| 摘要: |
| 目的 研究Homer1b/c蛋白在谷氨酸兴奋性毒性损伤诱发的细胞自噬中的作用及机制。方法 选用小鼠海马神经元HT22细胞，通过500 μmol/L L-谷氨酸处理建立细胞损伤模型。用siRNA慢病毒转染方式下调Homer1b/c表达，用10 μmol/L钙离子螯合剂BAPTA-AM、10 mmol/L内质网应激抑制剂4-PBA分别抑制细胞内钙离子释放和内质网应激后，使用蛋白质印迹法检测细胞中Homer1b/c蛋白，自噬效应蛋白[beclin-1、微管相关蛋白1轻链3（LC3）]及内质网应激标志蛋白[C/EBP同源蛋白（CHOP）、葡萄糖调节蛋白78（GRP 78）]的表达水平。结果 L-谷氨酸处理HT22细胞12 h后，细胞中beclin-1表达和LC3-Ⅱ/LC3-Ⅰ比值均较对照组升高（均P<0.05）；与转染对照组相比，下调Homer1b/c表达可降低细胞中beclin-1表达和LC3-Ⅱ/LC3-Ⅰ比值（均P<0.05）；抑制细胞内钙离子释放和内质网应激均能降低细胞中beclin-1表达和LC3-Ⅱ/LC3-Ⅰ比值（均P<0.05）；下调Homer1b/c表达后，抑制细胞内钙离子释放和内质网应激未能进一步降低细胞中beclin-1表达和LC3-Ⅱ/LC3-Ⅰ比值。结论 Homer1b/c能够调节谷氨酸兴奋性毒性损伤诱发的细胞自噬，其调节作用可能与内质网功能有关。 |
| 关键词: 海马神经元 自噬 Homer 钙稳态 内质网应激 兴奋性损伤 谷氨酸 |
| DOI：10.16781/j.CN31-2187/R.20230219 |
| 投稿时间：2023-04-19修订日期：2023-09-04 |
| 基金项目: |
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| Homer1b/c regulates autophagy in mouse hippocampal neuronal HT22 cells induced by glutamate excitotoxic injury through endoplasmic reticulum function |
| ZHANG Minmin△,ZHU Xuan△,SHEN Hongjian,LÜ, Nan,WU Xiongfeng,XU Xiaolong*,WU Tao |
(Neurovascular Center, The First Affiliated Hospital of Naval Medical University (Second Military Medical University), Shanghai 200433, China
△Co-first authors.
* Corresponding author) |
| Abstract: |
| Objective To study the role of Homer1b/c in cell autophagy induced by glutamate excitotoxic injury and its mechanism. Methods Mouse hippocampal neuronal HT22 cells were treated with 500 μmol/L L-glutamate to establish cell injury model. The expression of Homer1b/c in HT22 cells was down-regulated by small interfering RNA (siRNA) lentivirus transfection. 10 μmol/L 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM, a calcium ion chelating agent) and 10 mmol/L 4-phenylbutyric acid (4-PBA, an endoplasmic reticulum stress inhibitor) were used to inhibit intracellular calcium ion release and endoplasmic reticulum stress, respectively. Then, the expression levels of Homer1b/c, autophagy proteins (beclin-1 and microtubule-associated protein 1 light chain 3 [LC3]) and endoplasmic reticulum stress marker proteins (C/EBP homologous protein [CHOP] and glucose regulated protein 78 [GRP-78]) in cells were measured by Western blotting. Results Compared with the control group, the expression of beclin-1 and the ratio of LC3-Ⅱ/LC3-Ⅰ were significantly increased after the HT22 cells were treated with L-glutamate for 12 h (both P<0.05); down-regulation of Homer1b/c expression could reduce the expression of beclin-1 and the ratio of LC3-Ⅱ/LC3-Ⅰ (both P<0.05). Inhibition of intracellular calcium release and endoplasmic reticulum stress could reduce the beclin-1 expression and LC3-Ⅱ/LC3-Ⅰ ratio (both P<0.05). After down-regulation of Homer1b/c expression, inhibiting intracellular calcium release and endoplasmic reticulum stress failed to further reduce the beclin-1 expression and LC3-Ⅱ/LC3-Ⅰ ratio. Conclusion Homer1b/c can regulate cell autophagy induced by glutamate excitotoxic injury, and its regulatory effects may be related to the function of endoplasmic reticulum. |
| Key words: hippocampal neurons autophagy Homer calcium homeostasis endoplasmic reticulum stress excitatory injury glutamate |
