miR-370-3p/SESN2调控脂多糖诱导的小鼠肺泡上皮细胞凋亡的机制研究  

Mechanism of miR-370-3p/SESN2 regulating lipopolysaccharide-induced apoptosis of mouse alveolar epithelial cells

作　　者：武周游 李婷[1] 张腾伟 房巧燕 杨刘 黎巧 Wu Zhouyou;Li Ting;Zhang Tengwei;Fang Qiaoyan;Yang Liu;Li Qiao(First Department of Neonatology,Hunan Provincial Maternal and Child Health Care Hospital)

机构地区：[1]湖南省妇幼保健院新生儿一科,长沙410008

出　　处：《重庆医科大学学报》2024年第9期1121-1128,共8页Journal of Chongqing Medical University

基　　金：湖南省卫生健康委员会科研资助项目(编号:202102168412)。

摘　　要：目的:探讨miR-370-3p/Sestrin2(SESN2)调控脂多糖(lipopolysaccharide,LPS)诱导的小鼠肺泡上皮细胞(alveolar epithelial cells,AECs)凋亡的机制。方法:将C57BL/6J小鼠随机分为LPS+anti-NC组和LPS+miR-370-3p抑制剂(anti-miR-370-3p)组,每组12只。通过气管内注射LPS(5 mg/kg)来诱导急性肺损伤(acute lung injury,ALI)。分别通过小鼠尾静脉注射antimiR-370-3p或anti-NC。MLE12细胞随机分为对照组(Con+anti-NC)、Con+anti-miR-370-3p组、LPS+anti-NC组、LPS+antimiR-370-3p组、LPS+si-SESN2+anti-miR-370-3p组。使用定量实时PCR分析肺组织或细胞中miR-370-3p。通过测量线粒体形态学和线粒体膜电位考察miR-370-3p敲低对LPS诱导的线粒体功能障碍的保护作用。双荧光素酶报告分析证实了miR-370-3p和SESN2之间的靶向关系。结果:与LPS+anti-NC组相比,LPS+anti-miR-370-3p组肺组织中miR-370-3p表达、丝氨酸苏氨酸激酶3(receptor-interacting serine/threonine-protein kinase 3,RIPK3)、混合系列蛋白激酶样结构域(mixed lineage kinase domain-Like,MLKL)蛋白水平、肺部炎症评分明显降低(P<0.05),和肺组织中肺表面活性蛋白C(surfactant protein C,SPC)蛋白水平明显增加(P<0.001)。与LPS+anti-NC组相比,LPS+anti-miR-370-3p组MLE12细胞中RIPK3、MLKL蛋白表达、PI阳性细胞百分比明显降低(P<0.05),并且线粒体片段化和线粒体膜电位明显增加(P<0.05)。miR-370-3p对SESN2-WT报道基因的荧光素酶活性具有抑制作用。与LPS+anti-miR-370-3p组相比,LPS+si-SESN2+anti-miR-370-3p组线粒体片段化和线粒体膜电位均明显降低(P<0.05)。结论:miR-370-3p/SESN2轴通过介导线粒体断裂和损害线粒体功能促进LPS诱导的ALI期间AECs坏死性凋亡。Objective:To investigate the mechanism of miR-370-3p/Sestrin2(SESN2)regulating the apoptosis of mouse alveolar epi⁃thelial cells(AECs)induced by lipopolysaccharide(LPS).Methods:C57BL/6J mice were randomly divided into LPS+anti-NC group and LPS+miR-370-3p inhibitor(anti-miR-370-3p)group,with 12 mice in each group.LPS(5 mg/kg)was given by intratracheal in⁃jection to induce acute lung injury(ALI),and anti-miR-370-3p or anti-NC was injected via the caudal vein of mice.MLE12 cells were randomly divided into control group(Con+anti-NC),Con+anti-miR-370-3p group,LPS+anti-NC group,LPS+anti-miR-370-3p group,and LPS+si-SESN2+anti-miR-370-3p group.Quantitative real-time PCR was used to analyze miR-370-3p in lung tissue or cells.Mitochondrial morphology and mitochondrial membrane potential were measured to investigate the protective effect of miR-370-3p knockdown against LPS-induced mitochondrial dysfunction.Dual-luciferase reporter assay confirmed the targeting relation⁃ship between miR-370-3p and SESN2.Results:Compared with the LPS+anti-NC group,the LPS+anti-miR-370-3p group had sig⁃nificant reductions in the expression of miR-370-3p and the protein expression levels of receptor-interacting serine/threonine-protein kinase 3(RIPK3)and mixed lineage kinase domain-like(MLKL)in lung tissue,as well as a significant reduction in lung inflammation score(P<0.05)and a significant increase in the protein expression level of surfactant protein C(SPC)in lung tissue(P<0.001).Com⁃pared with the LPS+anti-NC group,the LPS+anti-miR-370-3p group had significant reductions in the protein expression levels of RIPK3 and MLKL in MLE12 cells and the percentage of PI-positive cells(P<0.05),as well as significant increases in mitochondrial fragmentation and mitochondrial membrane potential(P<0.05).MiR-370-3p could inhibit the luciferase activity of SESN2-WT re⁃porter gene.Compared with the LPS+anti-miR-370-3p group,the LPS+Si-SEN2+anti-miR-370-3p group had significant reductions in mitochondrial fragmentation and mitochondrial membrane

关 键 词：miR-370-3p Sestrin2 脂多糖 肺泡上皮细胞 坏死性凋亡 

分 类 号：R563[医药卫生—呼吸系统]