南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (1): 60-67.doi: 10.12122/j.issn.1673-4254.2023.01.08

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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

黄永祺，喻 伟，游月华   

  1. 南方医科大学附属深圳市龙华人民医院口腔科，广东 深圳 518109；南方医科大学口腔医学院，广东 广州 510515
  • 出版日期:2023-01-20 发布日期:2023-02-22

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

HUANG Yongqi, YU Wei, YOU Yuehua   

  1. Department of Stomatology, Longhua People's Hospital Affiliated to Southern Medical University, Shenzhen 518109, China; School of Stomatology,Southern Medical University, Guangzhou 510515, China
  • Online:2023-01-20 Published:2023-02-22

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摘要： 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mRNA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。

关键词: 槟榔碱；口腔黏膜下纤维化；外泌体；口腔成纤维细胞；巨噬细胞

Abstract: Objective To investigate the mechanism by which arecoline regulates the level of miR-155-5p in macrophage-secreted 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. DCFH-DA 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 M0 to 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 of α-SMA and type I collagen, and lowered the mRNA and protein expressions of SOCS1. In HOMF cells treated with exosomes from arecoline- treated THP-1 cells, overexpression of miR-155-5p significantly enhanced cell migration ability and increased cellular expressions of α-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.

Key words: arecoline; oral submucosal fibrosis; exosome; oral fibroblasts; macrophages