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[摘要]
目的 探究白屈菜红碱(CHE)对腺样囊性癌细胞(ACC2)生长的抑制作用及机制。方法 利用CCK8法、EdU法、Hoechst33342/PI双染色法、试剂盒法检测CHE对ACC2细胞活力、细胞增殖、细胞凋亡和活性氧(ROS)水平的影响;通过Western blotting技术检测CHE对Cleaved-Caspase 3、PARP、NF-κB、p-JNK、p-p38蛋白表达的影响;利用斑马鱼移植瘤模型检测CHE对斑马鱼体内ACC2细胞生长的抑制作用。结果 CCK-8结果显示:与对照组比较,2、3、4、5、6、7、8、9、10 μmol/L的CHE显著降低ACC2细胞的存活率(P<0.05、0.01),且呈浓度相关性; ROS检测结果显示:与对照组比较,5、8 μmol/L的CHE导致ACC2细胞内的ROS水平显著上升(P<0.05、0.01); EdU增殖检测结果表明:与对照组比较,5、8 μmol/L的CHE致使ACC2细胞的增殖能力显著下降(P<0.01);Hoechst/PI染色结果显示:与对照组比较,CHE 5、8 μmol/L组ACC2细胞凋亡率显著上升(P<0.01)。抗氧化剂N-乙酰半胱氨酸(NAC)显著抑制CHE诱导的ROS水平升高、细胞凋亡增加(P<0.01);Western blotting结果显示:2、5、8 μmol/L的CHE能够显著上调Cleaved-Caspase 3、PARP、NF-κB蛋白的表达(P<0.01),且呈现浓度相关性,5、8 μmol/L的CHE能够显著上调p-JNK的蛋白表达(P<0.01),8 μmol/L的CHE能够显著上调p-p38的蛋白表达(P<0.01);NAC显著降低由CHE导致的Cleaved-Caspase 3、PARP、NF-κB、p-JNK、p-p38蛋白表达增加(P<0.01),5、8 μmol/L CHE能够有效抑制斑马鱼体内肿瘤的生长(P<0.01)。结论 体外及斑马鱼移植瘤模型证明,CHE可以有效抑制ACC2细胞生长,其机制与提高细胞ROS水平,上调NF-κB、p-JNK、p-p38表达,从而抑制细胞增殖、诱导细胞凋亡相关。
[Key word]
[Abstract]
Objective To investigate the inhibitory effect of chelerythrine (CHE) on adenoid cystic carcinoma (ACC2) cells and its molecular mechanism. Methods CCK8, EdU, hoechst33342/PI double staining, and test kit method were used to detect the effects of CHE on ACC2 cell viability, cell proliferation, apoptosis and reactive oxygen species (ROS) level. The effects of CHE on the expression levels of Caspase-3, PARP, NF-κB, P-JNK and p-p38 were detected by Western blotting. The inhibitory effect of chelerythrine on the growth of ACC2 cells was detected by zebrafish xenograft tumor model. Results CCK-8 assay showed that: CHE can significantly inhibited the proliferation of ACC2 cells in a concentration dependent manner (P < 0.05 and 0.01) at the concentration of 2, 3, 4, 5, 6, 7, 8, 9, 10 μmol/L. The results showed that ROS in ACC2 cells was significantly increased at the concentration of 5 and 8 μmol/L (P < 0.05 and 0.01). EdU assay results showed that the proliferation of ACC2 cells was significantly decreased by CHE at the concentration of 5 and 8 μmol/L (P < 0.01). Hoechst/PI staining showed that apoptosis was significantly induced by CHE at the concentration of 5 and 8 μmol/L (P < 0.01). while N-acetylcysteine (NAC) significantly inhibited the increase of ROS and apoptosis induced by CHE (P < 0.01). Western blotting results showed that the expression of Cleaved Caspase 3, PARP and NF-κB were significantly up-regulated by 2, 5, and 8 mol/L CHE (P < 0.01), the expression of p-JNK was significantly up-regulated by 5 and 8 mol/L CHE (P < 0.01), and the protein expression of p-p38 was significantly up-regulated by 8 mol/L CHE (P < 0.01). NAC significantly decreased the expression of Cleaved Caspase 3, PARP, NF-κB, p-JNK, p-p38 protein induced by CHE (P < 0.01). CHE of 5 and 8 mol/L could effectively inhibit the growth of ACC2 cells in zebrafish (P < 0.01). Conclusion CHE can effectively inhibit the proliferation of ACC2 cells in vivo and zebrafish xenograft tumor model. Elevation of the ROS level induced by CHE, upregulation of NF-κB, p-JNK and p-p38 and then lead to the inhibition of proliferation and induction of apoptosis were involved in the inhibition of ACC2 cells in vivo and in vivo.
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[基金项目]
国家重点研发计划(2018YFC1707300);山东省重大科技创新工程项目(2019JZZY020905);齐鲁工业大学(山东省科学院)科教产融合创新试点工程项目(2020KJC-ZD08)