Li Zhen, Szentmáry Nóra, Fries Fabian N, Suiwal Shweta, Chai Ning, Seitz Berthold, Shi Lei, Amini Maryam, Stachon Tanja
Dr. Rolf M. Schwiete Center for Limbal Stem Cell and Congenital Aniridia Research, Saarland University, Kirrberger Str. 100, Homburg, Saarland, 66424, Germany.
Department of Ophthalmology, Saarland University Medical Center, Homburg, Saarland, Germany.
Ophthalmol Ther. 2024 Nov;13(11):2931-2950. doi: 10.1007/s40123-024-01032-8. Epub 2024 Sep 21.
In congenital aniridia caused by mutations in paired box 6 (PAX6), PAX6 influences the migration and differentiation of limbal epithelial cells (LECs), thereby playing a pivotal role in aniridia-associated keratopathy. The antidepressants ritanserin and duloxetine affect PAX6 expression in LECs. Limbal stromal cells, which support limbal epithelial stem cells, are crucial in the limbal stem cell niche. This study explores how ritanserin and duloxetine influence gene expression in primary human limbal stromal cells from subjects with congenital aniridia and from healthy subjects, in vitro.
Primary human limbal stromal cells from corneas affected by aniridia (AN-LSCs) (n = 8) and from healthy corneas (LSCs) (n = 8) were isolated and cultured in either low-glucose serum-free (LGSF) or normal-glucose serum-containing (NGSC) media. Cells were treated with 4 µM ritanserin or duloxetine for 24 h. Quantitative PCR (qPCR) and western blot were used to assess the expression of PAX6, FOSL2, TGF-β1, ACTA2A1, LUM, COL1A1, COL5A1, DSG1, FABP5 and ADH7.
In AN-LSCs with LGSF-medium, ritanserin increased PAX6 messenger RNA (mRNA) (p = 0.007) and decreased TGF-β1 and FOSL2 mRNA levels (P = 0.005, P = 0.038). In addition, TGF-β1 protein levels decreased with both treatments (P = 0.02, P = 0.007), and FABP5 protein level increased, using ritanserin (P = 0.019). In LSCs with LGSF-medium, ACTA2A1 mRNA levels decreased using ritanserin and duloxetine (P = 0.028; P = 0.031), while FABP5 mRNA levels increased with ritanserin treatment (P = 0.003). Also, duloxetine use reduced α-SMA protein (P = 0.013) and increased FABP5 protein levels (P = 0.029). In LSCs with NGSC-medium, ritanserin elevated LUM, FABP5 and ADH7 mRNA and protein levels (P = 0.025, P = 0.003, P = 0.047, P = 0.024, P = 0.013, P = 0.039).
The results of our study confirmed that the antipsychotropic drugs ritanserin and duloxetine alter PAX6 and TGF-β1 gene expression in AN-LSCs cultured in LGSF-medium. These drugs were found to have an impact on retinoic acid signaling pathways and keratocyte characteristic markers both in LSCs and AN-LSCs, using different culture media.
在由配对盒6（PAX6）基因突变引起的先天性无虹膜症中，PAX6影响角膜缘上皮细胞（LECs）的迁移和分化，从而在无虹膜相关角膜病变中起关键作用。抗抑郁药利坦色林和度洛西汀会影响LECs中PAX6的表达。支持角膜缘上皮干细胞的角膜缘基质细胞在角膜缘干细胞微环境中至关重要。本研究探讨利坦色林和度洛西汀如何在体外影响先天性无虹膜症患者和健康受试者的原代人角膜缘基质细胞中的基因表达。
从受无虹膜症影响的角膜（AN-LSCs）（n = 8）和健康角膜（LSCs）（n = 8）中分离出原代人角膜缘基质细胞，并在低葡萄糖无血清（LGSF）或正常葡萄糖含血清（NGSC）培养基中培养。细胞用4µM利坦色林或度洛西汀处理24小时。采用定量PCR（qPCR）和蛋白质印迹法评估PAX6、FOSL2、TGF-β1、ACTA2A1、LUM、COL1A1、COL5A1、DSG1、FABP5和ADH7的表达。
在LGSF培养基培养的AN-LSCs中，利坦色林增加了PAX6信使核糖核酸（mRNA）（p = 0.007），并降低了TGF-β1和FOSL2 mRNA水平（P = 0.005，P = 0.038）。此外，两种处理均使TGF-β1蛋白水平降低（P = 0.02，P = 0.007），而使用利坦色林时FABP5蛋白水平升高（P = 0.019）。在LGSF培养基培养的LSCs中，使用利坦色林和度洛西汀时ACTA2A1 mRNA水平降低（P = 0.028；P = 0.031），而利坦色林处理使FABP5 mRNA水平升高（P = 0.003）。此外，使用度洛西汀降低了α-SMA蛋白（P = 0.013）并增加了FABP5蛋白水平（P = 0.029）。在NGSC培养基培养的LSCs中，利坦色林提高了LUM、FABP5和ADH7 mRNA和蛋白水平（P = 0.025，P = 0.003，P = 0.047，P = 0.024，P = 0.013，P = 0.039）。
我们的研究结果证实，抗精神病药物利坦色林和度洛西汀改变了在LGSF培养基中培养的AN-LSCs中PAX6和TGF-β1基因的表达。发现这些药物在使用不同培养基时，对LSCs和AN-LSCs中的视黄酸信号通路和角膜细胞特征标志物均有影响。
