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我降解过程,涉及多个生物过程,其中氯喹(CQ)是最常见的抑制剂。然而,CQ是否影响以及如何影响腭发育尚不清楚。小鼠胚胎腭细胞(MEPCs)用CQ处理,观察细胞活力,凋亡,迁移,通过细胞增殖测定成骨分化,流式细胞仪分析,划痕试验,和茜素红染色。PI染色用于测量细胞周期分布。免疫荧光(IF)法和透射电镜法检测自噬体。自噬相关因子(LC3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,CDK2,CDK4,细胞周期蛋白D1和细胞周期蛋白E)均通过蛋白质印迹测量。CQ通过抑制细胞周期的G0/G1期以浓度和时间依赖性方式抑制MEPCs的增殖,细胞周期相关蛋白P21上调,CDK2,CDK4,细胞周期蛋白D1和细胞周期蛋白E下调。然后,我们检测到CQ还通过降低BCL-2/BAX比率和增加裂解的caspase-3以剂量依赖性方式诱导细胞凋亡。接下来,研究发现,CQ治疗以剂量依赖性方式降低了MEPCs的迁移和成骨作用。同时,CQ通过上调LC3II和P62表达来阻断自噬途径,从而激活P53途径。CQ激活P53,通过抑制自噬改变MEPCs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 D1, 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 D1, 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.