Department of Health Sciences, University of Milan, San Paolo Hospital, Milan, Italy.
Research Unit on Bioactive Molecules, Department of Biomedicinal Chemistry, Catalan Institute of Advanced Chemistry (IQAC/CSIC), Barcelona, Spain.
Naunyn Schmiedebergs Arch Pharmacol. 2017 Jul;390(7):741-751. doi: 10.1007/s00210-017-1374-3. Epub 2017 Apr 13.
Retinal degeneration and in particular retinitis pigmentosa (RP) is associated to ceramide (Cer) accumulation and cell death induction. Cer and sphingosine-1-phosphate (S1P) belong to the sphingolipids class and exert a pro-apoptotic and pro-survival activity, respectively. Our aim is to target sphingolipid metabolism by inhibiting S1P lyase that regulates one of the S1P degradation pathways, to reduce retinal photoreceptor damage. The murine 661W cone-like cell line was pretreated with THI, an inhibitor of S1P lyase and exposed to HO-induced oxidative stress. 661W cell viability and apoptosis were evaluated by Trypan Blue and TUNEL assay, respectively. Protein expression of mediators of the survival/death pathway (ERK1/2, Akt, Bcl-2, Bax) was analyzed by Western blotting. RT-PCR was performed to establish HO-1 transcript changes and LC-MS analysis to measure Cer intracellular content. THI rescues inhibitory HO-effect on 661W cell viability and impairs HO-induced apoptosis by increasing Bcl-2/Bax ratio. THI administration counteracts the oxidative stress effects of HO on 661W cells by activating the Nrf2/HO-1 pathway, regulating ERK and Akt phosphorylation levels, and decreasing Cer intracellular content. We conclude that sphingolipid metabolism manipulation can be considered a therapeutic target to promote photoreceptor survival.
视网膜变性，特别是色素性视网膜炎（RP）与神经酰胺（Cer）积累和细胞死亡诱导有关。Cer 和鞘氨醇-1-磷酸（S1P）属于鞘脂类，分别具有促凋亡和促生存活性。我们的目的是通过抑制调节 S1P 降解途径之一的 S1P 裂解酶来靶向鞘脂代谢，以减少视网膜光感受器的损伤。用 S1P 裂解酶抑制剂 THI 预处理 661W 锥状细胞样细胞系，并使其暴露于 HO 诱导的氧化应激下。通过台盼蓝和 TUNEL 测定法分别评估 661W 细胞活力和细胞凋亡。通过 Western blot 分析存活/死亡途径（ERK1/2、Akt、Bcl-2、Bax）的介质蛋白表达。通过 RT-PCR 确定 HO-1 转录物的变化，通过 LC-MS 分析测量细胞内 Cer 含量。THI 通过增加 Bcl-2/Bax 比值来挽救 HO 对 661W 细胞活力的抑制作用，并损害 HO 诱导的凋亡。THI 通过激活 Nrf2/HO-1 途径、调节 ERK 和 Akt 磷酸化水平以及降低细胞内 Cer 含量来抵消 HO 对 661W 细胞的氧化应激作用。我们得出结论，鞘脂代谢的操纵可以被认为是一种促进光感受器存活的治疗靶点。
