Girdin 的上调通过促进血小板吞噬作用来延缓内皮细胞凋亡。
Upregulation of girdin delays endothelial cell apoptosis via promoting engulfment of platelets.
机构信息
Department of Vascular Surgery, Beijing hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, P.R. China.
Department of Cardiology, Beijing hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, P.R. China.
出版信息
Mol Biol Rep. 2023 Oct;50(10):8111-8120. doi: 10.1007/s11033-023-08625-9. Epub 2023 Aug 7.
BACKGROUND
Endothelial cells are crucial in maintaining the homeostasis of the blood-brain barrier. Girders of actin filament (Girdin) and phosphor (p)-Girdin are essential for the engulfment of human brain microvascular endothelial cells (HBMECs) into platelets (PLTs), but the potential mechanism remains unclear and requires further study.
METHODS
Following PLT and cytochalasin D treatment, Hoechst 33,342 detected apoptosis. The transfection efficiency of the short hairpin RNA targeting Girdin (sh-Girdin) or overexpressing Girdin (OE-Girdin) was determined using western blotting. Sh-Girdin, OE-Girdin, mutated Girdin (m-Girdin), and microfilament binding region deleted Girdin (Del-Girdin) were transfected into HBMECs under PLT conditions. Subsequently, the engulfment of HBMECs by PLTs was detected by flow cytometry and transmission electron microscopy. Girdin and phosphorylated (p)-Girdin levels were quantified by western blot. The positive expression of Girdin was measured by immunohistochemistry (IHC). The localization of PLT, Girdin, and p-Girdin and the engulfment of HBMECs in PLTs were analyzed by confocal microscopy.
RESULT
Cytochalasin D overturned the inhibitory effect of PLT on cell apoptosis. OE-Girdin enhanced the fluorescent intensity of PLT-labelling and the engulfment of HBMECs by PLTs, while sh-Girdin, m-Girdin, and Del-Girdin ran reversely. OE-Girdin elevated the Girdin and p-Girdin levels, while sh-Girdin and Del-Girdin were the opposite, but m-Girdin did not affect the p-Girdin and Girdin levels.
CONCLUSION
Girdin and p-Girdin were co-located with PLTs in HBMECs. The over-expression of Girdin was identified as being associated with the increasing engulfment of PTLs. Girdin may be an effective target to alleviate endothelial cell apoptosis.
背景
内皮细胞对于维持血脑屏障的内稳态至关重要。桩蛋白（Girdin）及其磷酸化形式（p-Girdin）对于人脑血管内皮细胞（HBMECs）被血小板（PLTs）吞噬至关重要，但潜在机制尚不清楚，需要进一步研究。
方法
用 Hoechst 33,342 检测经 PLT 和细胞松弛素 D 处理后的细胞凋亡情况。通过 Western blot 确定针对 Girdin 的短发夹 RNA（sh-Girdin）或过表达 Girdin（OE-Girdin）的转染效率。在 PLT 条件下，将 sh-Girdin、OE-Girdin、突变型 Girdin（m-Girdin）和缺失微丝结合区的 Girdin（Del-Girdin）转染到 HBMECs 中。随后，通过流式细胞术和透射电子显微镜检测 HBMECs 被 PLTs 吞噬的情况。通过 Western blot 定量 Girdin 和磷酸化（p）-Girdin 的水平。通过免疫组织化学（IHC）检测 Girdin 的阳性表达。通过共聚焦显微镜分析 PLT、Girdin 和 p-Girdin 的定位以及 HBMECs 在 PLTs 中的吞噬情况。
结果
细胞松弛素 D 逆转了 PLT 对细胞凋亡的抑制作用。OE-Girdin 增强了 PLT 标记的荧光强度和 PLTs 对 HBMECs 的吞噬作用，而 sh-Girdin、m-Girdin 和 Del-Girdin 的作用则相反。OE-Girdin 升高了 Girdin 和 p-Girdin 的水平，而 sh-Girdin 和 Del-Girdin 则相反，但 m-Girdin 对 p-Girdin 和 Girdin 水平没有影响。
结论
Girdin 和 p-Girdin 与 HBMECs 中的 PLTs 共定位。Girdin 的过表达与 PLTs 吞噬作用的增加有关。Girdin 可能是减轻内皮细胞凋亡的有效靶点。
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