International Medical Center, Geriatric Department, National clinical research center of geriatric diseases, Xiangya Hospital of Central South University, 87 Xiangya Road, Changsha, 410008, Hunan, China.
Department of Cardiology, Changsha Central Hospital, Changsha, 410000, Hunan, China.
Inflammation. 2020 Aug;43(4):1397-1410. doi: 10.1007/s10753-020-01217-w.
The present study aimed to investigate the role of Forkhead box protein C2 (Foxc2) in oxidized low-density lipoprotein (ox-LDL)-induced macrophages and identify the potential mechanisms. RAW264.7 cells, the murine macrophage cell line, were stimulated by ox-LDL, and cell proliferation was examined. The levels of inflammation- and oxidative stress-related markers were detected using kits after induction with ox-LDL. Subsequently, the expression of Foxc2 was measured using Western blotting. After transfection with Foxc2 pcDNA3.1, intracellular lipid droplets were examined using oil red O staining. The levels of total cholesterol (TC), free cholesterol (FC), inflammatory cytokines, and oxidative stress markers were determined. Moreover, apoptosis of RAW264.7 cells was detected using flow cytometry, and apoptosis-related proteins were measured using Western blotting. Angiopoietin-like protein 2 (Angptl2) was predicted as a target gene of Foxc2. Therefore, the expression of Angptl2 was examined after Foxc2 overexpression in ox-LDL-induced RAW264.7 cells. Then, the changes of intracellular lipid droplets, TC, FC, inflammatory cytokines, oxidative stress factors, and cell apoptosis were detected after Angptl2 overexpression or co-transfection with Foxc2 and Angptl2 pcDNA3.1. The results revealed that ox-LDL induction inhibited proliferation of RAW264.7 cells and promoted the release of inflammatory factors. Importantly, the expression of Foxc2 was obviously decreased after stimulation by ox-LDL. Foxc2 overexpression suppressed lipid accumulation, TC, FC levels, inflammation, oxidative stress, and apoptosis induced by ox-LDL, whereas these inhibitory effects were relieved after co-transfection with Angptl2 pcDNA3.1. These findings demonstrated that Foxc2 can alleviate ox-LDL-induced lipid accumulation, inflammation, and apoptosis of macrophage via regulating the expression of Angptl2.
本研究旨在探讨叉头框蛋白 C2(Foxc2)在氧化型低密度脂蛋白(ox-LDL)诱导的巨噬细胞中的作用,并确定潜在的机制。用 ox-LDL 刺激 RAW264.7 细胞(鼠源巨噬细胞系),并检测细胞增殖情况。用试剂盒检测 ox-LDL 诱导后炎症和氧化应激相关标志物的水平。随后,用 Western blot 检测 Foxc2 的表达。用 Foxc2 pcDNA3.1 转染后,用油红 O 染色检测细胞内脂滴。测定总胆固醇(TC)、游离胆固醇(FC)、炎症细胞因子和氧化应激标志物的水平。此外,用流式细胞术检测 RAW264.7 细胞的凋亡情况,用 Western blot 检测凋亡相关蛋白。预测血管生成素样蛋白 2(Angptl2)为 Foxc2 的靶基因。因此,在 ox-LDL 诱导的 RAW264.7 细胞中转染 Foxc2 后,检测 Angptl2 的表达。然后,在 Angptl2 过表达或与 Foxc2 和 Angptl2 pcDNA3.1 共转染后,检测细胞内脂滴、TC、FC、炎症细胞因子、氧化应激因子和细胞凋亡的变化。结果表明,ox-LDL 诱导抑制 RAW264.7 细胞增殖,并促进炎症因子的释放。重要的是,ox-LDL 刺激后 Foxc2 的表达明显降低。Foxc2 过表达抑制 ox-LDL 诱导的脂滴积累、TC、FC 水平、炎症、氧化应激和细胞凋亡,而共转染 Angptl2 pcDNA3.1 后这些抑制作用减轻。这些发现表明,Foxc2 可以通过调节 Angptl2 的表达来减轻 ox-LDL 诱导的巨噬细胞脂质积累、炎症和凋亡。