Plumbagin, a novel TRPV2 inhibitor, ameliorates microglia activation and brain injury in a middle cerebral artery occlusion/reperfusion mouse model.

IF 6.8 2区 医学 Q1 PHARMACOLOGY & PHARMACY
Meihuizi Ding, Rui Han, Yiming Xie, Ziyi Wei, Shuwen Xue, Fan Zhang, Zhengyu Cao
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Abstract

Background and purpose: Transient receptor potential vanilloid 2 (TRPV2) is a Ca2+-permeable non-selective cation channel. Despite the significant roles of TRPV2 in immunological response, cancer progression and cardiac development, pharmacological probes of TRPV2 remain to be identified. We aimed to discover TRPV2 inhibitors and to elucidate their molecular mechanism of action.

Experimental approach: Fluorescence-based Ca2+ assay in HEK-293 cells expressing murine TRPV2 was used to identify plumbagin as a novel TRPV2 inhibitor. Patch-clamp, in silico docking and site-directed mutagenesis were applied to investigate the molecular mechanisms critical for plumbagin interaction. ELISA and qPCR were used to assess nitric oxide release and mRNA levels of inflammatory mediators, respectively. si-RNA interference was used to knock down TRPV2 expression, which was validated by western blotting. Neurological and histological analyses were used to examine brain injury of mice following middle cerebral artery occlusion/reperfusion (MCAO/R).

Key results: Plumbagin is a potent TRPV2 negative allosteric modulator with an IC50 value of 0.85 μM, exhibiting >14-fold selectivity over TRPV1, TRPV3 and TRPV4. Plumbagin suppresses TRPV2 activity by decreasing the channel open probability without affecting the unitary conductance. Moreover, plumbagin binds to an extracellular pocket formed by the pore helix and flexible loop between transmembrane helices S5 and S6 of TRPV2. Plumbagin effectively suppresses LPS-induced inflammation of BV-2 microglia and ameliorates brain injury of MCAO/R mice.

Conclusion and implications: Plumbagin is a novel pharmacological probe to study TRPV2 pathophysiology. TRPV2 is a novel molecular target for the treatment of neuroinflammation and ischemic stroke.

新型 TRPV2 抑制剂 Plumbagin 可改善大脑中动脉闭塞/再灌注小鼠模型中的小胶质细胞活化和脑损伤。
背景和目的:瞬时受体电位香草素 2(TRPV2)是一种钙离子渗透性非选择性阳离子通道。尽管 TRPV2 在免疫反应、癌症进展和心脏发育中发挥着重要作用,但 TRPV2 的药理探针仍有待确定。我们的目标是发现 TRPV2 抑制剂并阐明其分子作用机制:实验方法:在表达小鼠 TRPV2 的 HEK-293 细胞中进行基于荧光的 Ca2+ 分析,以确定 plumbagin 为新型 TRPV2 抑制剂。应用膜片钳、硅学对接和定点突变来研究 plumbagin 相互作用的关键分子机制。使用酶联免疫吸附和 qPCR 分别评估一氧化氮的释放和炎症介质的 mRNA 水平。通过神经学和组织学分析,研究了大脑中动脉闭塞/再灌注(MCAO/R)后小鼠的脑损伤:Plumbagin是一种强效的TRPV2负异位调节剂,IC50值为0.85 μM,对TRPV1、TRPV3和TRPV4的选择性大于14倍。Plumbagin 通过降低通道开放概率来抑制 TRPV2 的活性,而不影响单位电导。此外,Plumbagin 与 TRPV2 的孔螺旋和跨膜螺旋 S5 与 S6 之间的柔性环形成的细胞外口袋结合。Plumbagin 能有效抑制 LPS 诱导的 BV-2 小胶质细胞炎症,并能改善 MCAO/R 小鼠的脑损伤:Plumbagin是研究TRPV2病理生理学的新型药理学探针。TRPV2是治疗神经炎症和缺血性中风的新型分子靶点。
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来源期刊
CiteScore
15.40
自引率
12.30%
发文量
270
审稿时长
2.0 months
期刊介绍: The British Journal of Pharmacology (BJP) is a biomedical science journal offering comprehensive international coverage of experimental and translational pharmacology. It publishes original research, authoritative reviews, mini reviews, systematic reviews, meta-analyses, databases, letters to the Editor, and commentaries. Review articles, databases, systematic reviews, and meta-analyses are typically commissioned, but unsolicited contributions are also considered, either as standalone papers or part of themed issues. In addition to basic science research, BJP features translational pharmacology research, including proof-of-concept and early mechanistic studies in humans. While it generally does not publish first-in-man phase I studies or phase IIb, III, or IV studies, exceptions may be made under certain circumstances, particularly if results are combined with preclinical studies.
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