槟榔碱通过促进巨噬细胞分泌含miR-155-5p外泌体诱导人口腔成纤维细胞的活化

Arecoline induces activation of human oral fibroblasts by promoting macrophage secretion of exosomes containing miR-155-5p

目的探讨槟榔碱(ARE)通过调控巨噬细胞外泌体内miR-155-5p水平诱导人口腔黏膜成纤维细胞向成纤维表型转化的作用机制。方法以人单核细胞系(THP-1)与人口腔黏膜成纤维细胞系(HOMF)为研究对象。实验设为空白对照组、阴性对照组、ARE刺激组、ARE刺激后THP-1培养上清刺激组(CS)、ARE刺激后THP-1外泌体刺激组(EXO)、ARE刺激后THP-1无外泌体上清刺激组(NES)、miR-155-5p过表达组(miR-155-5p mimics)、ARE刺激后EXO处理对照组(NC+EXO-ARE)和miR-155-5p抑制联合ARE刺激后EXO处理(miR-155-5p inhibitor EXO-ARE)组。以流式细胞术检测THP-1细胞极化情况。Transwell细胞迁移实验检测HOMF细胞迁移能力。DCFH-DA检测细胞氧化应激水平。qRT-PCR检测细胞α-SMA、I型胶原和SOCS1的mRNA转录水平。免疫印迹实验检测细胞α-SMA、I型胶原与SOCS1蛋白表达。结果流式检测显示,相较于对照组,ARE组THP-1细胞由M0趋向M1极化。ARE对THP-1和HOMF细胞的刺激结果显示,相较于对照组,CS组与EXO组HOMF细胞迁移能力增强;细胞内氧化应激水平上调;细胞内miR-155-5p水平升高;细胞内α-SMA、I型胶原mRNA转录增多(1.90±0.08 vs 3.17±0.08;3.19±0.05 vs 5.65±0.01,P<0.05),SOCS1的m RNA转录减少(1.08±0.06 vs 0.34±0.02,P<0.05);α-SMA、I型胶原蛋白表达上调,SOCS1蛋白表达下调。miR-155-5p操纵实验显示,与阴性对照组相比,miR-155-5p mimics组HOMF细胞迁移能力增强,细胞内α-SMA和I型胶原蛋白表达上调;而与NC+EXO-ARE组相比,miR-155-5p inhibitor组HOMF细胞迁移能力减弱,细胞内α-SMA和I型胶原mRNA转录减少(5.84±0.08 vs 2.00±0.98;7.32±0.51 vs 2.33±0.43,P<0.05);α-SMA、I型胶原蛋白表达下调。结论ARE可上调THP-1胞内miR-155-5p并经外泌体途径转导和抑制HOMF胞内SOCS1蛋白表达,促进HOMF细胞的纤维化表型转化。

Objective To investigate the mechanism by which arecoline regulates the level of miR-155-5p in macrophagesecreted exosomes to induce the transformation of human oral mucosal fibroblasts(HOMFs)into fibroblast phenotype.Methods Exosomes were harvested from human monocytic cell line THP-1 with or without arecoline treatment.The effects of arecoline-treated THP-1 cell culture supernatant(CS),THP-1-derived exosomes(EXO),exosome-depleted THP-1 cell supernatant(NES),miR-155-5p overexpression,and miR-155-5p inhibitor on migration ability of arecoline-treated HOMF cells were examined using Transwell migration assay.The polarization of THP-1 cells was detected using flow cytometry.DCFHDA was used to detect the level of oxidative stress in the cells with different treatments.The mRNA and protein expressions ofα-SMA,type I collagen and SOCS1 in the cells were detected with qRT-PCR and Western blotting.Results Flow cytometry showed that arecoline-treated THP-1 cells exhibited obvious polarization from M0to M1.Both the supernatant and exosomes from arecoline-treated THP-1 cells significantly enhanced the migration ability of HOMF cells,increased intracellular oxidative stress,up-regulated the expressions of miR-155-5p and the mRNA and protein levels ofα-SMA and type I collagen,and lowered the mRNA and protein expressions of SOCS1.In HOMF cells treated with exosomes from arecoline-treated THP-1cells,overexpression of miR-155-5p significantly enhanced cell migration ability and increased cellular expressions ofα-SMA and type I collagen,and miR-155-5p inhibitor caused the opposite changes.Conclusion Arecoline can up-regulate miR-155-5p expression in THP-1 cells and inhibit the expression of SOCS1 protein in HOMF cells via the exosome pathway,thus promoting the fibrotic phenotype transformation of HOMF cells.