南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (10): 1796-1803.doi: 10.12122/j.issn.1673-4254.2023.10.19

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高同型半胱氨酸通过Notch1/Hes1信号通路促进HT22细胞自噬和凋亡

张竞文，何 静，米晓娟，许贤瑞，田 英，燕 茹   

  1. 宁夏医科大学基础医学院//国家卫生健康委员会代谢性心血管疾病研究重点实验室，总医院，宁夏 银川 750004
  • 出版日期:2023-10-20 发布日期:2023-11-02

High homocysteine level promotes autophagy and apoptosis of mouse hippocampal HT22 cells through the Notch1/Hes1 signaling pathway

ZHANG Jingwen, HE Jing, MI Xiaojuan, XU Xianrui, TIAN Ying, YAN Ru   

  1. School of Basic Medical Sciences, NHC Key Laboratory of Metabolic Cardiovascular Diseases Research, Ningxia Medical University, Yinchuan 750004, China; General Hospital of Ningxia Medical University, Yinchuan 750004, China
  • Online:2023-10-20 Published:2023-11-02

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摘要： 目的 探讨高同型半胱氨酸（HHcy）引起神经细胞损伤的相关机制。方法 采用HT22细胞，分为空白对照组（简称0组），高同型半胱氨酸组（Hcy 100 μmol/L，简称100组），高同型半胱氨酸维生素B干预组（Hcy 100 μmol/L +叶酸+维生素B12，简称100+fv组），空白对照+叶酸+维生素B12组（简称0+fv组）。通过透射电镜和流式细胞术观察自噬及凋亡；Western blotting、Real-time PCR 检测Hes1、Hes5、Notch1、Jagged1、Bcl-2、Bax、P62、LC3II/LC3I表达。结果 培养48 h后，高同型半胱氨酸组（100组）中死亡细胞明显增加（P<0.05）。流式细胞检测结果显示，高同型半胱氨酸组凋亡细胞比例均高于0组、100+fv组和0+fv组（P<0.05）。透射电镜观察显示，在高同型半胱氨酸组，线粒体肿胀、空泡化，自噬体增加。Western blot显示，在高同型半胱氨酸组，Bax/Bcl-2比值明显高于对照组和100+fv组（P<0.05）；P62相对值明显低于对照组（P<0.05）；LC3II/LC3I比值明显高于对照组和100+fv组（P<0.05）；HES1、HES5、Notch1和Jagged1蛋白表达明显低于对照组（P<0.05）。Real-time PCR 检测结果显示，在高同型半胱氨酸组，LC3 mRNA、Beclin1 mRNA明显高于对照组和100+fv组，P62mRNA明显低于100+fv组（P<0.05）；Hes1mRNA和Hes5 mRNA明显低于对照组、100+fv组和0+fv组（P<0.05）。Hes1 siRNA干扰后，Hes1 和Jagged1表达量明显低于对照组，Notch1表达量无显著性差异（P>0.05）。结论 高同型半胱氨酸能诱导HT22细胞自噬和凋亡增加，伴有Hes1和Jagged1基因表达下调。

关键词: 同型半胱氨酸；细胞；自噬；凋亡；Notch1；HES1；Jagged1

Abstract: Objective To explore the mechanism of neuronal injury caused by hyperhomocysteinemia. Methods Mouse hippocampal HT22 cells were treated with homocysteine (Hcy, 100 μmol/L), Hcy+folic acid+vitamin B12 (100+fv group) or folic acid +vitamin B12 (0+ fv group), and the changes in cell autophagy and apoptosis were detected using transmission electron microscope (TEM) and flow cytometry. The expressions of Hes1, Hes5, Notch1, Jagged1, Bcl-2, Bax, P62 and LC3 in the treated cells were detected with Western blotting and real-time PCR. Results Treatment with Hcy for 48 h significantly increased the number of dead cells in HT22 cell cultures. Flow cytometry showed that the percentage of apoptotic cells was significantly higher in cells treated with Hcy alone than in other treatment groups (P<0.05). TEM revealed obvious mitochondrial swelling and vacuolation and increased autophagy in Hcy-treated cells. Western blotting showed that the Bax/Bcl-2 ratio was significantly higher in Hcy-treated cells than in the blank control cells and cells in 100+fv group (P<0.05). The Hcy-treated cells showed a significantly lower relative expression of P62 than the blank control cells (P<0.05), a higher LC3II/LC3I ratio than the cells in the blank control and 100+fv groups (P<0.05), and lower expressions of HES1, HES5, Notch1 and Jagged1 proteins than the blank control cells (P<0.05). Interference with Hes1 siRNA significantly lowered the expression levels of Hes1 and Jagged1 without obviously affecting Notch1 expression in HT22 cells (P>0.05). Conclusion High Hcy levels promote autophagy and apoptosis and down-regulate Hes1 and Jagged1 expressions in HT22 cells.

Key words: homocysteine; hippocampal neurons; autophagy; apoptosis; Notch1; Hes1; Jagged1