Qin Qin
1,2,3 
, Yinru Wang
1, Yajun Liu
1, Weiwei An
2,4, Zifang He
1, Feifei Chen
1, Xinxuan Cheng
1, Deyan Chen
5 , Zhenggao Xie
1,2,3* 1 Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, Jiangsu, 210008, China
2 Nanjing Drum Tower Hospital, Clinical College of Nanjing Medical University, Nanjing, Jiangsu, 210008, China
3 Nanjing Drum Tower Hospital, Clinical College of Traditional Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, 210008, China
4 Yancheng Tinghu District People’s Hospital, Yancheng, Jiangsu, 224005, China
5 Key Laboratory of Infection and Immunity of Anhui Higher Education Institutes, Bengbu Medical University, No. 2600, Donghai Avenue, Bengbu, Anhui, 233030, China
Abstract
Introduction: Bacterial keratitis refers to a prevalent sight-threatening ophthalmologic infection. Owing to the challenge of antimicrobial resistance in treating bacterial keratitis, novel therapeutic strategies are needed. Resolvin D1 (RvD1), an endogenous lipid mediator, exhibits anti-inflammatory and immune-regulatory effects. The present study was aimed at investigating whether RvD1 alleviates lipopolysaccharide-induced inflammation to protect corneal fibroblasts and explore its potential mechanisms.
Methods: In this study, Raw264.7 cells were polarized towards M1 or M2 macrophages by the addition of lipopolysaccharides LPS or interleukin (IL)-4, respectively, and were treated with or without RvD1. Flow cytometry and Western blot were used to determine the expression of M1 and M2-related markers. EdU assay and trans-well assay were performed to detect the proliferation and migration ability of corneal fibroblasts. Bioinformatics analysis (GO and KEGG) of RNA-seq was applied to explore the RvD1-related signaling pathways. siRNA-c-Fos was further used to confirm the role of Fos expression in RvD1-mediated macrophage polarization. Flow cytometry and Western blotting analysis were performed to demonstrate that RvD1 alleviated LPS-induced inflammation by suppressing M1 macrophage polarization, facilitating M2 macrophage polarization, and increasing corneal fibroblast proliferation. Bioinformatics analysis identified PI3K-AKT, IL-17, and MAPK signaling pathways as potential targets of RvD1 in corneal inflammation.
Results: Enrichment analysis indicated that the RvD1 target gene showed a strong relationship to the regulation of macrophage polarization. RvD1 highly upregulated M2 macrophages by promoting c-Fos expression and enhanced the proliferation and migration of mouse corneal fibroblasts through modulating c-Fos expression.
Conclusion: Our findings reveal that RvD1 conferred protective effects against LPS-induced inflammation by enhancing M2 macrophage polarization through the promotion of c-Fos expression. Thus, RvD1 may be a potential therapeutic compound for enhancing corneal fibroblast proliferation and migration while attenuating inflammation.