Chloroquine regulates the proliferation and apoptosis of palate development on mice embryo by activating P53 through blocking autophagy in vitro
Chloroquine regulates the proliferation and apoptosis of palate development on mice embryo by activating P53 through blocking autophagy in vitro
复制标题
DOI：
10.1007/s11626-022-00704-8
复制
发表时间：
2022-08-10
影响因子：
2.1
通讯作者：
Du, Juan
中科院分区：
文献类型：
--
作者：
Chen, Jing;Yao, Yaxia;Du, Juan
唇腭裂是最常见的先天性发育缺陷之一。自噬是真核生物中高度保守的细胞自降解过程，涉及多个生物学过程，氯喹（CQ）是其中最常见的抑制剂。然而，CQ是否影响以及如何影响腭发育尚不清楚。采用细胞增殖实验、流式细胞术、划痕实验和茜素红染色等方法，观察CQ对小鼠胚胎腭突细胞（MEPCs）的增殖、凋亡、迁移和成骨分化的影响。PI染色检测细胞周期分布。免疫荧光法和透射电镜观察自噬体的形成。免疫印迹法检测自噬相关因子（LC 3和P62）、凋亡相关标志物（P53、caspase-3裂解的caspase-3、BAX和BCL-2）和细胞周期相关蛋白（P21、CDK 2、CDK 4、cyclin D1和cyclin E）。CQ可抑制MEPCs的增殖，使细胞周期阻滞于G 0/G1期，细胞周期相关蛋白P21表达上调，CDK 2、CDK 4、cyclin D1和cyclin E表达下调，呈浓度和时间依赖性。结果表明，CQ可通过降低BCL-2/BAX比值和增加caspase-3的裂解水平，诱导细胞凋亡，且呈剂量依赖性。接下来，研究了MEPC的迁移和成骨随着CQ处理以剂量依赖性方式降低。同时，CQ通过上调LC 3 II和P62的表达，激活P53通路，阻断自噬通路。CQ通过激活P53，通过抑制自噬改变MEPC的增殖和凋亡，从而影响MEPC的生物学特性。
Cleft lip and palate is one of the most frequent congenital developmental defects. Autophagy is a highly conserved process of cell self-degradation in eukaryotes, involving multiple biological processes in which chloroquine (CQ) is the most common inhibitor. However, whether CQ affects and how it affects palate development is unknown. Mouse embryonic palatal cells (MEPCs) were treated with CQ to observe cell viability, apoptosis, migration, osteogenic differentiation by cell proliferation assay, flow cytometric analysis, scratch assay, and alizarin red staining. PI staining was used to measure cell cycle distribution. Immunofluorescence (IF) assay and transmission electron microscopy were used to detect autophagosomes. The autophagy-related factors (LC3 and P62), apoptosis-related markers (P53, caspase-3 cleaved caspase-3, BAX, and BCL-2), and cell cycle-related proteins (P21, CDK2, CDK4, cyclin Dl, and cyclin E) were all measured by western blot. CQ inhibited the proliferation of MEPCs by arresting the G0/G1 phase of the cell cycle in a concentration- and time-dependent manner with cell cycle-related proteins P21 upregulated and CDK2, CDK4, cyclin Dl, and cyclin E downregulated. Then we detected CQ also induced cell apoptosis in a dose-dependent manner by decreasing the BCL-2/BAX ratio and increasing cleaved caspase-3. Next, it was investigated that migration and osteogenesis of MEPCs decreased with CQ treatment in a dose-dependent manner. Meanwhile, CQ blocked the autophagy pathway by upregulating LC3II and P62 expressions which activated the P53 pathway. CQ activates P53 which affects MEPC biological characteristics by changing the proliferation and apoptosis of MEPCs through inhibiting autophagy.