Kiełbasińska Aleksandra, Krysik Katarzyna, Janiszewska-Bil Dominika, Machaj Martyna, Lelek Zuzanna, Sułkowska Joanna, Nawotny-Czupryna Olga, Grabarek Beniamin Oskar
Department of Opthalmology, University Clinical Center Named After Prof. K. Gibiński of the Medical University of Silesia in Katowice, 40-514 Katowice, Poland.
Department of Ophthalmology, St. Barbara Hospital, Trauma Centre, 41-200 Sosnowiec, Poland.
Int J Mol Sci. 2025 Jun 4;26(11):5402. doi: 10.3390/ijms26115402.
The retinal pigment epithelium (RPE) plays a crucial role in maintaining retinal homeostasis, and dysregulation of the transforming growth factor-beta (TGF-β) signaling pathways contributes to retinal fibrosis and inflammatory diseases, including proliferative vitreoretinopathy (PVR). Tacrolimus (FK506), an immunosuppressant, has shown potential antifibrotic properties, but its effects on TGF-β-related genes and microRNAs (miRNAs) in RPE cells remain unclear. Human RPE (H-RPE) cells were treated with lipopolysaccharide (LPS) to induce inflammation and subsequently exposed to tacrolimus. Gene and miRNA expression profiling related to TGF-β signaling pathways were conducted using microarrays, followed by Quantitative Reverse-Transcription Polymerase Chain Reaction (RT-qPCR) validation. Protein levels were assessed via enzyme-linked immunosorbent assay (ELISA), and interactions were analyzed using STRING database network analysis. Tacrolimus modulated key components of the TGF-β pathway, upregulating TGF-β2, TGF-β3, SMAD2, and SMAD4 while downregulating TGF-βR1 and SMAD7. JAK/STAT and MAPK pathways were also affected, indicating broad regulatory effects. miRNA profiling identified hsa-miR-200a-3p, hsa-miR-589-3p, hsa-miR-21, and hsa-miR-27a-5p as key regulators. STRING analysis confirmed strong functional interactions within the TGF-β network. In conclusion, tacrolimus modulates both canonical (upregulation of SMAD2/4 and downregulation of SMAD7) and non-canonical (JAK/STAT and MAPK) TGF-β signaling pathways in LPS-stimulated RPE cells. These changes collectively suggest a dual anti-inflammatory and anti-fibrotic effect. The increased TGF-β2 and decreased SMAD7 levels, alongside altered miRNA expression (e.g., downregulation of miR-200a-3p), indicate that tacrolimus may inhibit key profibrotic mechanisms underlying PVR. These findings support the potential therapeutic repurposing of tacrolimus in PVR and warrant further in vivo validation.
视网膜色素上皮（RPE）在维持视网膜内环境稳定方面发挥着关键作用，而转化生长因子-β（TGF-β）信号通路失调会导致视网膜纤维化和炎症性疾病，包括增殖性玻璃体视网膜病变（PVR）。他克莫司（FK506）作为一种免疫抑制剂，已显示出潜在的抗纤维化特性，但其对RPE细胞中TGF-β相关基因和微小RNA（miRNA）的影响仍不清楚。用人视网膜色素上皮（H-RPE）细胞用脂多糖（LPS）处理以诱导炎症，随后使其暴露于他克莫司。使用微阵列进行与TGF-β信号通路相关的基因和miRNA表达谱分析，随后进行定量逆转录聚合酶链反应（RT-qPCR）验证。通过酶联免疫吸附测定（ELISA）评估蛋白质水平，并使用STRING数据库网络分析来分析相互作用。他克莫司调节TGF-β通路的关键成分，上调TGF-β2、TGF-β3、SMAD2和SMAD4，同时下调TGF-βR1和SMAD7。JAK/STAT和MAPK通路也受到影响，表明具有广泛的调节作用。miRNA谱分析确定hsa-miR-200a-3p、hsa-miR-589-3p、hsa-miR-21和hsa-miR-27a-5p为关键调节因子。STRING分析证实了TGF-β网络内的强功能相互作用。总之，他克莫司在LPS刺激的RPE细胞中调节经典的（上调SMAD2/4和下调SMAD7）和非经典的（JAK/STAT和MAPK）TGF-β信号通路。这些变化共同表明具有双重抗炎和抗纤维化作用。TGF-β2增加和SMAD7水平降低，以及miRNA表达改变（例如，miR-200a-3p下调）表明他克莫司可能抑制PVR潜在的关键促纤维化机制。这些发现支持他克莫司在PVR中潜在的治疗用途重新定位，并需要进一步的体内验证。
