人角膜纤维化可导致不透明和最终部分或完全视力丧失。目前,角膜移植是治疗严重角膜纤维化的唯一方法,同时存在排斥反应和供体短缺的风险.鞘脂(SPL)是已知的调节纤维化在各种组织和器官,包括角膜.我们以前报道过,SPL与两者密切相关,转化生长因子β(TGF-β)信号和角膜纤维化。这项研究的目的是研究鞘氨醇-1-磷酸(S1P)和S1P抑制对角膜纤维化中特定TGF-β和SPL家族成员的影响。分离健康的人角膜成纤维细胞(HCF),并在EMEM+FBS+VitC(构建体培养基)中在3D转移孔上培养4周。在构建培养基中制备以下处理:0.1ng/mLTGF-β1(β1),1μM鞘氨醇-1-磷酸(S1P),和5μM鞘氨醇激酶抑制剂2(I2)。测试了五组:(1)对照组(无治疗);抢救组;(2)β1/S1P;(3)β1/I2;预防组;(4)S1P/β1;(5)I2/β1。每种治疗施用2周,其中一种治疗并且切换到另一种治疗2周。使用蛋白质印迹分析,检查3D构建体的纤维化标志物的表达,SPL,和TGF-β信号通路成员。我们观察到潜在的TGF-β结合蛋白(LTBP)的纤维化表达和失活减少,TGF-β受体,母亲对十一项截瘫同系物(SMAD)的抑制作用,与S1P预防和抢救相比,I2预防和抢救治疗后的SPL信号,分别。此外,我们观察到I2预防和抢救组刺激后细胞迁移增加,划痕后12小时和18小时后,用S1P预防和挽救组刺激后,细胞迁移减少。我们已经证明I2治疗减少了纤维化并调节了LTBP的失活,TGF-β受体,SPLs,和典型的下游SMAD途径。为了充分揭示利用SphKI2作为角膜纤维化的新疗法的潜力,需要进一步的研究。
Human corneal fibrosis can lead to opacity and ultimately partial or complete vision loss. Currently, corneal transplantation is the only treatment for severe corneal fibrosis and comes with the risk of rejection and donor shortages. Sphingolipids (SPLs) are known to modulate fibrosis in various tissues and organs, including the
cornea. We previously reported that SPLs are tightly related to both, transforming growth factor beta (TGF-β) signaling and corneal fibrogenesis. The aim of this study was to investigate the effects of sphingosine-1-phosphate (S1P) and S1P inhibition on specific TGF-β and SPL family members in corneal fibrosis. Healthy human corneal fibroblasts (HCFs) were isolated and cultured in EMEM + FBS + VitC (construct medium) on 3D transwells for 4 weeks. The following treatments were prepared in a construct medium: 0.1 ng/mL TGF-β1 (β1), 1 μM sphingosine-1-phosphate (S1P), and 5 μM Sphingosine kinase inhibitor 2 (I2). Five groups were tested: (1) control (no treatment); rescue groups; (2) β1/S1P; (3) β1/I2; prevention groups; (4) S1P/β1; and (5) I2/β1. Each treatment was administered for 2 weeks with one treatment and switched to another for 2 weeks. Using Western blot analysis, the 3D constructs were examined for the expression of fibrotic markers, SPL, and TGF-β signaling pathway members. Scratch assays from 2D cultures were also utilized to evaluate cell migration We observed reduced fibrotic expression and inactivation of latent TGF-β binding proteins (LTBPs), TGF-β receptors, Suppressor of Mothers Against Decapentaplegic homologs (SMADs), and SPL signaling following treatment with I2 prevention and rescue compared to S1P prevention and rescue, respectively. Furthermore, we observed increased cell migration following stimulation with I2 prevention and rescue groups, with decreased cell migration following stimulation with S1P prevention and rescue groups after 12 h and 18 h post-scratch. We have demonstrated that I2 treatment reduced fibrosis and modulated the inactivation of LTBPs, TGF-β receptors, SPLs, and the canonical downstream SMAD pathway. Further investigations are warranted in order to fully uncover the potential of utilizing SphK I2 as a novel therapy for corneal fibrosis.