FTO 通过抑制 CBL 介导的 β-连环蛋白泛素化和降解来抑制 NLRP3 介导的细胞焦亡并减轻心肌缺血再灌注损伤,The FASEB Journal

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FTO 通过抑制 CBL 介导的 β-连环蛋白泛素化和降解来抑制 NLRP3 介导的细胞焦亡并减轻心肌缺血再灌注损伤
The FASEB Journal ( IF 4.4 ) Pub Date : 2023-05-18 , DOI: 10.1096/fj.202201793rr
Fei Sun ₁ , Cheng An ₁ , Can Liu ₁ , Ying Hu ₂ , Yue Su ₃ , Zhixiang Guo ₁ , Hong Che ₁ , Shenglin Ge ₁

Affiliation

心脏缺血/再灌注（I/R）损伤是一种复杂的病理事件，与细胞焦亡密切相关。本研究揭示了心脏 I/R 损伤期间 NLRP3 介导的细胞焦亡中脂肪量和肥胖相关蛋白 (FTO) 的调节机制。用氧-葡萄糖剥夺/复氧 (OGD/R) 刺激 H9c2 细胞。通过CCK-8和流式细胞术检测细胞活力和细胞焦亡。进行蛋白质印迹或 RT-qPCR 以分析靶分子表达。通过免疫荧光染色观察NLRP3和Caspase-1的表达。通过 ELISA 检测 IL-18 和 IL-1β 的产生。分别通过斑点印迹法和甲基化 RNA 免疫沉淀-qPCR 测定 CBL 的总 m6A 和 m6A 水平。IGF2BP3 和 CBL mRNA 之间的相互作用通过 RNA pull-down 和 RIP 测定得到证实。通过 Co-IP 评估 CBL 与 β-连环蛋白和 β-连环蛋白泛素化之间的蛋白质相互作用。在大鼠中建立心肌I/R模型。我们通过 TTC 染色确定梗塞面积，通过 H&E 染色确定病理变化。还评估了 LDH、CK-MB、LVFS 和 LVEF。FTO 和 β-catenin 被下调，而 CBL 被 OGD/R 刺激上调。FTO/β-连环蛋白过表达或 CBL 沉默抑制了 OGD/R 诱导的 NLRP3 炎症小体介导的细胞焦亡。CBL 通过泛素化和降解抑制 β-catenin 的表达。FTO 通过抑制 m6A 修饰降低了 CBL 的 mRNA 稳定性。CBL 介导的 β-连环蛋白泛素化和降解参与 FTO 诱导的心肌 I/R 损伤过程中的细胞焦亡抑制。

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FTO represses NLRP3-mediated pyroptosis and alleviates myocardial ischemia–reperfusion injury via inhibiting CBL-mediated ubiquitination and degradation of β-catenin

Cardiac ischemia/reperfusion (I/R) injury is a complicated pathological event, which has close association with pyroptosis. This study uncovered the regulatory mechanisms of fat mass and obesity-associated protein (FTO) in NLRP3-mediated pyroptosis during cardiac I/R injury. H9c2 cells were stimulated with oxygen–glucose deprivation/reoxygenation (OGD/R). Cell viability and pyroptosis were detected by CCK-8 and flow cytometry. Western blotting or RT-qPCR was performed to analyze target molecule expression. NLRP3 and Caspase-1 expression was observed by immunofluorescence staining. IL-18 and IL-1β production was detected by ELISA. The total m6A and m6A level of CBL was determined by dot blot assay and methylated RNA immunoprecipitation-qPCR, respectively. The interaction between IGF2BP3 and CBL mRNA was confirmed by RNA pull-down and RIP assays. The protein interaction between CBL and β-catenin and β-catenin ubiquitination were evaluated by Co-IP. Myocardial I/R model was established in rats. We determined infarct size by TTC staining and pathological changes by H&E staining. LDH, CK-MB, LVFS, and LVEF were also assessed. FTO and β-catenin were down-regulated, while CBL was up-regulated by OGD/R stimulation. FTO/β-catenin overexpression or CBL silencing restrained OGD/R-induced NLRP3 inflammasome-mediated pyroptosis. CBL repressed β-catenin expression via ubiquitination and degradation. FTO reduced the mRNA stability of CBL by inhibiting m6A modification. CBL-mediated ubiquitination and degradation of β-catenin were involved in FTO-induced pyroptosis inhibition during myocardial I/R injury. FTO inhibits NLRP3-mediated pyroptosis to attenuate myocardial I/R injury via repressing CBL-induced ubiquitination degradation of β-catenin.

更新日期：2023-05-23

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