红芪多糖通过线粒体途径诱导皮肤鳞状细胞癌细胞SCL-1凋亡的研究

Apoptosis of Cutaneous Squamous Cell Carcinoma Cell Line SCL-1 Induced by Hedysari Polysaccharide Through Mitochondrial Pathway

目的:观察红芪多糖对SCL-1细胞凋亡的调控作用,并探究其作用机制。方法:红芪多糖(25、50、100μg/mL)处理SCL-1细胞,0μg/mL红芪多糖处理的SCL-1细胞设为对照组;细胞计数法(CCK8)、膜联蛋白V-异硫氰酸荧光素-碘化丙锭(Annexin V-FITC/PI)双染法检测细胞增殖抑制率、细胞凋亡率,筛选50μg/mL浓度为红芪多糖最适浓度。JC-1染色法检测细胞线粒体膜电位,蛋白免疫印迹(WB)实验检测细胞中分裂的多聚DNA核糖聚合酶(Cleaved PARP)、Cleaved半胱氨酸天冬氨酸蛋白酶(Caspase)-3、Cleaved Caspase-9及细胞浆、线粒体中细胞色素C(Cytochrome c)、第二个线粒体衍生的caspases激活剂(Smac)、B细胞淋巴瘤/白血病2相关X蛋白(Bax)、B细胞淋巴瘤/白血病2家族促凋亡基因(Bak)的蛋白表达。结果:与对照组相比,红芪多糖(25、50、100μg/mL)组SCL-1细胞增殖抑制率显著升高(P<0.05),细胞凋亡率显著升高(P<0.05),确定50μg/mL红芪多糖为最适浓度。与对照组相比,红芪多糖组SCL-1细胞线粒体膜电位显著降低(P<0.05),线粒体凋亡相关蛋白Cleaved PARP、Cleaved Caspase-3、Cleaved Caspase-9表达显著升高(P<0.05),线粒体释放Cytochrome c、Smac蛋白表达显著升高(P<0.05),线粒体Bax、Bak蛋白表达显著升高(P<0.05)。结论:红芪多糖促进皮肤鳞癌细胞SCL-1凋亡,其机制为激活线粒体途径。

Objective:To observe the regulatory effect of Hedysari polysaccharide on SCL-1 cell apoptosis and explore its mechanism.Methods:SCL-1 cells were treated with radix Hedysari polysaccharide(25,50,100μg/mL);0μg/ml SCL-1 cells treated with hedysari polysaccharide were set as the control group.The cell proliferation inhibition rate and apoptosis rate were detected by cell counting method(CCK8)and annexin V-fluorescein isothiocyanate propidium iodide(annexin V-FITC/PI).50μg/mL was the optimum concentration of Hedysari polysaccharide.The mitochondrial membrane potential was detected by JC-1 staining.Protein expression of cleaved poly DNA ribose polymerase(cleaved PARP),cleaved cysteine aspartate protease(caspase)-3,cleaved caspase-9 and cytochrome C in cytoplasm,mitochondria,second mitochondrial derived caspases activator(SMAC),B-cell lymphoma/leukemia 2 related X protein(Bax),pro-apoptotic gene(bak)were detected by Western blotting(WB).Results:Compared with the control group,the proliferation inhibition rate and apoptosis rate of SCL-1 cells significantly increased in Hedysari polysaccharide(25,50,100μg/mL)group(P<0.05).50μg/mL hedysarum polysaccharide was affirmed the optimum concentration.Compared with the control group,the mitochondrial membrane potential decreased significantly(P<0.05),the expression of mitochondrial apoptosis related proteins cleaved PARP,cleaved caspase-3 and cleaved caspase-9 substantially increased(P<0.05),the expression of cytochrome c and Smac released by mitochondria obviously increased(P<0.05),and the expression of mitochondrial Bax and Bak protein significantly increased in hedysari polysaccharide group(P<0.05).Conclusion:Hedysari polysaccharide could promote the apoptosis of skin squamous cell carcinoma cell line SCL-1 by activating mitochondrial pathway.