归芪白术方有效小分子芦荟大黄素调控MDM2/p53信号通路促进胃癌细胞凋亡和周期阻滞的机制研究

Study on the Mechanism of the Active Small Molecule Aloe-Emodin from Guiqi Baizhu Formula in Inducing Gastric Cancer Cell Apoptosis and Cycle Arrest via the MDM2/p53 Signaling Pathway

关键词:  芦荟大黄素  MDM2/p53信号通路  细胞凋亡  细胞周期阻滞  归芪白术方

Key words:  aloe-emodin  MDM2/p53 signaling pathway  apoptosis  cell cycle arrest  Guiqi Baizhu Prescription

摘要:

目的 探讨归芪白术方有效小分子芦荟大黄素调控MDM2/p53信号通路促进胃癌细胞凋亡和周期阻滞的机制。方法 通过分子对接技术分析芦荟大黄素与MDM2蛋白的结合活性；运用CCK-8法检测胃癌细胞增殖能力；利用划痕实验检测细胞迁移能力；借助流式细胞术分析细胞周期及凋亡比例；通过Western Blot检测MDM2/p53信号通路相关蛋白MDM2、p53、Cyclin D1、c-Myc、Bcl-2、Bax表达水平；通过RT-qPCR技术检测MDM2/p53信号通路MDM2、p53、Cyclin D1、c-Myc、Bcl-2、Bax mRNA的表达水平。结果 分子对接结果显示,与川陈皮素和槲皮素相比,芦荟大黄素与MDM2具有较强结合能力；CCK-8法实验表明,与0 μM组相比,芦荟大黄素能浓度和时间依赖性地抑制HGC-27、AGS细胞增殖(P<0.05,P<0.01)；流式实验结果表明,与0 μM组相比,芦荟大黄素可使HGC-27细胞G1期比值随浓度增高呈剂量依赖性显著升高(P<0.01),12.5 μM和25 μM时作用显著,AGS细胞G1期比值也呈浓度依赖性升高(P<0.01)；划痕实验显示,与0 μM组相比,12.5 μM和25 μM的芦荟大黄素可抑制HGC-27、AGS细胞迁移能力；Western blot和qRT-PCR实验结果表明,与0 μM组相比,不同浓度芦荟大黄素干预HGC-27、AGS细胞后,能下调MDM2、CyclinD1、c-Myc、Bcl-2蛋白和基因的表达,并且上调p53和Bax蛋白和基因的表达(P<0.05,P<0.01)。结论 芦荟大黄素可以通过靶向MDM2/p53信号通路,抑制胃癌细胞增殖、诱导凋亡和周期阻滞。

Abstract:

OBJECTIVE This study aims to investigate how aloe-emodin, an active compound derived from the Guiqi Baizhu Prescription, promotes apoptosis and cell cycle arrest in gastric cancer cells by regulating the MDM2/p53 signaling pathway. METHODS This study employed a comprehensive methodological approach to investigate the therapeutic mechanisms of aloe-emodin from Guiqi Baizhu Prescription. Bioinformatics analysis was initially performed to detect differential expression of the MDM2 gene in gastric cancer tissues and evaluate its correlation with patient survival outcomes. Molecular docking technology was subsequently utilized to characterize the binding activity between aloe-emodin and the MDM2 protein. Functional assessments included measurement of gastric cancer cell proliferation using the CCK-8 assay and evaluation of migratory capacity through wound healing assays. Flow cytometric analysis was conducted to quantify cell cycle distribution profiles and apoptosis rates. Molecular mechanisms were further probed by Western blotting to determine protein expression levels of key MDM2/p53 signaling pathway components (MDM2, p53, Cyclin D1, c-Myc, Bcl-2, and Bax), complemented by RT-qPCR analysis of corresponding mRNA expression patterns for these targets. RESULTS Differential expression analysis revealed significantly elevated MDM2 levels in gastric cancer tissues (P<0.01), with survival analysis confirming its strong association with poor patient prognosis (P<0.01). Molecular docking demonstrated substantial binding affinity between aloe-emodin and MDM2. CCK-8 assays indicated concentration- and time-dependent inhibition of HGC-27 and AGS cell proliferation (P<0.05/P<0.01). Flow cytometry confirmed aloe-emodin-induced G1-phase cell cycle arrest and dose-dependent apoptosis enhancement (P<0.01). Wound healing assays showed concentration-dependent suppression of cell migration at 12.5 μM and 25 μM concentrations. Both Western blot and qRT-PCR analyses consistently revealed that aloe-emodin treatment downregulated MDM2, Cyclin D1, c-Myc, and Bcl-2 expression while upregulating p53 and Bax at protein and mRNA levels across tested concentrations (P<0.05/P<0.01). CONCLUSION Aloe-emodin from Guiqi Baizhu Prescription demonstrates anti-tumor effects in gastric cancer by targeting the MDM2/p53 pathway, effectively inhibiting cell proliferation, inducing apoptosis, and triggering cell cycle arrest.