Kiełbasińska Aleksandra, Krysik Katarzyna, Janiszewska-Bil Dominika, Machaj Martyna, Lelek Zuzanna, Szulik Mariola, Mickiewicz Patrycja, Lyssek-Boroń Anita, Grabarek Beniamin Oskar
Department of Opthalmology, University Clinical Center Named After Prof. K. Gibiński of the Medical University of Silesia in Katowice, Katowice 40-514, Poland.
Department of Ophthalmology, Faculty of Medicine, Academy of Silesia, Katowice 40-555, Poland.
Mediators Inflamm. 2025 Jul 1;2025:8586711. doi: 10.1155/mi/8586711. eCollection 2025.
The retinal pigment epithelium (RPE) is central to retinal health and immune regulation. In diseases, such as proliferative vitreoretinopathy (PVR), dysregulated RPE function, driven by aberrant signaling pathways like mitogen-activated protein kinase (MAPK), contributes to fibrotic membrane formation and retinal detachment. Tacrolimus, an immunosuppressive agent, has shown potential to modulate signaling beyond immune cells, but its effect on MAPK signaling in RPE cells remains unclear. This study aimed to investigate the impact of tacrolimus on MAPK pathway gene expression and microRNA (miRNA)-mediated regulation in human RPE (H-RPE) cells under inflammatory conditions induced by lipopolysaccharide (LPS). H-RPE cells were treated with LPS and tacrolimus, and cell viability was evaluated by 3- (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Transcriptomic profiling of 300 MAPK-related genes and corresponding miRNAs was performed using Affymetrix microarrays. Key targets were validated via quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). Gene interaction networks were analyzed with STRING. LPS significantly suppressed MAPK pathway gene and protein expression, including transforming growth factor beta 1 (TGF-β-1), mitogen-activated protein kinase kinase 7 (MAP2K7), mitogen-activated protein kinase 3 (MAPK3), and dual specificity phosphatase 4 (DUSP4). Tacrolimus reversed these effects in a time-dependent manner, restoring expression levels and modulating regulatory miRNAs (e.g., miR-3196, miR-27a/b-3p, miR-190a-3p, miR-149-3p). STRING analysis revealed a highly connected protein network, with MAPK3, MAPK8, and TRAF6 acting as central nodes. Tacrolimus modulates MAPK signaling in H-RPE cells by reversing LPS-induced suppression and regulating specific miRNAs. These findings suggest a potential therapeutic role for tacrolimus in mitigating inflammatory and fibrotic responses associated with PVR.
视网膜色素上皮（RPE）对于视网膜健康和免疫调节至关重要。在诸如增殖性玻璃体视网膜病变（PVR）等疾病中，由丝裂原活化蛋白激酶（MAPK）等异常信号通路驱动的RPE功能失调，会导致纤维化膜形成和视网膜脱离。他克莫司是一种免疫抑制剂，已显示出调节免疫细胞以外信号传导的潜力，但其对RPE细胞中MAPK信号传导的影响仍不清楚。本研究旨在探讨他克莫司在脂多糖（LPS）诱导的炎症条件下对人RPE（H-RPE）细胞中MAPK途径基因表达和微小RNA（miRNA）介导的调控的影响。用LPS和他克莫司处理H-RPE细胞，并通过3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐（MTT）法评估细胞活力。使用Affymetrix微阵列对300个MAPK相关基因和相应的miRNA进行转录组分析。通过定量逆转录聚合酶链反应（RT-qPCR）和酶联免疫吸附测定（ELISA）验证关键靶点。用STRING分析基因相互作用网络。LPS显著抑制MAPK途径基因和蛋白质表达，包括转化生长因子β1（TGF-β-1）、丝裂原活化蛋白激酶激酶7（MAP2K7）、丝裂原活化蛋白激酶3（MAPK3）和双特异性磷酸酶4（DUSP4）。他克莫司以时间依赖性方式逆转这些作用，恢复表达水平并调节调控性miRNA（例如，miR-3196、miR-27a/b-3p、miR-190a-3p、miR-149-3p）。STRING分析揭示了一个高度连接的蛋白质网络，其中MAPK3、MAPK8和TRAF6作为中心节点。他克莫司通过逆转LPS诱导的抑制作用和调节特定的miRNA来调节H-RPE细胞中的MAPK信号传导。这些发现表明他克莫司在减轻与PVR相关的炎症和纤维化反应方面具有潜在的治疗作用。
