Li Hui, Ma Yunxia, Li Junliang, Hou Siyu, Song Hui, Zhu Yazhou, Zhao Wei
College of Basic Medicine, Ningxia Medical University, 1160 Shengli Street, Ningxia Hui Autonomous Region, Xingqing, Yinchuan, 750004, P.R. China.
The First People's Hospital of Yinchuan, Ningxia Hui Autonomous Region, Yinchuan, 750000, China.
Mol Biol Rep. 2025 Jun 6;52(1):558. doi: 10.1007/s11033-025-10636-7.
Inhibition of hypoxia-induced apoptosis in cardiomyocytes is crucial for heart failure treatment. Previous research suggests that nuclear respiratory factor-1 (NRF-1) protects hypoxic cardiomyocytes against apoptosis. In the present study, we hypothesized that NRF-1 regulates the expression of Caspase 8 and FADD-like apoptosis regulator (CFLAR) and thus contributes to the regulation of apoptosis in hypoxic cardiomyocytes.
Chromatin immunoprecipitation (ChIP) and Dual-Glo luciferase assays confirmed that NRF-1 binds to the Cflar gene promoter and regulates its transcriptional activity. Furthermore, the interactions between NRF-1 and CFLAR and their effects on H9C2 cardiomyocytes apoptosis were tested under hypoxic conditions. Using the BioTek imaging system, we showed that CFLAR siRNA reversed the effects of NRF-1 overexpression on cell growth and death; CFLAR siRNA markedly increased the apoptosis rates and the activities of Caspase-3 and Caspase-8 in NRF-1-overexpressing cells. Conversely, in NRF-1-knockdown cells, CFLAR overexpression suppressed hypoxia-induced apoptosis. Western blot analysis showed that NRF-1-mediated regulation of CFLAR expression primarily influences the protein levels of cleaved Caspase-8 and tBid, without any significant differences in Bid, Bcl-2, and Bax expression.
We demonstrated that NRF-1 directly regulates CFLAR expression, thereby inhibiting the death receptor pathway, and ultimately, protects H9C2 cardiomyocytes from hypoxia-induced apoptosis. Our findings will provide novel insights into the molecular mechanisms underlying the protective role of NRF-1 and support its potential to serve as a therapeutic target for ameliorating heart failure.
抑制心肌细胞缺氧诱导的凋亡对心力衰竭治疗至关重要。先前的研究表明，核呼吸因子-1（NRF-1）可保护缺氧心肌细胞免受凋亡。在本研究中，我们假设NRF-1调节半胱天冬酶8（Caspase 8）和FADD样凋亡调节因子（CFLAR）的表达，从而有助于调节缺氧心肌细胞的凋亡。
染色质免疫沉淀（ChIP）和双荧光素酶报告基因检测证实NRF-1与Cflar基因启动子结合并调节其转录活性。此外，在缺氧条件下测试了NRF-1与CFLAR之间的相互作用及其对H9C2心肌细胞凋亡的影响。使用BioTek成像系统，我们发现CFLAR siRNA可逆转NRF-1过表达对细胞生长和死亡的影响；CFLAR siRNA显著增加了NRF-1过表达细胞中的凋亡率以及Caspase-3和Caspase-8的活性。相反，在NRF-1敲低的细胞中，CFLAR过表达抑制了缺氧诱导的凋亡。蛋白质印迹分析表明，NRF-1介导的CFLAR表达调节主要影响裂解的Caspase-8和tBid的蛋白质水平，而Bid、Bcl-2和Bax的表达没有任何显著差异。
我们证明NRF-1直接调节CFLAR表达，从而抑制死亡受体途径，并最终保护H9C2心肌细胞免受缺氧诱导的凋亡。我们的研究结果将为NRF-1保护作用的分子机制提供新的见解，并支持其作为改善心力衰竭治疗靶点的潜力。
