School of Pharmaceutical Sciences, Jiangnan University, Wuxi, 214122, China.
School of Medicine, Jiangnan University, Wuxi, 214122, China.
Acta Pharmacol Sin. 2022 Jun;43(6):1430-1440. doi: 10.1038/s41401-021-00780-8. Epub 2021 Oct 15.
Transient receptor potential channel TRPV4 and nicotinamide adenine dinucleotide phosphate oxidase (Nox2) are involved in oxidative stress that increases endothelial permeability. It has been shown that obesity enhances the physical association of TRPV4 and Nox2, but the role of TRPV4-Nox2 association in obesity has not been clarified. In this study we investigated the function of TRPV4-Nox2 complex in reducing oxidative stress and regulating abnormal vascular permeability in obesity. Obesity was induced in mice by feeding a high-fat diet (HFD) for 14 weeks. The physical interaction between TRPV4 and Nox2 was measured using FRET, co-immunoprecipitation and GST pull-down assays. The functional interaction was measured by rhodamine phalloidin, CM-HDCFDA in vitro, the fluorescent dye dihydroethidium (DHE) staining assay, and the Evans blue permeability assay in vivo. We demonstrated that TRPV4 physically and functionally associated with Nox2, and this physical association was enhanced in aorta of obese mice. Furthermore, we showed that interrupting TRPV4-Nox2 coupling by TRPV4 knockout, or by treatment with a specific Nox2 inhibitor Nox2 dstat or a specific TRPV4 inhibitor HC067046 significantly attenuated obesity-induced ROS overproduction in aortic endothelial cells, and reversed the abnormal endothelial cytoskeletal structure. In order to discover small molecules disrupting the over-coupling of TPRV4 and Nox2 in obesity, we performed molecular docking analysis and found that compound M12 modulated TRPV4-Nox2 association, reduced ROS production, and finally reversed disruption of the vascular barrier in obesity. Together, this study, for the first time, provides evidence for the TRPV4 physically interacting with Nox2. TRPV4-Nox2 complex is a potential drug target in improving oxidative stress and disruption of the vascular barrier in obesity. Compound M12 targeting TRPV4-Nox2 complex can improve vascular barrier function in obesity.
瞬时受体电位通道 TRPV4 和烟酰胺腺嘌呤二核苷酸磷酸氧化酶（Nox2）参与增加内皮通透性的氧化应激。已经表明肥胖增强了 TRPV4 和 Nox2 的物理关联，但 TRPV4-Nox2 关联在肥胖中的作用尚未阐明。在这项研究中，我们研究了 TRPV4-Nox2 复合物在降低肥胖中的氧化应激和调节异常血管通透性中的作用。通过用高脂肪饮食（HFD）喂养 14 周来诱导肥胖小鼠。使用 FRET、共免疫沉淀和 GST 下拉测定来测量 TRPV4 和 Nox2 之间的物理相互作用。通过体外 rhodamine phalloidin、CM-HDCFDA、荧光染料二氢乙啶（DHE）染色测定和体内 Evans 蓝通透性测定来测量功能相互作用。我们证明 TRPV4 与 Nox2 物理和功能上相互作用，并且这种物理相互作用在肥胖小鼠的主动脉中增强。此外，我们表明，通过 TRPV4 敲除、通过用特异性 Nox2 抑制剂 Nox2 dstat 或特异性 TRPV4 抑制剂 HC067046 治疗来中断 TRPV4-Nox2 偶联，显著减弱肥胖诱导的主动脉内皮细胞中 ROS 的过度产生，并逆转异常的内皮细胞细胞骨架结构。为了发现破坏肥胖中 TPRV4 和 Nox2 过度偶联的小分子，我们进行了分子对接分析，发现化合物 M12 调节 TRPV4-Nox2 关联，减少 ROS 产生，最终逆转肥胖中血管屏障的破坏。总之，这项研究首次提供了 TRPV4 与 Nox2 物理相互作用的证据。TRPV4-Nox2 复合物是改善肥胖中氧化应激和血管屏障破坏的潜在药物靶点。靶向 TRPV4-Nox2 复合物的化合物 M12 可改善肥胖中的血管屏障功能。
