化香树果序乙醇提取物调控RAS/MAPK通路对鼻咽癌细胞的影响

Effect of Ethanol Extract of Infructescence of Platycarya strobilacea on Nasopharyngeal Carcinoma Cells Through RAS/MAPK Pathway

目的：研究化香树果序醇提物（ethanol extract of infructescence of Platycarya strobilacea,EPS）的对鼻咽癌CNE1,CNE_2细胞的药理作用机制。方法：以鼻咽癌CNE1,CNE_2细胞为研究对象,以EPS 0.25,0.5,1.0,1.5,2.0 g·L^（-1）干预,设空白组,孵育24 h后,采用噻唑蓝（MTT）比色法检测EPS对CNE1,CNE_2细胞活力的影响。用1.0 g·L^（-1）EPS处理CNE1,CNE_2细胞24 h,倒置显微镜观察细胞形态变化,透射电镜观察细胞器的变化。流式细胞仪检测细胞凋亡程度,RNA高通量测序检测EPS处理后细胞中的差异基因。蛋白免疫印迹法检测HRas proto-oncogene（HRAS）蛋白细胞外调节蛋白激酶1/2（ERK1/2）和c-Fos proto-oncogene（c-Fos）蛋白表达水平。结果：与空白组比较,EPS能明显抑制CNE1,CNE_2细胞的增殖（P〈0.05）,且呈浓度依赖性。与空白组比较,EPS明显诱导CNE1,CNE_2细胞质出现大量空泡,空泡相互融合,不断增大,最后细胞膜破裂,透射电镜发现细胞核没有发生明显的变化,溶酶体空泡化明显,流式结果未发现明显凋亡,与细胞methuosis死亡特征一致。高通量测序结果显示与癌蛋白（RAS）/丝裂原活化蛋白激酶（MAPK）信号通路相关,与空白组比较HRAS,ERK1/2,c-Fos蛋白表达降低,磷酸化细胞外调节蛋白激酶1/2（p-ERK1/2）蛋白表达升高（P〈0.05）。结论：EPS可通过调控RAS/MAPK信号通路诱导人鼻咽癌细胞CNE1,CNE_2发生methuosis死亡,导致细胞相互融合,细胞内出现大量空泡。

Objective： To study the pharmacological mechanism of ethanol extract of infructescence of Platycarya strobilacea（ EPS） treatment on CNE1 and CNE_2 cells in nasopharyngeal carcinoma（ NPC）. Method：NPC cells（ CNE1 and CNE_2） were taken as the research objects and treated with 0（ blank group）,0. 25,0. 5,1. 0,1. 5,2. 0 g·L^（-1） for 24 h. The effect of EPS on viability of CNE1 and CNE_2 cells were detected by（ 4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide（ MTT） assay. After CNE1 and CNE_2 cells were treated with 1. 0 g·L^（-1） EPS for 24 h,the morphological changes of the cells were observed by inverted microscope.The changes of organelles were observed by transmission electron microscopy（ TEM）. Flow cytometry was used to detect the apoptosis of CNE1 and CNE_2 cells induced by EPS. The differential genes in the cells were detected by nucleotide high throughput sequencing,and the expression levels of HRas proto-oncogene（ HRAS）,extracellular regulated protein kinase 1/2（ ERK1/2） and c-Fos proto-oncogene（ c-Fos） proteins were detected by Western blot（ WB）. Result： As compared with blank control group,EPS could significantly inhibit the growth of CNE1 and CNE_2 cells in a dose-dependent manner（ P〈0. 05）. After being treated with EPS,CNE1 and CNE_2 cells showed a large number of vacuoles,and the vacuoles became confluent with each other and became larger and larger.Finally,the cell membrane ruptured and the nucleus did not change obviously. EPS could not promote CNE1 and CNE_2 cells apoptosis and NPC cells had obvious characteristic methuosis changes in the observation under phase contrast microscope. RNA sequencing results showed that renin-angiotensinsystem（ RAS）/mitogen-activated protein kinase（ MAPK） signaling pathway was involved. WB results showed that as compared with the blank control group,EPS could reduce the protein expression levels of HRAS,c-Fos and ERK1/2,but increase the protein expression levels of p-ERK1/2（ P〈0. 05）. Conclusion： EPS can induce methuosis death in human nasopharyngeal carcinoma cells CNE1 and CNE_2 through regulating RAS/MAPK signaling pathway,thus leading to confluence of cells and resulting in a large number of vacuoles.