Department of Pathophysiology, Guizhou Provincial Key Laboratory of Pathogenesis and Drug Research on Common Chronic Diseases, Guizhou Medical University, Guiyang 550025, Guizhou Province, China.
Department of Pathophysiology, College of Basic Medical Sciences, Guizhou Medical University, Guiyang 550025, Guizhou Province, China.
World J Gastroenterol. 2022 Jun 21;28(23):2569-2581. doi: 10.3748/wjg.v28.i23.2569.
Endoplasmic reticulum (ER) stress-related hepatocyte apoptosis is responsible for multiple hepatic diseases. Previous studies have revealed that endoplasmic reticulophagy (ER-phagy) promotes the selective clearance of damaged ER fragments during ER stress, playing a crucial role in maintaining ER homeostasis and inhibiting apoptosis. Family with sequence similarity 134 member B (FAM134B) is a receptor involved in ER-phagy that can form a complex with calnexin (CNX) and microtubule-associated protein 1 light chain 3 (LC3). The complex can mediate the selective isolation of ER fragments to attenuate hepatocyte apoptosis. However, the precise regulatory mechanisms remain unclear.
To elucidate the effect of FAM134B-mediated ER-phagy on ER stress-induced apoptosis in buffalo rat liver 3A (BRL-3A) rat hepatocytes and the potential regulatory mechanisms.
ER stress-related hepatocyte apoptosis was induced using dithiothreitol (DTT). Proteins related to ER stress and autophagy were measured with western blotting. Protein complex interactions with FAM134B were isolated by co-immunoprecipitation. ER-phagy was evaluated in immunofluorescence experiments. Cell cycle distribution and apoptosis were measured by flow cytometry. Mitochondrial Ca levels were evaluated by the co-localization of intracellular Ca-tracker and Mito-tracker. The small interfering RNA against was used to knockdown in BRL-3A cells.
ER stress-related and autophagy-related proteins in BRL-3A cells were elevated by both short and long-term DTT treatment. Furthermore, co-immunoprecipitation confirmed an interaction between FAM134B, CNX, FAM134B, and LC3 in BRL-3A cells. Immunofluorescence assays revealed that autolysosomes significantly decreased following short-term DTT treatment, but increased after long-term treatment. Mitochondrial Ca levels and apoptotic rates were dramatically elevated, and more cells were arrested in the G1 stage after short-term DTT treatment; however, these decreased 48 h later. Moreover, FAM134B downregulation accelerated mitochondrial apoptotic pathway activation and aggravated hepatocyte apoptosis under ER stress.
FAM134B-mediated ER-phagy attenuates hepatocyte apoptosis by suppressing the mitochondrial apoptotic pathway. Our findings provide new evidence highlighting the importance of FAM134B-mediated ER-phagy in attenuating hepatocyte apoptosis.
内质网（ER）应激相关的肝细胞凋亡是多种肝脏疾病的原因。先前的研究表明，内质网自噬（ER-phagy）在 ER 应激期间促进受损 ER 片段的选择性清除，在维持 ER 稳态和抑制细胞凋亡方面发挥着关键作用。家族与序列相似性 134 成员 B（FAM134B）是一种参与 ER-phagy 的受体，它可以与钙连蛋白（CNX）和微管相关蛋白 1 轻链 3（LC3）形成复合物。该复合物可以介导 ER 片段的选择性隔离，从而减轻肝细胞凋亡。然而，确切的调节机制尚不清楚。
阐明 FAM134B 介导的 ER-phagy 对牛磺酸大鼠肝 3A（BRL-3A）大鼠肝细胞中 ER 应激诱导的细胞凋亡的影响及其潜在的调节机制。
使用二硫苏糖醇（DTT）诱导 ER 应激相关的肝细胞凋亡。使用 Western 印迹法测量与 ER 应激和自噬相关的蛋白质。通过共免疫沉淀分离与 FAM134B 相互作用的蛋白质复合物。通过免疫荧光实验评估 ER-phagy。通过流式细胞术测量细胞周期分布和细胞凋亡。通过细胞内钙示踪剂和 Mito-tracker 的共定位评估线粒体 Ca 水平。使用针对的小干扰 RNA 转染 BRL-3A 细胞以敲低 。
DTT 短期和长期处理均可使 BRL-3A 细胞中的 ER 应激相关蛋白和自噬相关蛋白水平升高。此外，共免疫沉淀证实了 FAM134B、CNX、FAM134B 和 LC3 在 BRL-3A 细胞中的相互作用。免疫荧光实验显示，自噬溶酶体在短期 DTT 处理后显著减少，但在长期处理后增加。线粒体 Ca 水平和凋亡率显著升高，DTT 短期处理后更多细胞停滞在 G1 期，但 48 小时后下降。此外，FAM134B 下调加速了 ER 应激下线粒体凋亡途径的激活并加重了肝细胞凋亡。
FAM134B 介导的 ER-phagy 通过抑制线粒体凋亡途径来减轻肝细胞凋亡。我们的研究结果提供了新的证据，表明 FAM134B 介导的 ER-phagy 在减轻肝细胞凋亡方面具有重要意义。
