益气解毒方通过Wnt/β-catenin信号通路诱导鼻咽癌5-8F细胞凋亡

Yiqi Jiedu Formula Induced Apoptosis of Nasopharyngeal Carcinoma 5-8F Cells via Wnt/β-catenin Signaling Pathway

目的基于Wnt/β-catenin信号通路探讨益气解毒方(黄芪、黄连、白花蛇舌草等)对鼻咽癌5-8F细胞凋亡的作用机制。方法(1)将5-8F细胞分为空白对照组、益气解毒方不同浓度组(0.25、0.5、1.0、2.0 mg·mL^(-1))及顺铂组(4μg·mL^(-1)),采用实时无标记细胞功能分析仪(RTCA)监测细胞的增殖情况。(2)将5-8F细胞分为5组:空白对照组、益气解毒方1.0 mg·mL^(-1)组、HLY7810μmol·L^(-1)组、HLY7810μmol·L^(-1)+益气解毒方1.0 mg·mL^(-1)组、IWR-18μmol·L^(-1)组,采用RTCA监测细胞的增殖;药物干预36 h后,采用Annexin V-FITC/PI双荧光染色法检测细胞凋亡率;JC-1荧光探针法检测细胞线粒体膜电位;Western Blot法检测细胞β-catenin、Wnt5a/b、Bcl2及Bax蛋白的表达水平。结果与空白对照组比较,益气解毒方不同浓度组(0.25、0.5、1.0、2.0 mg·mL^(-1))的5-8F细胞增殖曲线均明显降低;益气解毒方1.0 mg·mL^(-1)组、IWR-18μmol·mL^(-1)组的细胞凋亡率显著升高(P<0.01),线粒体膜电位明显降低,β-catenin、Wnt5a/b及Bcl2蛋白表达明显下调(P<0.05,P<0.01),Bax蛋白表达显著上调(P<0.01),Bax/Bcl2比值显著升高(P<0.01)。与益气解毒方1.0 mg·mL^(-1)组比较,HLY7810μmol·L^(-1)+益气解毒方1.0 mg·mL^(-1)组的5-8F细胞增殖曲线明显升高,细胞凋亡率显著下降(P<0.01),线粒体膜电位明显升高,β-catenin、Wnt5a/b及Bcl2蛋白表达明显上调(P<0.05,P<0.01),Bax蛋白表达显著下调(P<0.01),Bax/Bcl2比值显著降低(P<0.01)。结论益气解毒方能够抑制鼻咽癌5-8F细胞增殖,并诱导细胞凋亡,其机制与抑制Wnt/β-catenin信号通路相关。

Objective To explore the mechanism of action of Yiqi Jiedu Formula(Radix Astragali seu Hedysari,Rhizoma Coptidis,and Herba Hedyotis)on apoptosis of 5-8F cells in nasopharyngeal carcinoma based on Wnt/β-catenin signaling pathway.Methods(1)The 5-8F cells were divided into the blank control group,different concentration of Yiqi Jiedu Formula groups(0.25,0.5,1.0,2.0 mg·mL^(-1))and cisplatin group(4μg·mL^(-1)),and the proliferation of cells was monitored by real-time cell analysis(RTCA).(2)5-8F cells were divided into five groups:the blank control group,the Yiqi Jiedu Formula 1.0 mg·mL^(-1)group,the HLY7810μmol·L^(-1)group,the HLY7810 mg·mL^(-1)+Yiqi Jiedu Formula 1.0 mg·mL^(-1)group and the IWR-18μmol·L^(-1)group,and cell proliferation was monitored by RTCA;after 36 hours of drug intervention,Annexin V-FITC/PI double fluorescence staining was used to detect the cell apoptosis rate;JC-1 fluorescent probe was used to detect the mitochondrial membrane potential;the protein expression levels ofβ-catenin,Wnt5a/b,Bcl2 and Bax were detected by Western Blot method.Results Compared with the blank control group,the proliferation curves of 5-8F cells in different concentration groups(0.25,0.5,1.0 and 2.0 mg·mL^(-1))of Yiqi Jiedu Formula were significantly decreased;the apoptosis rate of Yiqi Jiedu Formula 1.0 mg·mL^(-1)group and IWR-18μmol·L^(-1)group were significantly increased(P<0.01),the mitochondrial membrane potential was significantly decreased,the protein expression ofβ-catenin,Wnt5a/b and Bcl2 were significantly down-regulated(P<0.05,P<0.01),the protein expression of Bax was significantly up-regulated(P<0.01)and Bax/Bcl2 ratio was significantly increased(P<0.01).Compared with the Yiqi Jiedu Formula 1.0 mg·mL^(-1)group,the proliferation curve of 5-8F cells in the HLY7810μmol·L^(-1)+Yiqi Jiedu Formula 1.0 mg·mL^(-1)group was significantly increased,the apoptosis rate was significantly decreased(P<0.01),the mitochondrial membrane potential was significantly increased,and the protein expressiosn ofβ-catenin,Wnt5a/b and Bcl2 were significantly up-regulated(P<0.05,P<0.01),the protein expression of Bax was significantly down-regulated(P<0.01)and Bax/Bcl2 ratio was significantly decreased(P<0.01).Conclusion Yiqi Jiedu Formula inhibits the proliferation and induces apoptosis of nasopharyngeal carcinoma 5-8F cells,and the mechanism may be related to the inhibition of Wnt/β-catenin signaling pathway.