Tang Yixin, Cai Yaqin, Peng Fengling, Li Ming, Mo Zhongcheng
Guangxi Key Laboratory of Diabetic Systems Medicine, Department of Histology and Embryology, Guilin Medical University, Guilin, 541199, China.
Department of Cardiovascular Medicine, First Affiliated Hospital, Hengyang Medical School, University of South China, Hengyang, 421001, China.
Cardiovasc Drugs Ther. 2025 May 22. doi: 10.1007/s10557-025-07717-x.
Midkine (MK) has been shown to facilitate atherosclerotic plaque formation by downregulating the expression of ATP-binding cassette transporter A1 (ABCA1). However, the mechanism by which MK regulates ABCA1 to promote atherosclerosis remains incompletely understood. In this study, we sought to investigate the molecular mechanism by which MK's regulation of ABCA1 influences the pathogenesis of atherosclerosis.
Male apoE mice were subjected to a high-fat diet to establish an atherosclerosis model. The model mice received intraperitoneal injections of MK and activator protein-1 (AP-l) inhibitor SR11302. The ATP-binding cassette transporter A1 (ABCA1) and AP1 expression were detected using immunohistochemistry (IHC), quantitative polymerase chain reaction (qPCR), and western blotting (WB). RAW264.7 macrophages were incubated with oxidized low-density lipoprotein (ox-LDL) to generate foam cells. These foam cells were treated with MK, SR11302, JNK inhibitor SP600125, and PI3K inhibitor wortmannin. The expression of ABCA1, AP-1, JNK, and PI3K were detected using qPCR and WB. The cholesterol efflux and lipid accumulation of cells were analyzed using scintillation counting and oil red O staining, respectively.
MK-treated mice exhibited an accelerated development of atherosclerotic lesion (30% in the MK group vs. 20% in the control group), along with hepatic steatosis and lipid disorder. The expression of c-fos and AP-1 were up-regulated by MK in macrophages. Compared with the MK-treated group, inhibition of AP-1 using SR11302 or transfection with c-fos siRNA markedly enhanced the cholesterol efflux (12.73% in the MK + SR11302 group vs. 9.98% in the MK group, 12.73% in the MK + si-c-fos group vs. 10.02 % in the MK group), reduced lipid accumulation, and increased the protein levels of ABCA1 in macrophages. Compared to the MK-treated group, mice treated with both MK and SR11302 showed downregulated ABCA1 expression in aortic sinus lesions, a larger lesion area (22.59% vs. 18.54%), and significantly elevated levels of plasma total cholesterol (TC), low-density lipoprotein (LDL), and triglycerides (TG). These results suggest that MK-induced pharmacological inhibition of AP-1 augmented ABCA1 expression in plaques, ameliorated lipid disorders, and abrogated atherosclerosis progression in apoE mice. In addition, in vitro experiments revealed that the MK-induced up-regulation of c-fos expression was effectively suppressed by inhibitors of JNK and PI3K.
Our findings unveil a novel mechanistic pathway in atherosclerosis, whereby MK promotes the development of atherosclerosis by up-regulating AP-1 in macrophages via the PI3K/AKT/JNK signaling cascade.
中期因子（MK）已被证明可通过下调ATP结合盒转运蛋白A1（ABCA1）的表达促进动脉粥样硬化斑块形成。然而，MK调节ABCA1促进动脉粥样硬化的机制仍不完全清楚。在本研究中，我们试图探究MK对ABCA1的调节影响动脉粥样硬化发病机制的分子机制。
雄性载脂蛋白E基因敲除（apoE）小鼠接受高脂饮食以建立动脉粥样硬化模型。模型小鼠腹腔注射MK和活化蛋白-1（AP-1）抑制剂SR11302。采用免疫组织化学（IHC）、定量聚合酶链反应（qPCR）和蛋白质印迹法（WB）检测ATP结合盒转运蛋白A1（ABCA1）和AP-1的表达。将RAW264.7巨噬细胞与氧化型低密度脂蛋白（ox-LDL）孵育以生成泡沫细胞。这些泡沫细胞分别用MK、SR11302、JNK抑制剂SP600125和PI3K抑制剂渥曼青霉素处理。采用qPCR和WB检测ABCA1、AP-1、JNK和PI3K的表达。分别使用闪烁计数法和油红O染色分析细胞的胆固醇流出和脂质蓄积情况。
MK处理的小鼠动脉粥样硬化病变发展加速（MK组为30%，对照组为20%），同时伴有肝脂肪变性和脂质紊乱。MK使巨噬细胞中c-fos和AP-1的表达上调。与MK处理组相比，用SR11302抑制AP-1或用c-fos小干扰RNA（siRNA）转染可显著增强胆固醇流出（MK + SR11302组为12.73%，MK组为9.98%；MK + si-c-fos组为12.73%，MK组为10.02%），减少脂质蓄积，并增加巨噬细胞中ABCA1的蛋白水平。与MK处理组相比，同时用MK和SR11302处理的小鼠主动脉窦病变中ABCA1表达下调，病变面积更大（22.59%对18.54%），血浆总胆固醇（TC）、低密度脂蛋白（LDL）和甘油三酯（TG）水平显著升高。这些结果表明，MK诱导的AP-1药理抑制增强了斑块中ABCA1的表达，改善了脂质紊乱，并消除了apoE小鼠的动脉粥样硬化进展。此外，体外实验表明，JNK和PI3K抑制剂可有效抑制MK诱导的c-fos表达上调。
我们的研究结果揭示了动脉粥样硬化中一条新的机制途径，即MK通过PI3K/AKT/JNK信号级联上调巨噬细胞中的AP-1来促进动脉粥样硬化的发展。
