苦胆草多糖抗氧化及抗人宫颈癌HeLa细胞的作用及机制  

Antioxidant and Anti-tumor Effect and Mechanisms of Andrographis paniculata Polysaccharide on Human Cervical Cancer HeLa Cells

作　　者：黄丽金 李自霖 杨紫焰 王涵 陈贵元 HUANG Lijin;LI Zilin;YANG Ziyan;WANG Han;CHEN Guiyuan(Basic Medicine School,Dali University,Dali 671000,China;Provincial Key Laboratory of Entomology Biopharmaceutical R&D,Dali 671000,China)

机构地区：[1]大理大学基础医学院,云南大理671000 [2]云南省昆虫生物医药研发重点实验室,云南大理671000

出　　处：《中国实验方剂学杂志》2024年第18期80-88,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(31860252);云南省昆虫生物医药研发重点实验室2022年度开放项目(AG2022006)。

摘　　要：目的:探讨苦胆草多糖(APP)抗氧化及抗人宫颈癌HeLa细胞的作用及机制。方法:细胞功能试验通过细胞增殖与活性检测法(CCK-8)、划痕试验、Transwell试验检测APP(400、450、500 mg·L^(-1))对HeLa细胞的增殖、迁移、侵袭的作用;分子机制实验通过流式细胞术、实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)检测APP对HeLa细胞凋亡及周期相关调控mRNA及蛋白表达的影响;DPPH^(+)、OH^(-)、还原力实验检测APP的抗氧化活性。结果:与空白组比较,APP(400、450、500 mg·L^(-1)),均能明显抑制HeLa细胞的迁移、增殖、侵袭能力,具有时间、浓度依赖(P<0.05,P<0.01);流式细胞术碘化丙啶(PI)单染法检测检测细胞周期,结果显示,与空白组比较,APP(400、450、500 mg·L^(-1))作用HeLa细胞48 h后,HeLa细胞G2/M期所占比例升高,提示APP可将HeLa细胞阻滞在G_(2)/M期,流式细胞术异硫氰酸荧光素(Annexin V-FITC)/PI凋亡试剂盒检测细胞凋亡,与空白组比较,APP(400、450、500 mg·L^(-1))作用HeLa细胞48 h,早期凋亡和晚期凋亡的细胞比例均明显增加(P<0.05,P<0.01),并且具有浓度依赖性,提示APP促进HeLa细胞的凋亡;与空白组比较,APP(400、450、500 mg·L^(-1))作用48 h,细胞周期依赖性激酶-1(CDK-1)、细胞周期蛋白B_(1)(Cyclin B_(1))、B细胞淋巴瘤-2(Bcl-2)mRNA和蛋白表达均下降,胱天蛋白酶(Caspase)-3、Caspase-8、Caspase-9、Bcl-2相关X蛋白(Bax) mRNA和蛋白表达明显升高(P<0.05,P<0.01),以上指标均具有浓度依赖性,与流式细胞结果一致;体外抗氧化实验,APP(50~1 000 mg·L^(-1))具有清除DPPH^(+)、OH^(-)的能力,具有一定的抗氧化活性。结论:APP具有抗氧化活性并能够抑制HeLa细胞的活性,促进HeLa细胞凋亡的能力。Objective To explore the antioxidant and anti-human cervical cancer HeLa cell effect and mechanisms of Andrographis paniculata polysaccharide(APP).Method Cell function assays were conducted to assess the effects of APP(400,450,500 mg·L^(-1))on the proliferation,migration,and invasion of HeLa cells using the cell counting kit-8(CCK-8)assay,scratch assay,and Transwell assay.Molecular mechanism experiments were conducted to detect the effects of APP on HeLa cell apoptosis and cell cycle-related mRNA and protein expression using flow cytometry,real-time fluorescence-based quantitative polymerase chain reaction(Real-time PCR),and Western blot analysis.The antioxidant activity of APP was tested using DPPH^(+),OH^(-),and reducing power assays.Result Compared with the blank group,APP(400,450,500 mg·L^(-1))significantly inhibited the migration,proliferation,and invasion abilities of HeLa cells in a time-and dose-dependent manner(P<0.05,P<0.01).Flow cytometry with propidium iodide(PI)single staining was used to detect cell cycle.The results showed that compared with the blank group,the proportion of HeLa cells in the G_(2)/M phase increased after 48 hours of treatment with APP(400,450,500 mg·L^(-1)),indicating that APP can arrest HeLa cells in the G_(2)/M phase.Flow cytometry with fluorescein isothiocyanate(Annexin V-FITC)/PI apoptosis kit was used to detect cell apoptosis.Compared with the blank group,the proportion of early and late apoptotic HeLa cells increased in a dose-dependent manner after 48 hours of APP(400,450,500 mg·L^(-1))treatment(P<0.05,P<0.01),indicating that APP promotes HeLa cell apoptosis.The results of Real-time PCR and Western blot assay showed that compared with the blank group,after 48 hours of APP(400,450,500 mg·L^(-1))treatment resulted in decreased mRNA and protein expression of cell cycle-dependent kinase-1(CDK-1),Cyclin B_(1),and B-cell lymphoma-2(Bcl-2),and increased mRNA and protein expression of cysteine aspartate protease(Caspase)-3,Caspase-8,Caspase-9,and Bcl-2-associated X protein(Bax

关 键 词：苦胆草多糖 宫颈癌HELA细胞 抗肿瘤 抗氧化 凋亡 

分 类 号：R2-0[医药卫生—中医学] R22R242R285.5R711.74