SP1-mediated SYN1 promotes hemin-induced damage in PC12 cells in vitro and exacerbates blood-barrier disruption and brain injury after intracerebral hemorrhage in vivo

IF 2.9 4区医学 Q2 PATHOLOGY

Pathology, research and practice Pub Date : 2024-11-05 DOI:10.1016/j.prp.2024.155705

Haiping Zhang , Wei Li

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引用次数: 0

Abstract

Background

Intracerebral hemorrhage (ICH) is one deadly subtype of stroke with high morbidity and mortality. Oxidative stress and inflammation are major factors contributing to blood-brain barrier (BBB) dysfunction, which induces perihematoma edema and exacerbates ICH-induced secondary brain injury. SYN1 is a crucial neuronal phosphoprotein that plays a pivotal role in neuronal development. Our study was devised to clarify the function of SYN1 in hemin-induced cell injury in PC12 cells and brain injury in ICH rat models.

Methods

For in vitro assay, PC12 cells were first stimulated by 150 µM hemin for 4 h and then transfected with si-SYN1 and pcDNA3.1-SP1. Cell viability was tested through lactate dehydrogenase (LDH) release and CCK-8 assays. Cell apoptosis was observed through TUNEL staining. Inflammatory factors (IL-1β, IL-6, and TNF-α) were evaluated through western blotting. Oxidative markers (GSH, MDA, and SOD) were detected by adopting commercial kits. The binding sites of SP1 on the SYN1 promoter were predicted by using the JASPAR database and were validated through performing CHIP and luciferase reporter assays. SP1 and SYN1 expression in PC12 cells was investigated by RT-qPCR. For in vivo assay, rats were administrated intracerebroventricularly with si-SYN1 at 72 h before ICH induction. SYN1 and SP1 expression in ICH rats was assessed by RT-qPCR. Neurological deficits, brain edema, and BBB disruption at 24 h following ICH were assessed by conducting neurobehavioral tests, brain water content examination, and Evans blue extravasation assay.

Results

In vitro, hemin stimulation elevated SYN1 expression, attenuated cell viability, enhanced apoptosis, and induced inflammation and oxidative stress in PC12 cells, which were offset by SYN1 knockdown. SP1 bound to the SYN1 promoter region and positively regulated SYN1 expression. Overexpressing SP1 antagonized the impacts of SYN1 downregulation on hemin-induced damage in PC12 cells. In vivo, SYN1 and SP1 expression was upregulated in ICH rat models. Administration of si-SYN1 effectually improved neurological function, attenuated brain edema, and ameliorated BBB disruption following ICH.

Conclusion

SP1-mediated SYN1 probably participates in the process of neuronal damage, BBB disruption, and brain injury following ICH.

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SP1 介导的 SYN1 可促进体外 PC12 细胞由血氨诱导的损伤，并加剧体内脑出血后的血屏障破坏和脑损伤。

背景：脑出血（ICH）是中风的一种致命亚型，发病率和死亡率都很高。氧化应激和炎症是导致血脑屏障（BBB）功能障碍的主要因素，而血脑屏障功能障碍会诱发血肿周围水肿，加重 ICH 引起的继发性脑损伤。SYN1 是一种重要的神经元磷蛋白，在神经元发育过程中起着关键作用。我们的研究旨在阐明 SYN1 在海明诱导的 PC12 细胞损伤和 ICH 大鼠模型脑损伤中的功能：体外检测：首先用 150 µM hemin 刺激 PC12 细胞 4 小时，然后转染 si-SYN1 和 pcDNA3.1-SP1。通过乳酸脱氢酶（LDH）释放和 CCK-8 检测法检测细胞活力。通过 TUNEL 染色观察细胞凋亡。炎症因子（IL-1β、IL-6 和 TNF-α）通过 Western 印迹法进行评估。氧化标志物（GSH、MDA 和 SOD）采用商业试剂盒进行检测。利用JASPAR数据库预测了SP1在SYN1启动子上的结合位点，并通过CHIP和荧光素酶报告实验进行了验证。通过 RT-qPCR 检测了 PC12 细胞中 SP1 和 SYN1 的表达。为了进行体内检测，在诱导 ICH 前 72 小时给大鼠脑室内注射 si-SYN1 。通过 RT-qPCR 评估 ICH 大鼠体内 SYN1 和 SP1 的表达。通过神经行为测试、脑含水量检查和埃文斯蓝外渗试验，评估 ICH 后 24 小时的神经功能缺损、脑水肿和 BBB 破坏情况：结果：在体外，SYN1刺激可提高PC12细胞中SYN1的表达，降低细胞活力，促进细胞凋亡，并诱导炎症和氧化应激。SP1 与 SYN1 启动子区域结合并正向调节 SYN1 的表达。过表达 SP1 可拮抗 SYN1 下调对 PC12 细胞中海明诱导的损伤的影响。在体内，SYN1 和 SP1 在 ICH 大鼠模型中表达上调。给予 si-SYN1 能有效改善 ICH 后的神经功能、减轻脑水肿并改善 BBB 破坏：结论：SP1 介导的 SYN1 可能参与了 ICH 后神经元损伤、BBB 破坏和脑损伤的过程。

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来源期刊

Pathology, research and practice 医学-病理学

CiteScore

5.00

自引率

3.60%

发文量

405

审稿时长

24 days

期刊介绍： Pathology, Research and Practice provides accessible coverage of the most recent developments across the entire field of pathology: Reviews focus on recent progress in pathology, while Comments look at interesting current problems and at hypotheses for future developments in pathology. Original Papers present novel findings on all aspects of general, anatomic and molecular pathology. Rapid Communications inform readers on preliminary findings that may be relevant for further studies and need to be communicated quickly. Teaching Cases look at new aspects or special diagnostic problems of diseases and at case reports relevant for the pathologist''s practice.