Yu Jie, Mu Xiu'e, Guan Chang, Wang Yaqin, Li Hongying
Cardiovascular Care Unit, The First Hospital of Hebei Medical University, Shijiazhuang 050000, China.
Department of Anesthesiology, The First Hospital of Hebei Medical University, Shijiazhuang 050000, China.
Tissue Cell. 2024 Dec;91:102555. doi: 10.1016/j.tice.2024.102555. Epub 2024 Sep 7.
The present study aims to investigate the biological function of Tyrobp in myocardial ischemia-reperfusion injury (MIRI) and to clarify its potential reaction mechanisms.
AC16 cells were induced by oxygen-glucose deprivation/reoxygenation (OGD/R) to simulate the MIRI in vitro. The cell transfection technology was used to downregulate Tyrobp, followed by assessment of cell damage, apoptosis and cytokines production via Cell Counting Kit (CCK)-8 assay, lactate dehydrogenase (LDH) release assay, TUNEL and ELISA assays, respectively. Immunofluorescence assay was performed to assess GSDMD. Corresponding proteins were detected via western blotting, and Co-immunoprecipitation (Co-IP) assay was used to validate proteins interaction.
Tyrobp was upregulated in OGD/R-exposed AC16 cells, and Tyrobp deficiency significantly alleviated OGD/R-caused cell viability loss, LDH release and cell apoptosis in AC16 cells. Meanwhile, Tyrobp deficiency inhibited the activation of NLRP3 inflammasome, reduced the production of cytokines and inhibited GSDMD intensity and GSDMD-N expression. Additionally, Tyrobp could interact with Syk and regulate Syk/NF-κB signaling. The rescue experiments showed that the above effects of Tyrobp deficiency on OGD/R-exposed AC16 cells were partly weakened by Syk overexpression.
Tyrobp deficiency alleviated MIRI by inhibiting NLRP3-mediated inflammation and pyroptosis through regulating Syk, providing a novel target for the treatment of MIRI.
本研究旨在探讨酪氨酸结合蛋白（Tyrobp）在心肌缺血再灌注损伤（MIRI）中的生物学功能，并阐明其潜在的反应机制。
采用氧糖剥夺/复氧（OGD/R）诱导AC16细胞，以在体外模拟MIRI。利用细胞转染技术下调Tyrobp，随后分别通过细胞计数试剂盒（CCK）-8法、乳酸脱氢酶（LDH）释放法、TUNEL法和ELISA法评估细胞损伤、细胞凋亡和细胞因子产生。进行免疫荧光测定以评估Gasdermin D（GSDMD）。通过蛋白质免疫印迹法检测相应蛋白质，并使用免疫共沉淀（Co-IP）测定法验证蛋白质相互作用。
在OGD/R处理的AC16细胞中Tyrobp上调，并且Tyrobp缺陷显著减轻了OGD/R诱导的AC16细胞活力丧失、LDH释放和细胞凋亡。同时，Tyrobp缺陷抑制了NLRP3炎性小体的激活，减少了细胞因子的产生，并抑制了GSDMD强度和GSDMD-N表达。此外，Tyrobp可与脾酪氨酸激酶（Syk）相互作用并调节Syk/核因子κB（NF-κB）信号通路。挽救实验表明，Syk过表达部分削弱了Tyrobp缺陷对OGD/R处理的AC16细胞的上述影响。
Tyrobp缺陷通过调节Syk抑制NLRP3介导的炎症和细胞焦亡，从而减轻MIRI，为MIRI的治疗提供了新靶点。
