淫羊藿甙促进活性氧表达诱导甲状腺癌B-CPAP细胞凋亡的研究

摘要

背景与目的：淫羊藿甙(icariin，ICA)，是从檗科淫羊藿属植物中提取的重要黄酮类活性化合物。研究表明，ICA对肺癌和胃癌等肿瘤有很好的抑制作用，有望开发为疗效较好的抗肿瘤药，但其作为有前景抗肿瘤药物在甲状腺癌中的研究较少，其作用机制罕见报道。该研究拟探究不同浓度ICA对甲状腺癌B-CPAP细胞系增殖凋亡、细胞内活性氧(reactive oxygen species，ROS)及抗氧化酶系统的影响，探究ICA对甲状腺癌活性影响机制是否具有浓度-时间依赖性。方法：采用细胞计数试剂盒(cell counting kit-8,CCK-8)检测不同浓度ICA对B-CPAP细胞系增殖的影响，采用流式细胞仪技术检测ICA对细胞凋亡及细胞内ROS的影响，采用超氧化物歧化酶(superoxide dismutase，SOD)试剂盒检测ICA对细胞内SOD表达影响，采用丙二醛(malondialdehyd，MDA)检测试剂盒检测ICA对细胞内MDA表达的影响，采用蛋白[质]印迹法(Western blot)检测Bcl-2及γ-HA2X蛋白的表达。结果：ICA处理48 h后B-CPAP细胞活性降低，细胞凋亡率上升，呈剂量-时效依赖性(P＜0.01)。ICA 50和200 mg/L处理组ROS生成量依次是对照组(Control组)的(2.12±0.14)倍和(2.41±0.12)倍。ICA促进细胞膜脂质化产物MDA的累积，降低抗氧化酶SOD活性，活性比Control组下降(9.35±1.45)%和(21.5±1.52)%。ICA 200 mg/L处理组抗凋亡蛋白Bcl-2表达降低(13.64±1.71)%。结论：ICA具有良好的抑制甲状腺癌B-CPAP细胞活性的作用，并呈现剂量依赖性，其主要作用途径可能是促进细胞内ROS高表达，抑制SOD表达，抗凋亡蛋白Bcl-2低表达，导致细胞不可逆损伤，从而诱导细胞凋亡。%Background and purpose:Icariin (ICA) is the important active flavonoids extracted from Berberidaceaeepimedium. It has been shown to be effective in suppressing cancers including lung cancer and gastric cancer. Thus, it is expected to be developed for cancer treatment. However, there were few studies on icariin as a promising anticancer drug for the treatment of thyroid cancer. The mechanisms underlying anticancer effects of ICA in thyroid cancer are rarely reported. This study was to explore the proliferation and apoptosis, intracellular ROS and antioxidant enzyme systems of the thyroid carcinoma cell line B-CPAP treated with different concentrations of ICA. It aimed to explore the mechanism underlying anticancer effects of ICA, and to determine whether it is concentration- or time-dependent manner.Methods:The proliferation of B-CPAP cell line treated with different concentrations of ICA was detected by cell counting kit-8 (CCK-8). The cell apoptosis and intracellular ROS were observed by flow cytometry. The expression of intracellular superoxide dismutase and intracellular malondialdehyde were measured by SOD detection kit and MDA assay kit, respectively. Bcl-2 and γ-HA2X were detected by Western blot.Results:ICA reduced B-CPAP cell activity, increased the rate of apoptosis in a dose- and time-dependent manner after 48 h (P<0.01). The ROS of ICA 50 mg/L and 200 mg/L groups were (2.12±0.14)-fold and (2.41±0.12)-fold of the control group, respectively. ICA promoted accumulation of malondialdehyde, and reduced antioxidant enzyme SOD activity. The SOD activity was decreased by (9.35±1.45)% (ICA 50 mg/L group) and (21.5±1.52)% (ICA 200 mg/L group) compared with the control group, respectively. The anti-apoptotic protein Bcl-2 in ICA 200 mg/L group was decreased by (13.64±1.71)%compared with the control group.Conclusion:Icariin inhibited activity of thyroid cancer B-CPAP cells in a dose- and time-dependent manner. It plays an important role in promoting intracellular ROS expression, inhibiting superoxide dismutase expression and decreasing Bcl-2, which leads to irreversible damage to the cell, thereby inducing apoptosis.