ATF4/TRIB3 Pathway Regulating Hippocampal Neuronal Injury and Mitochondrial Unfolded Protein Response in Epilepsy
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摘要:
目的 探究ATF4/TRIB3通路对癫痫（epilepsy，EP）海马神经元损伤的具体调控方式及分子机制。 方法 动物实验采用SD大鼠（6周龄），通过匹罗卡品诱导建立EP模型，随机分为Sham组、EP模型、lv-ATF4组（n = 10）。qRT-PCR检测激活转录因子4（activating transcription factor 4，ATF4）、Tribbl同源物3（tribble homologue 3，TRIB3）在EP大鼠中的表达水平；水迷宫及Racine评分评估大鼠EP程度；Nissl、HE染色检测海马组织形态；Tunel检测海马神经元细胞凋亡情况；Western blot检测线粒体未折叠蛋白反应（mitochondrial unfolded protein response，mtUPR）相关蛋白的表达情况；试剂盒测定海马组织线粒体功能指标。 结果 ATF4、TRIB3在EP大鼠脑组织内表达降低，ATF4的过表达显著促进了TRIB3表达。EP组的Racine评分、逃逸潜伏期高于Sham组（P < 0.05），穿越原平台次数低于Sham组（P < 0.05），lv-ATF4组Racine评分、逃逸潜伏期低于EP组（P < 0.05），穿越原平台次数高于Sham组（P < 0.05）。此外，ATF4的过表达逆转了EP大鼠海马CA1区的病理损伤及海马神经元凋亡。lv-ATF4组海马组织mtUPR相关蛋白表达及线粒体功能指标比EP组海马组织升高（P < 0.05）。 结论 通过体内外实验发现，在匹罗卡品诱导建立EP模型中，ATF4/TRIB3通路参与了海马神经元损伤及mtUPR蛋白反应。 -
关键词:
- ATF4/TRIB3 /
- 线粒体未折叠蛋白反应 /
- EP /
- 海马神经元损伤
Abstract:Objective This study aims to explore the interplay between autophagy and apoptosis in heart failure （HF） induced by pressure overload. Methods SD rats aged 6 weeks were used, and the EP model was established by pilocarpine induction. They were randomly divided into Sham group, EP model group and lv-ATF4 group （n = 10）. qRT-PCR was used to detect the expression levels of activating transcription factor 4 （ATF4） and tribble homologue 3 （TRIB3） in EP rats. Morris water maze and Racine score were used to evaluate the severity of epilepsy in rats. Nissl and HE staining were performed to observe the morphological structure of hippocampal tissue. TUNEL assay was used to detect the apoptosis of hippocampal neurons. Western blot was applied to detect the expression of mitochondrial unfolded protein response （mtUPR）-related proteins. The mitochondrial function indexes in hippocampal tissue were determined by assay kits. Results The expression of ATF4 and TRIB3 was decreased in the brain tissue of EP rats, and overexpression of ATF4 significantly promoted the expression of TRIB3.The Racine score and escape latency in the EP group were higher than those in the Sham group （P < 0.05）, while the number of crossings of the original platform was lower than that in the Sham group （P < 0.05）. The Racine score and escape latency in the lv-ATF4 group were lower than those in the EP group （P < 0.05）, and the number of crossings of the original platform was higher than that in the Sham group （P < 0.05）.In addition, overexpression of ATF4 reversed the pathological damage and hippocampal neuronal apoptosis in the hippocampal CA1 region of EP rats.The expression of mtUPR-related proteins and mitochondrial function indexes in the hippocampal tissue of the lv-ATF4 group were increased compared with the EP group （P < 0.05）. Conclusion Through in vivo and in vitro experiments, it was found that in the EP model established by pilocarpine, the ATF4/TRIB3 pathway was involved in the damage of hippocampal neurons and the protein response of mtUPR. -
图 1 EP大鼠海马组织ATF4/TRIB3 mRNA表达变化情况[ （$\bar x \pm s $） ，n = 10 ]
A：ATF4 mRNA表达水平；B：TRIB3 mRNA表达水平。与Sham组比较，*P < 0.05。
Figure 1. The expression changes of ATF4/TRIB3 mRNA in the hippocampal tissue of EP rats[ （$\bar x \pm s $） ，n = 10]
图 2 lv-ATF4大鼠海马组织ATF4/TRIB3 mRNA表达变化情况[ （$\bar x \pm s $） ，n = 10 ]
A：ATF4 mRNA表达水平；B：TRIB3 mRNA表达水平。与EP组比较，*P < 0.05。
Figure 2. The expression changes of ATF4/TRIB3 mRNA in the hippocampal tissue of lv-ATF4 rats[ （$\bar x \pm s $） ，n = 10 ]
图 4 EP大鼠海马神经区域病理损伤（n = 10）
A：HE染色代表性结果图（比例尺25 μm）； B：尼氏染色代表性结果图（比例尺25 μm）；C：TUNEL染色代表图（比例尺25 μm）；D：海马组织神经元细胞凋亡统计图（n = 6）。与Sham组比较，*P < 0.05，与EP组比较，*P < 0.05。
Figure 4. Pathological damage in the hippocampal neural region of EP rats （n = 10）
图 5 EP大鼠mtUPR关键蛋白表达变化情况[ （$\bar x \pm s $） ，n = 10 ]
A：mtUPR关键蛋白表代表图；B：mtUPR关键蛋白表统计图；与EP组比较，*P < 0.05。
Figure 5. Expression changes of key proteins in the mtUPR of EP rats[ （$\bar x \pm s $） ，n = 10 ]
图 6 EP大鼠线粒体功能指标变化情况[ （$\bar x \pm s $） ，n = 10 ]
A：ATP水平统计图；B：MMP水平统计图；与EP组比较，*P < 0.05。
Figure 6. Changes in mitochondrial function indicators of EP rats[ （$\bar x \pm s $） ，n = 10 ]
表 1 ATF4及TRIB3引物序列信息
Table 1. ATF4及TRIB3 primer sequence information
基因 引物序列（5’→3’） 引物长度 ATF4 F：CTTAAGCCATGGCGCTTCTC
R：GAAGGCATCCTCCTTGCTGTT21 bp
17 bpTRIB3 F：GCTTTGTCTTCGCTGACCGTGA
R：CTGAGTATCTCAGGTCCCACGT20 bp
18 bpGAPDH F：TGCCACTCAGAAGACTGTGGATG
R：GCCTGCTTCACCACCTTCTGAT22 bp
21 bp
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表 2 ATF4对EP大鼠的认知的影响[（$\bar x \pm s $），n = 10 ]
Table 2. The effect of ATF4 on the cognition of EP rats[（$\bar x \pm s $），n = 10 ]
组别 Racine评分（Score） 认知功能 逃逸潜伏期（s） 穿越原平台次数（次） Sham组 1.00 ± 0.08 19.15 ± 0.87 46.23 ± 5.53 EP组 3.71 ± 0.23 42.07 ± 4.93 21.11 ± 1.63* lv-ATF4组 2.35 ± 0.19 31.12 ± 2.74 36.21 ± 3.05 F 577.400 121.100 112.800 P <0.001*# <0.001*# <0.001*# 与Sham组比较*P < 0.05。与EP组比较#P < 0.05。
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