NDUFA13轻度下调激活STAT3/Nrf2/Nox4信号通路调控内皮细胞氧化应激保护动脉粥样硬化病变机制的研究

The causative role of mitochondrial oxidative stress in endothelial cells injury that initiates atherosclerotic lesion has drawn great attentions. The data from our group have shown that NDUFA13 can generate a moderate increase in ROS production which activates ROS dependent protective signaling. Interestingly, we have observed altered NDUFA13 expression in atherosclerotic lesion in mice, accordingly we studied on Tie2Cre-NDUFA13flox/flox mice（ECs specificity）and showed that heterozygous mice were resistant to high-fat diet induced plaque formation compared with wild type mice. We also detected protein interaction between STAT3 and Nrf2 with co-IP experiment, which was accompanied by nuclear translocation of Nrf2 and increased Nox4 expression, all of which indicate STAT3/Nrf2 signal is involved in transcriptional regulation of Nox4. In contrast, homozygous mice, due to a severe loss of NDUFA13, had significantly worsend atherosclerotic lesions, with an increased expression of Nox2. These data support the notion that NDUFA13 is involved in atherosclerotic lesions formation by modulating the expression of Nox family proteins and hence the oxidative status in ECs. Thus, the research proposal aims to elucidate: (1) whether slight down-regulation of NDUFA13 expression in atherosclerotic Apoe-/- mice can also protect atherosclerosis;(2) whether the down-regulation of Nox4 expression level blocks the protective effects offered by a moderate decrease in NDUFA13 level of ECs;(3) Luciferase essay will be applied to verify that the STAT3/Nrf2 interaction plays a key role in transcriptional activity for Nox4.

内皮细胞（ECs）线粒体氧化应激参与动脉粥样硬化病变。既往研究显示线粒体蛋白NDUFA13轻度下调有细胞保护作用，与STAT3信号相关，我们前期研究发现单纯ECs的NDUFA13表达轻度下调时，高脂饮食所诱导斑块减轻，伴随STAT3和Nrf2的相互结合、Nrf2入核以及Nox4表达增高，提示NDUFA13/STAT3/Nrf2信号在转录水平对Nox4的调控。表明：NDUFA13/STAT3调控Nrf2转录活性而影响Nox4表达并参与动脉粥样硬化病变。为此本课题组利用Tie2Cre，NDUFA13-flox，Nox-flox小鼠及体外实验探讨：①轻度ECs NDUFA13下调是否同样具有保护Apoe-/-小鼠动脉粥样斑块的作用；②下调Nox4表达水平是否干扰ECs中NDUFA13轻度下调对血管病变的保护作用；③证实STAT3/Nrf2相互作用结合在Nox4启动子区发挥的转录调控作用。

内皮细胞功能不全是动脉粥样硬化的开端，是动脉粥样硬化病变过程最早能被识别的病理改变。我们发现：在普通饮食喂养的Apoe-/-缺陷小鼠主动脉内皮细胞部位NDUFA13变化不明显，而在西方饮食诱导的小鼠主动脉内皮部位可见NDUFA13表达降低，并且这个趋势在斑块负荷较重的区域尤为明显。为了定量分析NDUFA13轻度下调保护内皮细胞过程中的作用Nox2和Nox4蛋白表达水平，我们培养Tie2-Cre NDUFA13WT/WT，Tie2-Cre NDUFA13WT/flox和Tie2-Cre NDUFA13flox/flox小鼠主动脉内皮细胞（Endothelial Cells，ECs），给予ox-LDL诱导氧化应激，结果显示：① 在NDUFA13轻度降低时，ECs中H2O2生成轻度增多，然而超氧阴离子(O2·-)较对照组增加明显减少；此时伴随着Nox2仅轻度表达上调，而Nox4则明显增加；② 当NDUFA13重度下调（纯合子小鼠ECs）时，H2O2和超氧阴离子(O2·-)同时生成显著增多，此时Nox2显著增加。进一步的研究发现，在Nox4表达适量增高时，增加内皮细胞中具有保护作用的H2O2适度增高，抑制NFκb等炎症转录因子表达，起到保护内皮功能以及抗动脉粥样硬化作用；但是，当NDUFA13严重下调时，使NDUFA13/STAT3/Nrf2通路过度激活，导致内皮细胞内H2O2激增，引发NF-κb过度激活，造成内皮细胞氧化应激加重，Nox2表达上调，进而引发ROS induced ROS Generation，造成内皮细胞内ROS大量产生，严重的内皮细胞氧化应激促进了动脉粥样硬化的进展。