Lentivirus-Mediated Missense Mutation in HtrA1 Leads to Activation of the TGF-β/Smads Pathway and Increased Apoptosis of Mouse Brain Microvascular Endothelial Cells via the Oxidative Stress Pathway.

IF 2.5 4区 医学 Q3 NEUROSCIENCES
Shi-Na Song, Hui-Juan Li, Jian-Lin Liang, Qian-Qian Ren, Chang-Xin Li, Sui-Yi Xu
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Abstract

Background: The aim of this study was to investigate the possible molecular mechanisms underlying cerebral small vessel disease caused by a missense mutation in the high-temperature serine peptidase A1 gene, HtrA1 (NM_002775.4, Exon4, c.905G>A, p.Arg302Gln). Stable strain models were constructed using wild-type and mutant HtrA1 overexpression lentiviral vectors to infect mouse brain microvascular endothelial cells (bEnd.3 cells).

Methods: HtrA1 mRNA and protein expression were analyzed by Western blot and quantitative real-time polymerase chain reaction. Western blot technique was also used to evaluate the expression of transforming growth factor (TGF)-β/Smads-related signaling pathway proteins and the oxidative stress pathway protein nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4). The level of reactive oxygen species (ROS) was evaluated using dichloro-dihydro-fluorescein diacetate (DCFH-DA) fluorescent probes.

Results: HtrA1 mRNA and protein expression levels were found to be decreased in mutant cells, whereas the levels of ROS, the TGF-β/Smads proteins, and the caspase3 and cleaved-caspase3 apoptotic proteins were increased.

Conclusions: Lentivirus-mediated missense mutation in HtrA1 leads to activation of the TGF-β/Smads pathway and to increased apoptosis of mouse brain microvascular endothelial cells via the oxidative stress pathway. Further in vivo studies are required to explore the connections between different signaling pathways in animals, and to identify potential molecular targets for clinical therapy.

慢病毒介导的HtrA1错sense突变通过氧化应激途径激活TGF-β/Smads通路并增加小鼠脑微血管内皮细胞凋亡
背景:本研究的目的是探讨高温丝氨酸肽酶A1基因HtrA1 (NM_002775.4, Exon4, c.905G> a, p.Arg302Gln)错义突变引起脑血管疾病的可能分子机制。利用野生型和突变型HtrA1过表达慢病毒载体感染小鼠脑微血管内皮细胞(bEnd),构建稳定的菌株模型。3细胞)。方法:采用Western blot和实时定量聚合酶链反应分析HtrA1 mRNA和蛋白的表达。Western blot检测转化生长因子(TGF)-β/ smads相关信号通路蛋白和氧化应激通路蛋白烟酰胺腺嘌呤二核苷酸磷酸氧化酶4 (NOX4)的表达。采用双乙酸二氯-二氢荧光素(DCFH-DA)荧光探针检测小鼠体内活性氧(ROS)水平。结果:突变细胞中HtrA1 mRNA和蛋白表达水平降低,ROS、TGF-β/Smads蛋白、caspase3和cleaved-caspase3凋亡蛋白表达水平升高。结论:慢病毒介导的HtrA1错sense突变可通过氧化应激途径激活TGF-β/Smads通路,增加小鼠脑微血管内皮细胞凋亡。需要进一步的体内研究来探索动物中不同信号通路之间的联系,并确定临床治疗的潜在分子靶点。
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来源期刊
CiteScore
2.80
自引率
5.60%
发文量
173
审稿时长
2 months
期刊介绍: JIN is an international peer-reviewed, open access journal. JIN publishes leading-edge research at the interface of theoretical and experimental neuroscience, focusing across hierarchical levels of brain organization to better understand how diverse functions are integrated. We encourage submissions from scientists of all specialties that relate to brain functioning.
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