Hu Zongqiang, Zhao Yingpeng, Jiang Jie, Li Wang, Su Gang, Li Li, Ran Jianghua
First People's Hospital of Kunming City, Kunming, China.
The Calmette Affiliated Hospital of Kunming Medical University, Kunming, China.
Environ Toxicol. 2023 Aug;38(8):1860-1873. doi: 10.1002/tox.23813. Epub 2023 May 20.
This study aims to explore the effect of liver stem cells (LSCs)-derived exosomes and the miR-142a-5p carried by them on the process of fibrosis by regulating macrophages polarization.
In this study, CCL was used to establish liver fibrosis model. The morphology and purity of exosomes (EVs) were verified by transmission electron microscopy, western blotting (WB) and nanoparticle tracing analysis (NTA). Real-time quantitative PCR (qRT-PCR), WB and enzyme-linked immunoadsorption (ELISA) were used to detect liver fibrosis markers, macrophage polarization markers and liver injury markers. Histopathological assays were used to verify the liver injury morphology in different groups. The cell co-culture model and liver fibrosis model were constructed to verify the expression of miR-142a-5p and ctsb.
Immunofluorescence of LSCs markers CK-18, epithelial cell adhesion molecule (EpCam), and AFP showed that these markers were up-regulated in LSCs. In addition, we evaluated the ability of LSCs to excrete EVs by labeling LSCs-EVs with PKH67. We found that CCL and EVs were simultaneously treated at 50 and 100 μg doses, and both doses of EVs could reduce the degree of liver fibrosis in mice. We tested markers of M1 or M2 macrophage polarization and found that EVs reduced M1 marker expression and promoted M2 marker expression. Further, ELISA was used to detect the secreted factors related to M1 and M2 in tissue lysates, which also verified the above views. Further analysis showed that the expression of miR-142a-5p increased significantly with the increase of EVs treatment concentration and time. Further, in vitro and in vivo LSCs-EVs regulate macrophage polarization through miR-142a-5p/ctsb pathway and affect the process of liver fibrosis.
Our data suggest that EVs-derived miR-142-5p from LSCs improves the progression of liver fibrosis by regulating macrophage polarization through ctsb.
本研究旨在探讨肝干细胞（LSCs）来源的外泌体及其携带的miR-142a-5p通过调节巨噬细胞极化对纤维化进程的影响。
本研究中，采用CCL建立肝纤维化模型。通过透射电子显微镜、蛋白质印迹法（WB）和纳米颗粒追踪分析（NTA）验证外泌体（EVs）的形态和纯度。采用实时定量聚合酶链反应（qRT-PCR）、WB和酶联免疫吸附测定（ELISA）检测肝纤维化标志物、巨噬细胞极化标志物和肝损伤标志物。采用组织病理学检测验证不同组的肝损伤形态。构建细胞共培养模型和肝纤维化模型以验证miR-142a-5p和组织蛋白酶B（ctsb）的表达。
LSCs标志物细胞角蛋白18（CK-18）、上皮细胞黏附分子（EpCam）和甲胎蛋白（AFP）的免疫荧光显示这些标志物在LSCs中上调。此外，我们通过用PKH67标记LSCs-EVs评估LSCs分泌EVs的能力。我们发现以50和100μg剂量同时处理CCL和EVs，两种剂量的EVs均可降低小鼠肝纤维化程度。我们检测了M1或M2巨噬细胞极化标志物，发现EVs降低M1标志物表达并促进M2标志物表达。此外，ELISA用于检测组织裂解物中与M1和M2相关的分泌因子，这也验证了上述观点。进一步分析表明，miR-142a-5p的表达随EVs处理浓度和时间的增加而显著增加。此外，体外和体内LSCs-EVs通过miR-142a-5p/ctsb途径调节巨噬细胞极化并影响肝纤维化进程。
我们的数据表明，LSCs来源的EVs衍生的miR-142-5p通过ctsb调节巨噬细胞极化改善肝纤维化进程。
