Department of Endocrinology, Putuo People's Hospital, School of Medicine, Tongji University, Shanghai, 200060, China.
Department of Endocrinology, Shanghai Tongji Hospital, School of Medicine, Tongji University, Shanghai, China; Ouyang Road Community Health Service Center, Hongkou District, Shanghai, China.
Exp Cell Res. 2024 Jul 1;440(1):114103. doi: 10.1016/j.yexcr.2024.114103. Epub 2024 Jun 5.
Elevated homocysteine (Hcy) levels have been recognized as significant risk factor for cardiovascular and cerebrovascular diseases, closely related to endothelial injury. While expression of Ciliary Neurotrophic Factor (CNTF) significantly increases during Hcy-induced vascular endothelial cell injury, the precise molecular pathways through which CNTF operates remain to be clarified. To induce vascular endothelial cell injury, human umbilical vein endothelial cells (HUVECs) were treated with Hcy. Cell viability and apoptosis in HUVECs were assessed using the CCK-8 assay and flow cytometry. Western blot analysis determined the expression levels of the JAK2-STAT3 pathway, inflammation-related factors (IL-1β, NLRP3, ICAM-1, VCAM-1), and apoptosis-related factors (cleaved Caspase-3 and Bax). Immunofluorescence staining and western blotting were employed to examine CD31 and α-SMA expression. Knockdown of CNTF was achieved using lentiviral interference, and its effects on inflammation and cell injury were evaluated. Chromatin immunoprecipitation (ChIP) and dual luciferase reporter analysis were conducted to investigate the interaction between the MAFK and CNTF promoters. Our results indicated that Hcy induced high expression of CNTF and activated the JAK2-STAT3 signaling pathway, thereby upregulating factors associated with inflammation and cell apoptosis. Inhibiting CNTF alleviated Hcy-induced inflammation and cell injury. MAFK was identified as a transcription factor promoting CNTF transcription, and its overexpression exacerbated inflammation and cell injury in Hcy-treated HUVECs through the CNTF-JAK2-STAT3 axis, which could be reversed by knocking down CNTF. Activation of MAFK leads to CNTF upregulation, which activates the JAK2-STAT3 signaling pathway, regulating inflammation and inducing injury in Hcy-exposed vascular endothelial cells. Targeting CNTF or its upstream regulator MAFK may represent potential therapeutic strategies for mitigating endothelial dysfunction associated with hyperhomocysteinemia and cardiovascular diseases.
同型半胱氨酸（Hcy）水平升高已被认为是心血管和脑血管疾病的重要危险因素，与内皮损伤密切相关。虽然睫状神经营养因子（CNTF）的表达在 Hcy 诱导的血管内皮细胞损伤时显著增加，但 CNTF 作用的确切分子途径仍有待阐明。为了诱导血管内皮细胞损伤，用 Hcy 处理人脐静脉内皮细胞（HUVEC）。通过 CCK-8 测定和流式细胞术评估 HUVEC 中的细胞活力和细胞凋亡。Western blot 分析确定 JAK2-STAT3 通路、炎症相关因子（IL-1β、NLRP3、ICAM-1、VCAM-1）和凋亡相关因子（裂解的 Caspase-3 和 Bax）的表达水平。免疫荧光染色和 Western blot 用于检测 CD31 和 α-SMA 的表达。使用慢病毒干扰敲低 CNTF，并评估其对炎症和细胞损伤的影响。进行染色质免疫沉淀（ChIP）和双荧光素酶报告基因分析以研究 MAFK 和 CNTF 启动子之间的相互作用。我们的结果表明，Hcy 诱导 CNTF 的高表达并激活 JAK2-STAT3 信号通路，从而上调与炎症和细胞凋亡相关的因子。抑制 CNTF 减轻了 Hcy 诱导的炎症和细胞损伤。MAFK 被鉴定为促进 CNTF 转录的转录因子，其过表达通过 CNTF-JAK2-STAT3 轴加剧了 Hcy 处理的 HUVEC 中的炎症和细胞损伤，通过敲低 CNTF 可逆转该作用。MAFK 的激活导致 CNTF 的上调，其激活 JAK2-STAT3 信号通路，调节炎症并诱导 Hcy 暴露的血管内皮细胞损伤。靶向 CNTF 或其上游调节因子 MAFK 可能代表减轻与高同型半胱氨酸血症和心血管疾病相关的内皮功能障碍的潜在治疗策略。
