摘要
目的:基于核苷酸结合寡聚化结构域样受体蛋白3/天冬氨酸蛋白水解酶-1(NLRP3/Caspase-1)信号通路探讨熊果酸促进肝癌SMMC-7721细胞焦亡作用机制。方法:培养肝癌SMMC-7721细胞,以熊果酸0、30、40、50μmol/L干预,电镜观察细胞焦亡时的形态变化;噻唑蓝比色(MTT)法测定细胞24 h体外增殖的影响;Hoechst 33242染色法荧光显微镜下观察细胞焦亡情况;集落形成实验(Clonogenicity assay)观察熊果酸对人肝癌SMMC-7721细胞集落形成能力的影响;流式细胞术分析熊果酸对人肝癌SMMC-7721细胞周期的影响;免疫印迹(Western-Blot)法检测Caspase-1、NLRP3和GSDMD-N蛋白表达。结果:不同浓度熊果酸作用肝癌SMMC-7721细胞24 h后,显示熊果酸可抑制肝癌SMMC-7721细胞增殖,且随着药物浓度的增加,抑制作用显著增强(P<0.01);随着熊果酸浓度的不断增加,细胞焦亡数量显著增加(P<0.01);随着熊果酸浓度的不断增加,细胞集落形成能力明显下降(P<0.05);30μmol/L的熊果酸作用于肝癌SMMC-7721细胞,细胞出现不同程度的肿胀,细胞膜破裂且有孔洞形成等典型的焦亡形态学特征;不同浓度熊果酸干预肝癌SMMC-7721细胞24 h,可上调NLRP3、Caspase-1、GSDMD-N蛋白表达(P<0.05)。结论:熊果酸能抑制人肝癌SMMC-7721细胞增殖、集落形成,促进肝癌细胞焦亡;熊果酸诱导肝癌SMMC-7721细胞焦亡可能与其上调Caspase-1和NLRP3蛋白表达有关。
Objective: To explore the mechanism of ursolic acid(UA) in promoting the pyroptosis of hepatocellular carcinoma SMMC-7721 cells based on the NLRP3/Caspase-1 signaling pathway. Methods: Hepatocellular carcinoma SMMC-7721 cells were cultured and treated with the different concentrations of UA(0, 30, 40, 50 μmol/L). Morphological changes of the cells were observed by electron microscope. MTT assay was used to measure the cell proliferation for 24 hours in vitro. Hoechst 33242 staining method was used to observe the pyroptosis under fluorescence microscope. Clonogenic assay was performed to detect the effect of UA on the colony formation ability of hepatocellular carcinoma SMMC-7721 cells, and flow cytometry was employed to analyze the cell cycle changes after UA intervening in the cells. Western blot was adopted to determine the protein expressions of pyroptosis-related Caspase-1, NLRP3 and GSDMD-N in hepatocellular carcinoma SMMC-7721 cells treated with UA. Results: After different concentrations of UA acted on hepatocellular carcinoma SMMC-7721 cells for 24 hours, the cell proliferation was inhibited, and with the increase of UA concentration, the inhibitory effect was enhanced(P<0.01). In addition, with the increase of UA concentration, the number of cells undergoing pyroptosis was elevated(P<0.01), and the ability of cell colony formation was decreased(P<0.05). In UA 30 μmol/L group, different degrees of cell swelling was found, with typical morphological features of pyroptosis such as membranolysis and hole formation. After treatment with different concentrations of UA for 24 hours, the protein expressions of NLRP3, Caspase-1 and GSDMD-N in hepatocellular carcinoma SMMC-7721 cells were up regulated(P<0.05). Conclusion: UA could inhibit the proliferation and colony formation of hepatocellular carcinoma SMMC-7721 cells, and promote the pyroptosis. The mechanism might be related to up regulating the expressions of Caspase-1 and NLRP3 proteins.
作者
杨晨
黄峰
晁旭
雷莉妍
Yang Chen;Huang Feng;Chao Xu;Lei Li Yan(The Second Affiliated Hospital of Shaanxi University of Chinese Medicine,Xianyang 712000;Shaanxi University of Chinese Medicine,Xianyang 712046)
出处
《中药药理与临床》
CAS
CSCD
北大核心
2022年第4期101-107,共7页
Pharmacology and Clinics of Chinese Materia Medica
基金
国家自然科学基金项目(编号:8177413)
陕西中医药大学学科创新团队建设项目(项目编号:2019-YS05)。
