Li Bo, Lu Ying, Wang Rong, Xu Tao, Lei Xiaolu, Jin Huan, Gao Xiaohong, Xie Ye, Liu Xiaohong, Zeng Junwei
Department of Physiology, Zunyi Medical University, Zunyi, 563000, Guizhou, China.
Neurochem Res. 2023 Feb;48(2):519-536. doi: 10.1007/s11064-022-03776-w. Epub 2022 Oct 30.
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.
最近的报告表明，海马体中异常的miR-29c表达与一些神经退行性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方面的潜在作用尚不清楚。在本研究中，HT22细胞用含有miR-29c或miR-29c海绵的慢病毒感染。应用细胞计数试剂盒-8（CCK8）和乳酸脱氢酶（LDH）检测试剂盒评估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）。采用Hoechst 33258染色和TUNEL检测评估细胞凋亡。通过PCR或WB检测凋亡途径中关键mRNA/蛋白（TNFR1、Bcl-2、Bax、TRADD、FADD、caspase-3、-8和-9）的表达。此外，通过WB检测微管相关蛋白-2（MAP-2）、神经生长相关蛋白43（GAP-43）和突触素-1（SYN-1）的蛋白表达。结果，我们发现在TNF-α处理的HT22细胞中，miR-29c过表达可提高细胞活力、减轻LDH释放、减少ROS产生并抑制MMP去极化。此外，发现在TNF-α处理的HT22细胞中，miR-29c过表达可降低凋亡率，同时降低Bax、裂解的caspase-3、裂解的caspase-9的表达，并增加Bcl-2的表达。然而，miR-29c海绵表现出相反的作用。此外，在TNF-α处理的HT22细胞中，miR-29c过表达可降低TNFR1、TRADD、FADD和裂解的caspase-8的表达。然而，在转染miR-29c海绵的HT22细胞中，TNF-α诱导的TNFR1、TRADD、FADD和裂解的caspase-8的表达显著加剧。最后，miR-29c可逆转TNF-α诱导的MAP-2、GAP-43和SYN-1的表达降低，但miR-29c海绵则使其加剧。总体而言，我们的研究表明，miR-29c通过减轻ROS产生和减少神经元凋亡来保护HT22细胞免受TNF-α诱导的损伤。因此，miR-29c可能是治疗TNF-α积累和毒性相关脑部疾病的潜在治疗剂。
