地参多糖对非小细胞肺癌A549细胞抗肿瘤作用及机制

Anti-tumor Effect and Mechanism of Lycopus lucidus Polysaccharide on Non-small Cell Lung Cancer A549 Cells

目的:观察地参多糖(LLP)的体外抗肿瘤活性及机制。方法:细胞增殖与活性检测(CCK-8)法检测LLP(0,5,10,15,20 g·L^(-1))对A549细胞不同作用时间(24,48,72 h)的增殖抑制作用;采用细胞划痕,transwell实验检测LLP(10,20 g·L^(-1))作用24,48 h后A549细胞的迁移侵袭能力;碘化丙啶(PI)单染法检测LLP(10,20 g·L^(-1))对A549细胞周期的影响;异硫氰酸荧光素(Annexin V-FITC)/PI凋亡试剂盒检测LLP(10,20 g·L^(-1))诱导A549细胞的凋亡作用;实时荧光定量聚合酶链式反应(Realtime PCR)检测LLP(10,20 g·L^(-1))对A549细胞中半胱氨酸天冬氨酸蛋白水解酶-3(Caspase-3),半胱氨酸天冬氨酸蛋白水解酶-8(Caspase-8),半胱氨酸天冬氨酸蛋白酶-9(Caspase-9),细胞周期依赖性激酶-1(CDK-1),细胞周期蛋白B(1 Cyclin B1)mRNA表达的影响;蛋白免疫印迹法(Western blot)检测LLP对A549细胞中Caspase-3,Caspase-8,Caspase-9,B淋巴细胞瘤-2(Bcl-2)相关X蛋白(Bax),CDK-1,细胞周期依赖性激酶4(CDK-4),细胞周期依赖性激酶-6(CDK-6),Cyclin B1,细胞周期蛋白D1(Cyclin D1)蛋白表达的影响。结果:与空白组比较,LLP组A549细胞的增殖、迁移和侵袭能力均降低(P<0.05,P<0.01);DNA合成准备期/DNA合成前期(G0/G1期)比例升高(P<0.05);凋亡率升高(P<0.05,P<0.01);Caspase-3,Caspase-8,Caspase-9 mRNA表达水平升高(P<0.05,P<0.01),CDK-1,Cyclin B1 mRNA表达水平降低(P<0.05,P<0.01);Caspase-3,Caspase-8,Caspase-9,Bax蛋白表达水平升高(P<0.05,P<0.01),Bcl-2,CDK-1,CDK-4,CDK-6,Cyclin B1,Cyclin D1蛋白表达水平降低(P<0.05,P<0.01)。结论:LLP可抑制A549细胞的增殖,使其周期阻滞在G0/G1期,同时可能包含DNA合成后期/DNA分裂期(G2/M期),并通过线粒体凋亡途径和死亡受体途径诱导细胞凋亡。

Objective:To study the anti-tumor activity and mechanism of Lycopus lucidus polysaccharide(LLP)in vitro.Method:Cell counting kit-8(CCK-8)assay was used to detect the inhibitory effect of LLP(0,5,10,15,20 g·L^(-1))on the proliferation of A549 cells at different time points(24,48,72 h).The migration and invasion abilities of A549 cells were detected by wound healing assay and transwell assay after LLP(10,20 g·L^(-1))treatment for 24,48 h.Propidium iodide(PI)single staining was applied to determine the effect of LLP of different concentrations(10,20 g·L^(-1))on the cell cycle of A549.The apoptosis of A549 cells induced by LLP(10,20 g·L^(-1))was detected by Annexin V-FITC/PI kit.Real-time fluorescence-based quantitative polymerase chain reaction(Real-time PCR)was adopted to measure effect of LLP(10,20 g·L^(-1))on gene expression of cysteine aspartate protease-3(Caspase-3),cysteine aspartate protease-8(Caspase-8),cysteine aspartate protease-9(Caspase-9),cyclin-dependent kinase-1(CDK-1),and Cyclin B1 in A549 cells.Western blot was used to detect the effect of LLP on protein expression of Caspase-3,Caspase-8,Caspase-9,B-cell lymphoma 2(Bcl-2)-associated X protein(Bax),CDK-1,cyclin-dependent kinase-4(CDK-4),cyclindependent kinase-6(CDK-6),Cyclin B1,and Cyclin D1 in A549 cells.Result:Compared with the blank group,the LLP group showed decreased proliferation,migration,and invasion of A549 cells(P<0.05,P<0.01),increased proportion of G0/G1 phase(P<0.05),enhanced apoptosis rate(P<0.05,P<0.01),elevated mRNA expression of Caspase-3,Caspase-8,and Caspase-9(P<0.05,P<0.01),reduced mRNA expression of CDK-1 and Cyclin B1(P<0.05,P<0.01),up-regulated protein expression of Caspase-3,Caspase-8,Caspase-9,and Bax(P<0.05,P<0.01),and down-regulated protein expression of Bcl-2,CDK-1,CDK-4,CDK-6,Cyclin B1,and Cyclin D1(P<0.05,P<0.01).Conclusion:LLP can inhibit the proliferation of A549 cells,block the cell cycle in the G0/G1 phase(also G2/M phase),and induce cell apoptosis via the mitochondrial apoptosis pathway and death receptor pathway.