摘要
目的研究紫草素对人结肠癌细胞系SNU⁃407增殖和自噬的影响,及其潜在的分子机制。方法通过细胞计数试剂盒⁃8(CCK⁃8)检测细胞存活率,用Hoechst 33258染色法和流式细胞术分析细胞生长情况,采用蛋白质印迹法(Western Blot)分析不同紫草素浓度(0μmol/L、10μmol/L、20μmol/L和30μmol/L)处理前后自噬相关的E1连接酶7(ATG7)、丝裂原活化蛋白激酶(MAPK)和死亡受体(Caspase⁃8,Caspase⁃3,PARP)蛋白表达情况。结果紫草素以时间和剂量依赖性方式诱导SNU⁃407细胞凋亡和自噬,在紫草素处理的SNU⁃407细胞中观察到ATG7表达水平显著升高;与0μmol/L的紫草素相比,10μmol/L、20μmol/L和30μmol/L紫草素处理后SNU⁃407细胞的ATG7/自噬相关的E1连接酶5(ATG5)相对表达量分别显著增加[(176±9)%、(212±13)%和(293±11)%]。与GFPi(阴性对照质粒pSUPER_GFP siRNA)阴性对照组相比,ATG被沉默的shATG7(ATG7的RNA干扰质粒pSUPER_ATG7 siRNA)组LC3⁃Ⅱ/LC3⁃Ⅰ比值显著低于GFPi阴性对照组[(8.25±1.36)比(97.45±1.98),P<0.001],ATG7沉默后抑制了紫草素诱导的SNU⁃407细胞自噬;荧光显微镜结果和流式细胞术分析证实紫草素诱导结肠癌细胞凋亡;蛋白质印迹法印迹分析显示紫草素通过MAPK活化诱导结肠癌细胞凋亡,激活死亡受体(FADD)调节细胞凋亡蛋白(Caspase⁃8,Caspase⁃3,PARP)表达。此外,紫草素通过增加ATG7的表达水平,进而下调促进细胞自噬的p62表达。结论紫草素通过激活ATG7信号通路诱导人结肠癌细胞系SNU⁃407细胞凋亡和自噬.
Objective To elucidate the effects of shikonin on proliferation and autophagy of human colon cancer cell line SNU⁃407,and to explore its molecular mechanism of potential action.Methods Cell Counting Kit⁃8(CCK⁃8)was used to detect cell viability,Hoechst 33258 staining and flow cytometry were used to analyze cell growth,and Western Blot was used to analyze different shikonin concentrations(0μmol/L,10μmol/L,20μmol/L and 30μmol/L)autophagy⁃related E1 ligase 7(ATG7),mito⁃gen⁃activated protein kinase(MAPK)and death receptor(Caspase⁃8,Caspase⁃3,PARP)protein expression before and after treatment Happening.Results The results showed that shikonin induced apoptosis of SNU⁃407 cells in a time⁃and dose⁃dependent manner,and ATG7 expression level was significantly increased in shikonin⁃treated SNU⁃407 cells.Compared with 0μmol/L shiko⁃nin,the relative expression of ATG7/autophagy⁃related E1 ligase 5(ATG5)in SNU⁃407 cells was significantly increased by(176±9)%,(212±13)%and(293±11)%after treatment with 10μmol/L,20μmol/L and 30μmol/L shikonin.Compared with GFPi nega⁃tive control group(the negative control plasmid pSUPER_GFP siRNA),the LC3⁃Ⅱ/LC3⁃Ⅰratio of the shATG7 group(ATG7 RNA interference plasmid pSUPER_ATG7 siRNA)was significantly lower than that of the GFPi negative control group[(8.25±1.36)vs.(97.45±1.98),P<0.001],silencing of ANU7 inhibited shikonin⁃induced apoptosis of SNU⁃407 cells.Furtherly,fluores⁃cence microscopy results and flow cytometry analysis confirmed that shikonin induced apoptosis in colorectal cancer cells.In addi⁃tion,Western blotting analysis showed that shikonin induced apoptosis of colon cancer cells through MAPK activation,and activat⁃ed death receptor(FADD)to regulate the expression of apoptosis proteins(Caspase⁃8,Caspase⁃3,PARP).In addition,shikonin in⁃creases the expression level of ATG7,thereby down⁃regulating the expression of p62 that promotes autophagy.Conclusion Based on the above results,we suggest that shikonin promotes
作者
孙艳华
李明
徐建立
董路
曹学彬
孟小晶
SUN Yanhua;LI Ming;XU Jianli;DONG Lu;CAO Xuebin;MENG Xiaojing(Department of Gastrointestinal Hernia,Cangzhou People’s Hospital,Cangzhou,Hebei 061000,China)
出处
《安徽医药》
CAS
2020年第12期2336-2342,I0001,共8页
Anhui Medical and Pharmaceutical Journal
基金
2019年度河北省医学科学研究重点课题计划(20191249)。
