Inhibition of SLC40A1 represses osteoblast formation via inducing iron accumulation and activating the PERK/ATF4/CHOP pathway mediated oxidative stress

This study aimed to investigate the effects of solute carrier family 40 member 1 (SLC40A1) on iron accumulation, oxidative stress and differentiation in osteoblasts and potential mechanisms. Mouse preosteoblastic MC3T3-E1 cells were transfected with the SLC40A1 overexpression vector (oeSLC40A1) and siRNA (siSLC40A1), then cell differentiation was induced via ascorbic acid and β-glycerophosphate. Besides, Ferrostatin-1 (ferroptosis inhibitor) and GSK2606414 (PERK inhibitor) were added with siSLC40A1. Fe²⁺, malondialdehyde (MDA), and reactive oxygen species (ROS) were higher but reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio was lower after siSLC40A1 transfection, while reduced Fe²⁺ and ROS but elevated GSH/GSSG ratio was observed after oeSLC40A1 transfection. Alkaline phosphatase (ALP) staining, Alizarin Red S (ARS) staining, osteopontin (OPN) and bone morphogenetic protein 2 (BMP2) were lower after siSLC40A1 transfection but were greater after oeSLC40A1 transfection. Furthermore, SLC40A1 negatively regulated the PERK/ATF4/CHOP pathway. Further exploration revealed that Fe²⁺, MDA, ROS, and the PERK/ATF4/CHOP pathway were attenuated, while GSH/GSSG ratio, ALP staining, ARS staining, and OPN expression were increased after ferrostatin-1 treatment in the siSLC40A1-transfected cells. Similar trends were observed with respect to GSK2606414 treatment with siSLC40A1. SLC40A1 inhibition suppresses osteoblast formation by facilitating iron accumulation and activating the PERK/ATF4/CHOP pathway-mediated oxidative stress.

本研究旨在探究溶质载体家族40成员1（Solute Carrier Family 40 Member 1, SLC40A1）对成骨细胞中铁蓄积、氧化应激及分化的影响及其潜在作用机制。实验以小鼠前成骨细胞MC3T3-E1为研究对象，分别转染SLC40A1过表达载体（oeSLC40A1）与小干扰RNA（siSLC40A1），随后通过抗坏血酸与β-甘油磷酸钠诱导细胞分化。此外，在转染siSLC40A1的细胞中分别加入铁抑素-1（Ferrostatin-1，铁死亡抑制剂）与GSK2606414（PERK抑制剂）。实验结果显示，转染siSLC40A1后，细胞内Fe²+、丙二醛（MDA）与活性氧（ROS）水平升高，而还原型谷胱甘肽/氧化型谷胱甘肽（GSH/GSSG）比值降低；转染oeSLC40A1后则呈现相反趋势，即Fe²+与ROS水平降低，GSH/GSSG比值升高。碱性磷酸酶（ALP）染色、茜素红S（ARS）染色结果，以及骨桥蛋白（OPN）与骨形态发生蛋白2（BMP2）的表达水平，在转染siSLC40A1后均下调，而转染oeSLC40A1后则上调。进一步机制探索表明，SLC40A1负调控PERK/ATF4/CHOP通路。更为深入的分析发现，在转染siSLC40A1的细胞中给予铁抑素-1处理后，细胞内Fe²+、MDA、ROS水平以及PERK/ATF4/CHOP通路的激活均被抑制，而GSH/GSSG比值、ALP染色强度、ARS染色结果以及OPN的表达水平均得到提升。采用GSK2606414处理转染siSLC40A1的细胞后，也观察到了相似的变化趋势。综上，抑制SLC40A1可通过促进铁蓄积、激活PERK/ATF4/CHOP通路介导的氧化应激，进而抑制成骨细胞形成。