先导化合物NHWL06031通过抑制METTL3/NF-κB轴阻止内皮细胞焦亡和改善内皮功能

The lead compound NHWL06031 inhibits endothelial cell pyroptosis and improves endothelial function via the METTL3/NF-κB axis

摘要:

目的]观察NHWL06031对氧化型低密度脂蛋白(ox-LDL)诱导的血管内皮细胞焦亡和功能障碍的影响,同时探究甲基转移酶样蛋白3/核因子κB(METTL3/NF-κB)轴在其中的作用。 [方法]采用ox-LDL处理人脐静脉内皮细胞(HUVEC)24 h,并分别给予NHWL06031及MCC950干预。通过CCK-8法和乳酸脱氢酶(LDH)试剂盒检测细胞活力和损伤程度,Hoechst33342/DCFH-DA双染法检测活性氧(ROS)的生成水平,细胞划痕实验评估内皮细胞迁移能力,Western blot检测焦亡相关蛋白及内皮功能相关蛋白表达。结合METTL3、NF-κB慢病毒过表达及METTL3 siRNA、NF-κB siRNA转染,检测细胞焦亡及内皮功能相关指标,明确NHWL06031对METTL3/NF-κB轴的调控作用。 [结果]与对照组相比,ox-LDL处理24 h的HUVEC细胞活力显著降低,LDH释放增加(P＜0.01),NOD样受体蛋白 3(NLRP3)、Gasdermin家族成员D N端结构域(GSDMD-N)、cleaved Caspase-1、白细胞介素18(IL-18)及细胞间黏附分子1(ICAM-1)、血管细胞黏附分子1(VCAM-1)蛋白表达上调(P＜0.05),细胞迁移受到抑制,ROS生成增多；NHWL06031及MCC950可显著逆转ox-LDL的上述作用(P＜0.05)。ox-LDL处理上调METTL3及NF-κB p-p65的表达(P＜0.05),NHWL06031干预下调二者水平(P＜0.01)；过表达METTL3、NF-κB均加重细胞损伤及焦亡相关蛋白表达,NHWL06031可部分逆转前述作用(P＜0.05)；si-NF-κB显著阻止ox-LDL诱导的细胞活力下降、LDH释放及ROS生成(P＜0.001),明显下调焦亡和内皮功能相关蛋白表达(P＜0.05),并恢复细胞迁移能力。 [结论]先导化合物NHWL06031通过抑制METTL3/NF-κB轴,阻止ox-LDL诱导的血管内皮细胞焦亡,从而改善内皮功能。

Abstract:

Aim To examine the effect of NHWL06031 on oxidized low density lipoprotein (ox-LDL)-induced pyroptosis and vascular endothelial dysfunction, and to explore the role of methyltransferase-like 3/nuclear factor-kappa B (METTL3/NF-κB) axis in this process. Methods Human umbilical vein endothelial cells (HUVEC) were treated with oxLDL for 24 h, followed by intervention with NHWL06031 or MCC950. CCK-8 assay and lactate dehydrogenase (LDH) detection kits were used to evaluate cell viability and cellular injury. Hoechst 33342/DCFH-DA double staining was applied to detect reactive oxygen species (ROS) production. Cell scratch assay assessed endothelial migratory capacity, and Western blot was performed to detect pyroptosis- and endothelial function-related proteins. By combining lentiviral overexpression of METTL3 or NF-κB with transfection of METTL3 siRNA and NF-κB siRNA, we detected indicators related to cell pyroptosis and endothelial function to clarify the regulatory effect of NHWL06031 on the METTL3/NF-κB axis. Results Compared with the control group, ox-LDL treatment for 24 h significantly reduced cell viability, increased LDH release (P＜0.01), upregulated protein expression of NOD-like receptors protein 3 (NLRP3), gasdermin D N-terminal domain (GSDMD-N), cleaved Caspase-1, interleukin-18 (IL-18), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) (P＜0.05), inhibited cell migration, and promoted ROS production in HUVEC. These effects were significantly reversed by NHWL06031 and MCC950 (P＜0.05). ox-LDL increased METTL3 and NF-κB p-p65 expression (P＜0.05), which were downregulated by NHWL06031 intervention (P＜0.01). Overexpression of METTL3 or NF-κB aggravated cell injury and pyroptosis-related protein expression, and these effects were partially reversed by NHWL06031 (P＜0.05). Knockdown of NF-κB significantly prevented ox-LDL-induced reduction in cell viability, LDH release and ROS production (P＜0.001), downregulated pyroptosis and endothelial function-related proteins (P＜0.05), and restored cell migration. Conclusion The lead compound NHWL06031 protects against ox-LDL-induced vascular endothelial cell pyroptosis and improves endothelial function by inhibiting the METTL3/NF-κB axis.