脂多糖刺激巨噬细胞分泌含miR-155-5p的外泌体促进肝星状细胞的活化及迁移  

Lipopolysaccharide stimulates macrophages to secrete exosomes containing miR-155-5p to promote activation and migration of hepatic stellate cells

作　　者：林嘉宜 娄安妮[1] 李旭[1,2] LIN Jiayi;LOU Anni;LI Xu(Department of Emergency Medicine,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China;Key Laboratory of First Aid and Trauma Research,Ministry of Education,Hainan Medical College,Haikou 571199,China)

机构地区：[1]南方医科大学南方医院急诊科,广东广州510515 [2]海南医学院急救与创伤研究教育部重点实验室,海南海口571199

出　　处：《南方医科大学学报》2023年第6期994-1001,共8页Journal of Southern Medical University

基　　金：广州市科技计划项目(201904010482);广东省自然科学基金(2021A1515012595,2017B020209003);急救与创伤研究教育部重点实验室(海南医学院)开放课题基金(Grant.KLET-202102)。

摘　　要：目的探索脂多糖(LPS)刺激下的巨噬细胞来源的外泌体对肝星状细胞的激活及迁移能力的影响及分子机制。方法以100 ng/mL丙二醇甲醚醋酸(PMA)处理人THP-1巨噬细胞24 h,诱导其分化为巨噬细胞,给予脂多糖刺激后收集巨噬细胞的培养上清,运用超速离心法提取外泌体并加以鉴定。荧光定量PCR(qRT-PCR)检测外泌体中miR-155-5p的表达。采用Transwell共培养体系观察巨噬细胞分泌的外泌体对肝星状细胞LX2增殖、氧化应激、迁移和I型胶原等纤维化标志物表达的影响。同时,LX2转染miR-155-5p-mimics及miR-155-5p-inhibitors分别过表达和干扰miR-155-5p,从正反两方面去验证miR-155-5p对LX2功能的影响。Western blot检测SOCS1以及其下游信号通路蛋白的表达。结果与对照相比,经脂多糖刺激后巨噬细胞的外泌体处理的LX2细胞增殖迁移能力增强,氧化应激水平和I型胶原等纤维化标志物的表达明显增高(P<0.05)。巨噬细胞经脂多糖处理后分泌的外泌体中miR-155-5p的表达显著增高(P<0.01)。miR-155-5p过表达的LX2中增殖和迁移能力增强,氧化应激水平和I型胶原等纤维化标志物的表达明显增高(P<0.05);而干扰miR-155-5p表达发挥相反的作用。进一步研究表明miR-155-5p抑制SOCS1的表达,促进p-Smad2/3、Smad 2/3和RhoA蛋白表达(P<0.05)。结论脂多糖刺激下巨噬细胞来源的外泌体miR-155-5p促进肝星状细胞的活化、迁移、氧化应激及胶原生成。Objective To observe the effect of exosomes secreted by lipopolysaccharides(LPS)-stimulated macrophages on hepatic stellate cell activation and migration and explore the underlying molecular mechanism.Methods Human monocyte THP-1 cells were induced to differentiate into macrophages using propylene glycol methyl ether acetic acid(PMA,100 ng/mL,24 h)followed by stimulation with LPS,and the culture supernatant of macrophages was collected for extraction of the exosomes by ultracentrifugation.The expression of miR-155-5p in the exosomes was detected using qRT-PCR.A Transwell coculture system was used to observe the effects of the macrophage-derived exosomes on LX2 cell(a hepatic stellate cell line)proliferation,migration,oxidative stress and the expression of fibrosis biomarkers,which were also observed in LX2 cells transfected with miR-155-5p-mimics or miR-155-5p-inhibitors.Western blotting was used to detect the expressions of SOCS1 and its downstream signal pathway proteins.Results Treatment with the exosomes from LPS-stimulated macrophages significantly enhanced the proliferation and migration ability of LX2 cells and increased the levels of oxidative stress and expressions of the fibrosis markers such as type I collagen(P<0.05).The expression of miR-155-5p in the exosomes secreted by macrophages was significantly increased after LPS treatment(P<0.01).LX2 cells overexpressing miR-155-5p also exhibited significantly enhanced proliferation and migration with increased oxidative stress levels and expression of type I collagen(P<0.05),and interference of miR-155-5p expression produced the opposite effects.Western blotting showed that miR-155-5p overexpression obviously inhibited SOCS1 expression and promoted p-Smad2/3,Smad2/3 and RhoA protein expressions in LX2 cells(P<0.05).Conclusion LPS stimulation of the macrophages increases miR-155-5p expression in the exosomes to promote activation and migration and increase oxidative stress and collagen production in hepatic stellate cells.

关 键 词：脂多糖 巨噬细胞 外泌体 miR-155-5p 肝星状细胞 肝纤维化 

分 类 号：R575.2[医药卫生—消化系统]