Li Cunshu, Chang Ye, Li Yuan, Chen Shuang, Chen Yintao, Ye Ning, Dai Dongxue, Sun Yingxian
Department of Cardiology, The First Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China.
Int J Mol Med. 2017 May;39(5):1242-1254. doi: 10.3892/ijmm.2017.2938. Epub 2017 Mar 28.
The present study was aimed to investigate the role of reactive oxygen species (ROS) on advanced glycation end product (AGE)-induced proliferation and migration of vascular smooth muscle cells (VSMCs) and whether Bcl-2‑associated athanogene 3 (BAG3) is involved in the process. Primary rat VSMCs were extracted and cultured in vitro. Cell viability was detected by MTT assay and cell proliferation was detected by EdU incorporation assay. Cell migration was detected by wound healing and Transwell assays. BAG3 was detected using qPCR and western blot analysis. Transcriptional and translational inhibitors (actinomycin D and cycloheximide, respectively) were used to study the effect of AGEs on the expression of BAG3 in VSMCs. Lentiviral plasmids containing short hairpin RNA (shRNA) against rat BAG3 or control shRNA were transduced into VSMCs. Cellular ROS were detected by 2',7'-dichlorofluorescein diacetate (DCFH-DA) staining. Mitochondrial membrane potential was detected by tetramethylrhodamine methyl ester (TMRE) staining. AGEs significantly increased the expression of BAG3 in a dose-and time-dependent manner. Furthermore, AGEs mainly increased the expression of BAG3 mRNA by increasing the RNA synthesis rather than inhibiting the RNA translation. BAG3 knockdown reduced the proliferation and migration of VSMCs induced by AGEs. BAG3 knockdown reduced the generation of ROS and sustained the mitochondrial membrane potential of VSMCs. Reduction of ROS production by N-acetylcysteine (NAC), a potent antioxidant, also reduced the proliferation and migration of VSMCs. On the whole, the present study demonstrated for the first time that AGEs could increase ROS production and promote the proliferation and migration of VSMCs by upregulating BAG3 expression. This study indicated that BAG3 should be considered as a potential target for the prevention and/or treatment of vascular complications of diabetes.
本研究旨在探讨活性氧（ROS）在晚期糖基化终产物（AGE）诱导的血管平滑肌细胞（VSMC）增殖和迁移中的作用，以及Bcl-2相关抗凋亡基因3（BAG3）是否参与该过程。提取原代大鼠VSMC并进行体外培养。通过MTT法检测细胞活力，通过EdU掺入法检测细胞增殖。通过伤口愈合实验和Transwell实验检测细胞迁移。使用qPCR和蛋白质印迹分析检测BAG3。分别使用转录抑制剂（放线菌素D）和翻译抑制剂（环己酰亚胺）研究AGEs对VSMC中BAG3表达的影响。将含有针对大鼠BAG3的短发夹RNA（shRNA）或对照shRNA的慢病毒质粒转导至VSMC中。通过2',7'-二氯荧光素二乙酸酯（DCFH-DA）染色检测细胞内ROS。通过四甲基罗丹明甲酯（TMRE）染色检测线粒体膜电位。AGEs以剂量和时间依赖性方式显著增加BAG3的表达。此外，AGEs主要通过增加RNA合成而非抑制RNA翻译来增加BAG3 mRNA的表达。敲低BAG3可降低AGEs诱导的VSMC增殖和迁移。敲低BAG3可减少ROS的产生并维持VSMC的线粒体膜电位。强效抗氧化剂N-乙酰半胱氨酸（NAC）减少ROS产生也降低了VSMC的增殖和迁移。总体而言，本研究首次证明AGEs可通过上调BAG3表达增加ROS产生并促进VSMC的增殖和迁移。本研究表明，BAG3应被视为预防和/或治疗糖尿病血管并发症的潜在靶点。
