MiR-29c 抑制小鼠海马 HT22 细胞系中 TNF-α 诱导的 ROS 产生和细胞凋亡,Neurochemical Research

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MiR-29c 抑制小鼠海马 HT22 细胞系中 TNF-α 诱导的 ROS 产生和细胞凋亡
Neurochemical Research ( IF 3.7 ) Pub Date : 2022-10-30 , DOI: 10.1007/s11064-022-03776-w
Bo Li ₁ , Ying Lu ₁ , Rong Wang ₁ , Tao Xu ₁ , Xiaolu Lei ₁ , Huan Jin ₁ , Xiaohong Gao ₁ , Ye Xie ₁ , Xiaohong Liu ₁ , Junwei Zeng ₁

Affiliation

最近的报告表明，海马中异常的 miR-29c 表达与一些神经退行性疾病和神经精神疾病的病理生理学有关。然而，miR-29c 在调节海马神经元功能中的潜在作用尚不清楚。在这项研究中，HT22 细胞感染了含有 miR-29c 或 miR-29c 海绵的慢病毒。应用细胞计数试剂盒 8 (CCK8) 和乳酸脱氢酶 (LDH) 测定试剂盒评估 TNF-α 给药前后的细胞活力和毒性。用荧光探针测量活性氧 (ROS) 生成和线粒体膜电位 (MMP)。Hoechst 33258 染色和 TUNEL 测定用于评估细胞凋亡。关键 mRNA/蛋白质（TNFR1、Bcl-2、Bax、TRADD、FADD、caspase-3、-8和-9)在凋亡通路中的表达通过PCR或WB检测。此外，WB检测微管相关蛋白2（MAP-2）、神经生长相关蛋白43（GAP-43）和突触蛋白1（SYN-1）的蛋白表达。结果，我们发现 miR-29c 过表达可以提高细胞活力，减弱 LDH 释放，减少 ROS 产生并抑制 TNF-α 处理的 HT22 细胞中的 MMP 去极化。此外，在 TNF-α 处理的 HT22 细胞中，发现 miR-29c 过表达会降低细胞凋亡率，同时降低 Bax、cleaved caspase-3、cleaved caspase-9 的表达，并增加 Bcl-2 的表达。然而，miR-29c 海绵表现出相反的效果。此外，在 TNF-α 处理的 HT22 细胞中，miR-29c 过表达可降低 TNFR1、TRADD、FADD 和 cleaved caspase-8 的表达。然而，在用miR-29c海绵转染的HT22细胞中，TNF-α诱导的TNFR1、TRADD、FADD和cleaved caspase-8的表达显着加剧。最后，TNF-α 诱导的 MAP-2、GAP-43 和 SYN-1 表达降低被 miR-29c 逆转，但被 miR-29c 海绵加剧。总体而言，我们的研究表明，miR-29c 通过减轻 ROS 的产生和减少神经元细胞凋亡来防止 TNF-α 诱导的 HT22 细胞损伤。因此，miR-29c 可能是 TNF-α 积累和毒性相关脑部疾病的潜在治疗剂。我们的研究表明，miR-29c 通过减轻 ROS 的产生和减少神经细胞凋亡来防止 TNF-α 诱导的 HT22 细胞损伤。因此，miR-29c 可能是 TNF-α 积累和毒性相关脑部疾病的潜在治疗剂。我们的研究表明，miR-29c 通过减轻 ROS 的产生和减少神经细胞凋亡来防止 TNF-α 诱导的 HT22 细胞损伤。因此，miR-29c 可能是 TNF-α 积累和毒性相关脑部疾病的潜在治疗剂。

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MiR-29c Inhibits TNF-α-Induced ROS Production and Apoptosis in Mouse Hippocampal HT22 Cell Line

Recent reports have suggested that abnormal miR-29c expression in hippocampus have been implicated in the pathophysiology of some neurodegenerative and neuropsychiatric diseases. However, the underlying effect of miR-29c in regulating hippocampal neuronal function is not clear. In this study, HT22 cells were infected with lentivirus containing miR-29c or miR-29c sponge. Cell counting kit-8 (CCK8) and lactate dehydrogenase (LDH) assay kit were applied to evaluate cell viability and toxicity before and after TNF-α administration. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. Hoechst 33258 staining and TUNEL assay were used to evaluate cell apoptosis. The expression of key mRNA/proteins (TNFR1, Bcl-2, Bax, TRADD, FADD, caspase-3, -8 and -9) in the apoptosis pathway was detected by PCR or WB. In addition, the protein expression of microtubule-associated protein-2 (MAP-2), nerve growth-associated protein 43 (GAP-43) and synapsin-1 (SYN-1) was detected by WB. As a result, we found that miR-29c overexpression could improve cell viability, attenuate LDH release, reduce ROS production and inhibit MMP depolarization in TNF-α-treated HT22 cells. Furthermore, miR-29c overexpression was found to decrease apoptotic rate, along with decreased expression of Bax, cleaved caspase-3, cleaved caspase-9, and increased expression of Bcl-2 in TNF-α-treated HT22 cells. However, miR-29c sponge exhibited an opposite effects. In addition, in TNF-α-treated HT22 cells, miR-29c overexpression could decrease the expressions of TNFR1, TRADD, FADD and cleaved caspase-8. However, in HT22 cells transfected with miR-29c sponge, TNF-α-induced the expressions of TNFR1, TRADD, FADD and cleaved caspase-8 was significantly exacerbated. At last, TNF-α-induced the decreased expression of MAP-2, GAP-43 and SYN-1 was reversed by miR-29c but exacerbated by miR-29c sponge. Overall, our study demonstrated that miR-29c protects against TNF-α-induced HT22 cells injury through alleviating ROS production and reduce neuronal apoptosis. Therefore, miR-29c might be a potential therapeutic agent for TNF-α accumulation and toxicity-related brain diseases.

更新日期：2022-10-30

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