摘要
目的探讨乙酰紫草素对人喉癌Hep-2细胞增殖和凋亡的影响及其相关的分子机制。方法体外培养Hep-2细胞,按照处理因素不同,将实验对象分为时间处理组[以25μmol/L(近IC50值)乙酰紫草素分别处理细胞0,3,6,12,24 h)]及浓度处理组(以0、10、20、30、40、50μmol/L乙酰紫草素处理细胞24 h),细胞增殖与毒性实验(cell counting Kit-8,CCK-8)检测不同浓度乙酰紫草素对处理不同时间人喉癌HEP-2细胞的杀伤作用;流式细胞术检测乙酰紫草素处理后Hep-2细胞凋亡情况;Western blotting检测细胞凋亡相关蛋白和Wnt/β-catenin信号通路相关蛋白的表达。结果不同浓度(10~50μmol/L)乙酰紫草素能够以时间和浓度依赖性抑制喉癌Hep-2细胞的增殖;半数抑制浓度为(26.83±2.47)μmol/L;IC50浓度乙酰紫草素处理Hep-2细胞3、6、12及24 h,细胞凋亡率分别为(15.25±1.74)%,(28.75±2.36)%,(36.51±3.78)%及(43.78±3.69)%,各处理组与对照组(0 h)相比,差异均具有统计学意义(F=43.58,P<0.05);乙酰紫草素能够通过上调Bax,Cyt-c,Caspase-3表达(F=12.67、23.47、9.52,P值均<0.05),下调Bcl-2,Caspase-9表达(F=22.29、15.62,P<0.05)来诱导喉癌Hep-2细胞线粒体依赖性凋亡;乙酰紫草素能够降低Wnt1,β-catenin,c-myc和Cyclin D1蛋白的表达(F=32.67、19.57、13.88、28.14,P值均<0.05)从而有效抑制喉癌Hep-2细胞中的Wnt/β-catenin信号通路。结论乙酰紫草素能够有效抑制人喉癌Hep-2细胞增殖,并能够有效诱导Hep-2细胞发生线粒体依赖性凋亡,其具体机制可能是通过调控Wnt/β-catenin信号通路来实现的。
Objective To investigate the effects of acetylshikonin on proliferation and apoptosis of human laryngeal carcinoma(Hep-2)cells and the related molecular mechanisms.Methods Hep-2 cells were cultured in vitro,the subjects were divided into groups according to different time[25μmol/L(near IC50 value)cells were treated 0,3,6,12,24 h respectively]and treatment group according to different concentrations(cells were treated by acetyl shikonin with 0,10,20,30,40,50μmol/L for 24 h),Cell counting kit-8(CCK-8)was used to detect the killing effect of different concentrations of acetoshikonin on Hep-2 cells treated with different time.Flow cytometry was used to detect the apoptosis of Hep-2 cells treated with acetylshikonin.Western blotting was used to detect the expression of apoptosis-related proteins and Wnt/β-catenin signaling pathway-related proteins.Results Acetylshikonin can inhibit the proliferation of laryngeal cancer Hep-2 cells in a time and concentration-dependent manner,with a half inhibitory concentration of(26.83±2.47)μmol/L.The IC50 concentration of acetylshikonin treated Hep-2 cells for 3,6,12 and 24 h,the apoptosis rates were(15.25±1.74)%,(28.75±2.36)%,(36.51±3.78)%and(43.78±36.9)%,compared with the control group(0 h),the difference between each treatment group was statistically significant(F=43.58,P<0.05);Acetyl shikonin can up-regulate the expression of Bax,Cyt-c,and Caspase-3(F=12.67,23.47,9.52,all P<0.05),and down-regulate the expression of Bcl-2,Caspase-9(F=22.29,15.62,both P values<0.05)to induce mitochondrial-dependent apoptosis in laryngeal cancer Hep-2 cells;Acetyl shikonin can reduce the expression of Wnt1,β-catenin,c-myc and Cyclin D1 proteins(F=32.67,19.57,13.88,28.14,all P values<0.05),thereby effectively inhibiting Wnt/β-catenin signaling pathway.Conclusion Acetylshikonin can effectively inhibit the proliferation of human laryngeal carcinoma Hep-2 cells and induce mitochondria-dependent apoptosis in Hep-2 cells.The specific mechanism may be achieved by regulating Wnt/β-catenin signaling pathway.
作者
张萍
潘芳名
肖洪彬
Zhang Ping;Pan Fangming;Xiao Hongbin(Department of Otolaryngology,Xiaogan Hospital Affiliated to Wuhan University of Science and Technology,Xiaogan 432000,China;Heilongjiang University of Traditional Chinese Medicine,Harbin 150040,China)
出处
《国际免疫学杂志》
CAS
2020年第4期366-370,共5页
International Journal of Immunology
基金
国家自然科学基金面上项目(81473555)。
