickkopf-3 (DKK3) on endothelial barrier permeability, angiogenesis, and tight junctions in LPS-stimulated HPMECs.
METHODS: RT-qPCR was required for detecting DKK3 and miR-98-3p expression. The angiogenesis of HPMECs was evaluated by tube formation assays. Monolayer permeability of HPMECs was examined by Transwell rhodamine assays. The protein expression of DKK3 and tight junctions in HPMECs was measured via western blotting. Luciferase reporter assay was used to verify the interaction between miR-98-3p and DKK3.
RESULTS: LPS treatment inhibited angiogenetic ability while increasing the permeability of HPMECs. DKK3 expression was upregulated while miR-98-3p level was reduced in LPS-treated HPMECs. DKK3 knockdown alleviated HPMEC injury triggered by LPS stimulation. MiR-98-3p targeted DKK3 in HPMECs. Overexpression of miR-98-3p protects HPMECs from the LPS-induced endothelial barrier dysfunction, and the protective effect was reversed by DKK3 overexpression.
CONCLUSIONS: MiR-98-3p ameliorates LPS-evoked pulmonary microvascular endothelial barrier dysfunction in sepsis-associated ALI by targeting DKK3.
METHODS: RT-qPCR was required for detecting DKK3 and miR-98-3p expression. The angiogenesis of HPMECs was evaluated by tube formation assays. Monolayer permeability of HPMECs was examined by Transwell rhodamine assays. The protein expression of DKK3 and tight junctions in HPMECs was measured via western blotting. Luciferase reporter assay was used to verify the interaction between miR-98-3p and DKK3.
RESULTS: LPS treatment inhibited angiogenetic ability while increasing the permeability of HPMECs. DKK3 expression was upregulated while miR-98-3p level was reduced in LPS-treated HPMECs. DKK3 knockdown alleviated HPMEC injury triggered by LPS stimulation. MiR-98-3p targeted DKK3 in HPMECs. Overexpression of miR-98-3p protects HPMECs from the LPS-induced endothelial barrier dysfunction, and the protective effect was reversed by DKK3 overexpression.
CONCLUSIONS: MiR-98-3p ameliorates LPS-evoked pulmonary microvascular endothelial barrier dysfunction in sepsis-associated ALI by targeting DKK3.
摘要：
背景：内皮屏障功能障碍对于脓毒症诱导的急性肺损伤（ALI）的发病机制至关重要。脂多糖（LPS）刺激的人肺微血管内皮细胞（HPMECs）被广泛用作脓毒症相关ALI的细胞模型，以探索内皮屏障功能障碍。据报道，Dickkopf（DKK）家族蛋白在各种疾病中介导内皮功能。本研究探讨了Dickkopf-3（DKK3）对内皮屏障通透性的影响，血管生成,和LPS刺激的HPMECs中的紧密连接。
方法：检测DKK3和miR-98-3p表达需要RT-qPCR。HPMEC的血管生成通过管形成测定来评价。通过Transwell罗丹明测定法检查HPMEC的单层渗透性。通过蛋白质印迹法测量HPMEC中DKK3和紧密连接的蛋白质表达。荧光素酶报告基因测定用于验证miR-98-3p与DKK3之间的相互作用。
结果：LPS处理抑制血管生成能力，同时增加HPMECs的通透性。在LPS处理的HPMECs中,DKK3表达上调,而miR-98-3p水平降低。DKK3敲低减轻了LPS刺激引发的HPMEC损伤。MiR-98-3p靶向HPMEC中的DKK3。miR-98-3p过表达保护HPMECs免受LPS诱导的内皮屏障功能障碍,DKK3过表达逆转了保护作用。
结论：MiR-98-3p通过靶向DKK3改善脓毒症相关ALI中LPS诱发的肺微血管内皮屏障功能障碍。
方法：检测DKK3和miR-98-3p表达需要RT-qPCR。HPMEC的血管生成通过管形成测定来评价。通过Transwell罗丹明测定法检查HPMEC的单层渗透性。通过蛋白质印迹法测量HPMEC中DKK3和紧密连接的蛋白质表达。荧光素酶报告基因测定用于验证miR-98-3p与DKK3之间的相互作用。
结果：LPS处理抑制血管生成能力，同时增加HPMECs的通透性。在LPS处理的HPMECs中,DKK3表达上调,而miR-98-3p水平降低。DKK3敲低减轻了LPS刺激引发的HPMEC损伤。MiR-98-3p靶向HPMEC中的DKK3。miR-98-3p过表达保护HPMECs免受LPS诱导的内皮屏障功能障碍,DKK3过表达逆转了保护作用。
结论：MiR-98-3p通过靶向DKK3改善脓毒症相关ALI中LPS诱发的肺微血管内皮屏障功能障碍。
