Department of Otolaryngology-Head and Neck Surgery, Shandong Provincial Hospital, Shandong University, Jinan, Shandong, 250012, China.
School of Basic Medical Science, Jining Medical University, Jining, Shandong, China.
Arch Biochem Biophys. 2023 Oct 15;748:109766. doi: 10.1016/j.abb.2023.109766. Epub 2023 Oct 9.
FAM134B, the initial endoplasmic reticulum (ER)-phagy receptor identified, facilitates ER-phagy during ER stress. The malfunction of FAM134B has been demonstrated to have a crucial role in the pathological mechanisms of diverse human ailments. However, the role of FAM134B-mediated ER-phagy in ototoxicity, particularly in cisplatin-induced ototoxicity, remains unclear. The present study endeavors to investigate whether FAM134B is expressed in House Ear Institute-Organ of Corti 1 (HEI-OC1) and C57BL/6 murine cochlear hair cells (HCs), and to explore its potential function in cisplatin-mediated ototoxicity, with the aim of discovering new insights that can mitigate or forestall the irreversible adverse effect of cisplatin.
Immunofluorescence (IF) staining was used to test the expression pattern of FAM134B, levels of C/EBP-homologous protein (CHOP), autophagy, and co-localization ratio of lysosomes and ER. Western blotting was employed to measure changes in expression levels of FAM134B, LC3B, ER stress-related proteins, LAMP1 and apoptotic mediators. Cell apoptosis was examined using transferase dUTP nick end labeling (TUNEL) assay and flow cytometry.
In the present investigation, it was observed that FAM134B exhibited a diffuse expression pattern in the cytoplasm and nuclei of control HEI-OC1 cells. Following cisplatin administration, FAM134B was found to accumulate and form distinct dots around the nuclei, concomitant with increased levels of ER-phagy, ER stress, unfolded protein response (UPR), and cell apoptosis. Additionally, knockdown of FAM134B resulted in reduced ER-phagy, mitigated ER stress and UPR, and decreased apoptotic activity in HEI-OC1 cells following cisplatin exposure.
Collectively, the findings of this study demonstrate that FAM134B-mediated ER-phagy enhances the susceptibility of HCs to ER stress and apoptosis in response to cisplatin-induced stress. This suggests a sequential progression of ER-phagy, ER stress and apoptosis following cisplatin stimulus, and implies the potential therapeutic benefit of inhibiting of FAM134B-mediated ER-phagy in the prevention of cisplatin-related ototoxicity.
FAM134B 是最初被鉴定的内质网（ER）自噬受体，它在 ER 应激期间促进 ER 自噬。已经证明 FAM134B 的功能障碍在多种人类疾病的病理机制中起着关键作用。然而，FAM134B 介导的 ER 自噬在耳毒性中的作用，特别是在顺铂诱导的耳毒性中，尚不清楚。本研究旨在探讨 FAM134B 是否在 House Ear Institute-Organ of Corti 1（HEI-OC1）和 C57BL/6 鼠耳蜗毛细胞（HCs）中表达，并探讨其在顺铂介导的耳毒性中的潜在功能，以期发现新的见解，可以减轻或阻止顺铂的不可逆的不良影响。
免疫荧光（IF）染色用于测试 FAM134B 的表达模式、C/EBP 同源蛋白（CHOP）、自噬和溶酶体与内质网的共定位比例。Western blot 用于测量 FAM134B、LC3B、与 ER 应激相关的蛋白质、LAMP1 和凋亡介质的表达水平的变化。使用转移酶 dUTP 缺口末端标记（TUNEL）测定法和流式细胞术检测细胞凋亡。
在本研究中，观察到 FAM134B 在对照 HEI-OC1 细胞的细胞质和核中表现出弥散表达模式。顺铂给药后，发现 FAM134B 在核周围聚集并形成明显的点，同时 ER 自噬、ER 应激、未折叠蛋白反应（UPR）和细胞凋亡增加。此外，FAM134B 敲低导致 ER 自噬减少，减轻 ER 应激和 UPR，并减少顺铂暴露后 HEI-OC1 细胞的凋亡活性。
总的来说，这项研究的结果表明，FAM134B 介导的 ER 自噬增强了 HCs 对顺铂诱导应激的 ER 应激和细胞凋亡的敏感性。这表明 ER 自噬、ER 应激和细胞凋亡在顺铂刺激后存在一个连续的进展过程，并暗示抑制 FAM134B 介导的 ER 自噬在预防顺铂相关耳毒性方面具有潜在的治疗益处。
