[1]王利莹,陆玉成,王丽娟,等.欧当归内酯A对人胶质瘤细胞U251细胞凋亡及自噬的影响[J].新乡医学院学报,2022,39(3):201-207.[doi:10.7683/xxyxyxb.2022.03.001]
　WANG Liying,LU Yucheng,WANG Lijuan,et al.Effect of levistilide A on apoptosis and autophagy of human glioma cell line U251[J].Journal of Xinxiang Medical University,2022,39(3):201-207.[doi:10.7683/xxyxyxb.2022.03.001]
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欧当归内酯A对人胶质瘤细胞U251细胞凋亡及自噬的影响
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
39
期数:
2022年3
页码:
201-207
栏目:
基础研究
出版日期:
2022-03-05

文章信息/Info

Title:
Effect of levistilide A on apoptosis and autophagy of human glioma cell line U251
作者:
王利莹1; 陆玉成2; 王丽娟3; 王蒙恩1; 韦志永4; 车峰远3
（1.潍坊医学院临床医学院，山东　潍坊　261000；2.临沂市人民医院生物样本库，山东　临沂　276000；3.临沂市人民医院中心实验室，山东　临沂　276000；4.临沂市人民医院病理科，山东　临沂　276000）
Author(s):
WANG Liying1; LU Yucheng2; WANG Lijuan3; WANG Meng′en1; WEI Zhiyong4; CHE Fengyuan3
(1.School of Clinical Medicine,Weifang Medical University,Weifang 261000,Shandong Province,China;2.Biological Sample Library,Linyi People′s Hospital,Linyi 276000,Shandong Province,China;3.Central Laboratory,Linyi People′s Hospital,Linyi 276000,Shandong Province,China;4.Department of Pathology,Linyi People′s Hospital,Linyi 276000,Shandong Province,China)
关键词:
胶质瘤细胞; 欧当归内酯A; 凋亡; 自噬
Keywords:
glioma cell; levistilide A; apoptosis; autophagy
分类号:
R739.41
DOI:
10.7683/xxyxyxb.2022.03.001
文献标志码:
A
摘要:
目的　探讨欧当归内酯A（LA）对人胶质瘤细胞U251凋亡及自噬的影响。方法　将对数生长期的U251细胞随机分为空白组、30 μmol·L-1LA组、60 μmol·L-1LA组、90 μmol·L-1LA组，分别用含有0、30、60、90 μmol·L-1的LA培养基培养24、48、72 h，应用细胞计数试剂盒（CCK-8）检测4组细胞的增殖能力，应用流式细胞术检测4组细胞培养24 h时的细胞凋亡率。将对数生长期的U251细胞分为空白组、3-甲基腺嘌呤（3-MA）组、90 μmol·L-1LA组、30 μmol·L-1LA+3-MA组、60 μmol·L-1LA+3-MA组、90 μmol·L-1LA+3-MA组，空白组不加LA和3-MA，其余5组分别加入含0、90、30、60、90 μmol·L-1 LA和1、0、1、1、1 mmol·L-1 3-MA的培养基培养24 h,采用CCK-8检测各组细胞活性。应用Western blot法检测空白组、30 μmol·L-1LA组、60 μmol·L-1LA组、90 μmol·L-1LA组细胞中p53、B淋巴细胞瘤-2蛋白(Bcl-2)、Bcl-2相关X蛋白(Bax)、caspase-3、微管相关蛋白1A/1B-轻链3抗体（LC3）-Ⅰ及LC3-Ⅱ蛋白相对表达量。 结果　培养24、48、72 h时，30 μmol·L-1LA组、60 μmol·L-1LA组和90 μmol·L-1LA组的细胞增殖能力显著低于空白组，60 μmol·L-1LA组和90 μmol·L-1LA组细胞增殖能力显著低于30 μmol·L-1LA组，90 μmol·L-1LA组细胞增殖能力显著低于60 μmol·L-1LA组（P<0.05）。空白对照组培养24、48、72 h时的细胞增殖能力比较差异无统计学意义（P>0.05）。30 μmol·L-1LA组培养72 h时的细胞增殖能力显著高于培养24、48 h时（P<0.05），培养24 h与48 h 时的细胞增殖能力比较差异无统计学意义（P>0.05）；60 μmol·L-1LA组培养48、72 h时的细胞增殖能力显著低于培养24 h时（P<0.05），培养48 h与72 h时的细胞增殖能力比较差异无统计学意义（P>0.05）。90 μmol·L-1LA组培养48、72 h时的细胞增殖能力显著低于培养24 h时，培养72 h时的细胞增殖能力显著低于48 h时（P<0.05）。30 μmol·L-1LA组、60 μmol·L-1LA组、90 μmol·L-1LA组细胞凋亡率显著高于空白组，60 μmol·L-1LA组、90 μmol·L-1LA组细胞凋亡率显著高于30 μmol·L-1LA组（P<0.05）；60 μmol·L-1LA组与90 μmol·L-1LA组细胞凋亡率比较差异无统计学意义（P>0.05）。90 μmol·L-1LA组、90 μmol·L-1LA+3-MA组细胞活性低于空白组、3-MA组、30 μmol·L-1LA+3-MA组和60 μmol·L-1LA+3-MA组， 90 μmol·L-1LA组细胞活性低于90 μmol·L-1LA+3-MA组（P<0.05）；空白组、3-MA组、30 μmol·L-1LA+3-MA组和60 μmol·L-1LA+3-MA组细胞活性比较差异无统计学意义（P>0.05）。30 μmol·L-1 LA组、60 μmol·L-1 LA组和90 μmol·L-1 LA组U251细胞中p53和Bax蛋白相对表达量高于空白组，30 μmol·L-1 LA组U251细胞中caspase-3蛋白相对表达量低于空白组，90 μmol·L-1 LA组U251细胞中Bcl-2蛋白相对表达量低于空白组，90 μmol·L-1 LA组U251细胞中caspase-3蛋白相对表达量高于空白组（P<0.05）；30 μmol·L-1 LA组和60 μmol·L-1 LA组U251细胞中Bcl-2蛋白相对表达量及60 μmol·L-1 LA组U251细胞中caspase-3蛋白相对表达量与空白组比较差异无统计学意义（P>0.05）。90 μmol·L-1 LA组U251细胞中p53、Bcl-2蛋白相对表达量低于30 μmol·L-1LA组和60 μmol·L-1LA组，90 μmol·L-1 LA组U251细胞中Bax、caspase-3蛋白相对表达量高于30 μmol·L-1LA组，90 μmol·L-1 LA组U251细胞中caspase-3蛋白相对表达量高于60 μmol·L-1LA组（P<0.05）。90 μmol·L-1 LA组与60 μmol·L-1LA组U251细胞中Bax蛋白相对表达量比较差异无统计学意义（P>0.05）。 60 μmol·L-1 LA组U251细胞中Bax、caspase-3蛋白相对表达量高于30 μmol·L-1组（P<0.05）。60 μmol·L-1 LA组与 30 μmol·L-1组U251细胞中p53、Bcl-2蛋白相对表达量比较差异无统计学意义（P>0.05）。4组U251细胞中LC3-Ⅰ蛋白相对表达量比较差异无统计学意义（P>0.05）。60 μmol·L-1LA组、90 μmol·L-1LA组U251细胞中LC3-Ⅱ蛋白相对表达量及LC3-Ⅱ/LC3-Ⅰ比值显著高于空白组和30 μmol·L-1LA组（P<0.05）；30 μmol·L-1LA组与空白组U251细胞中LC3-Ⅱ 蛋白相对表达量及LC3-Ⅱ/LC3-Ⅰ 比值比较差异无统计学意义（P>0.05）； 60 μmol·L-1 LA组与90 μmol·L-1LA组U251细胞中LC3-Ⅱ 蛋白相对表达量及LC3-Ⅱ/LC3-Ⅰ 比值比较差异无统计学意义（P>0.05）。结论　LA能够通过增加凋亡蛋白p53、Bax、caspase-3和减少Bcl-2蛋白的表达，以及增加自噬蛋白LC3-Ⅱ蛋白的表达，抑制U251细胞增殖，促进U251细胞凋亡和自噬。
Abstract:
Objective　To investigate the effect of levistilide A（LA）on the apoptosis and autophagy of human glioma cell U251.Methods　The U251 cells in logarithmic growth phase were randomly divided into blank group,30 μmol·L-1 LA group,60 μmol·L-1 LA group and 90 μmol·L-1 LA group.The cells were cultured in medium containing 0,30,60 and 90 μmol· L-1LA for 24,48 and 72 h,respectively.The proliferation ability of U251 cells in the four groups was detected by using cell counting kit-8 (CCK-8)，and the apoptosis rate of U251 cells at 24 h of culture in the four groups was detected by using flow cytometry.The U251 cells in logarithmic growth phase were divided into blank group,3-methyladenine (3-MA) group,90 μmol·L-1LA group,30 μmol·L-1LA+3-MA group,60 μmol·L-1LA+3-MA group and 90 μmol·L-1LA+3-MA group.The blank group did not add LA and 3-MA,the other 5 groups were cultured for 24 h in medium containing 0,90,30,60,90 μmol·L-1 LA and 1,0,1,1,1 mmol· L-13-MA,respectively,and the activity of cells in the six groups was detected by CCK-8.The relative expression levels of p53,B-cell lymphoma-2(Bcl-2),Bcl2-associated X(Bax),caspase-3,microtubule associated protein 1A/1b light chain 3(LC3)-Ⅰ and LC3-Ⅱ protein in the blank group,30 μmol·L-1 LA group,60 μmol·L-1 LA group and 90 μmol·L-1 LA group were detected by Western blot.Results　At 24,48，72 h of culture,the cell proliferation ability in the 30 μmol·L-1 LA group,60 μmol·L-1 LA group and 90 μmol·L-1 LA group was significantly lower than that in the blank group(P<0.05);the cell proliferation ability in the 60 μmol·L-1 LA group and 90 μmol·L-1 LA group was significantly lower than that in the 30 μmol·L-1 LA group(P<0.05);the cell proliferation ability in the 90 μmol·L-1 LA group was significantly lower than that in the 60 μmol·L-1 LA group (P<0.05).There was no significant difference in cell proliferation ability in the blank control group among 24,48 and 72 h of culture(P>0.05);the cell proliferation ability in the 30 μmol· L-1 LA group at 72 h of culture was significantly higher than that at 24 h and 48 of culture (P<0.05);there was no significant difference in cell proliferation ability between 24 h and 48 h of culture (P>0.05).The cell proliferation ability in the 60 μmol· L-1LA group at 48 h and 72 h of culture was significantly lower than that at 24 h of culture (P<0.05),and there was no significant difference in cell proliferation ability between 48 h and 72 h of culture (P>0.05).The cell proliferation ability in the 90 μmol· L-1LA group at 48 h and 72 h of culture was significantly lower than that at 24 h of culture,and which at 72 h of culture was significantly lower than that at 48 h of culture(P<0.05).The apoptosis rate of cells in the 30 μmol·L-1 LA group,60 μmol·L-1 LA group and 90 μmol·L-1 LA group was significantly higher than that in the blank group;the apoptosis rate of cells in the 60 μmol·L-1 LA group and 90 μmol·L-1 LA group was significantly higher than that in the 30 μmol·L-1 LA group (P<0.05).There was no significant difference in apoptosis rate of cells between the 60 μmol·L-1 LA group and 90 μmol·L-1 LA group (P>0.05).The cell activity in the 90 μmol·L-1 LA group,90 μmol·L-1 LA +3-MA group was significantly lower than that in the blank group,3-MA group,30 μmol·L-1 LA +3-MA group and 60 μmol·L-1 LA +3-MA group(P<0.05);the cell activity in the 90 μmol·L-1 LA group was significantly lower than that in the 90 μmol·L-1 LA +3-MA group (P<0.05).There was no significant difference in the cell activity among the blank group,3-MA group,30 μmol·L-1 LA +3-MA group and 60 μmol·L-1 LA +3-MA group (P>0.05).The relative expression level of p53 and Bax protein in U251 cells in the 30 μmol·L-1LA group,60 μmol·L-1LA group and 90 μmol·L-1 LA group was significantly higher than that in blank group,the relative expression level of caspase-3 protein in U251 cells in the 30 μmol·L-1 LA group was significantly lower than that in the blank group,the relative expression of Bcl-2 protein in U251 cells in the 90 μmol·L-1 LA group was significantly lower than that in the blank group,the relative expression of caspase-3 protein in the 90 μmol·L-1 LA group was significantly higher than that in the blank group (P<0.05).The relative expression of Bcl-2 protein in the 30 μmol·L-1 LA group and 60 μmol·L-1 LA group,and the relative expression of caspase-3 protein in the 60 μmol·L-1 LA group had no significant difference compared with those in the blank group (P>0.05).The relative expression levels of p53 and Bcl-2 proteins in U251 cells in the 90 μmol·L-1LA group were significantly lower than those in the 30 μmol·L-1LA group and 60 μmol·L-1LA group(P<0.05).The relative expression levels of Bax and caspase-3 protein in U251 cells in the 90 μmol·L-1LA group were significantly higher than those in the 30 μmol·L-1LA group(P<0.05);the relative expression levels of caspase-3 in U251 cells in the 90 μmol·L-1LA group were significantly higher than those in the 60 μmol·L-1LA group (P< 0.05).There was no significant difference in the relative expression of Bax protein between the 90 μmol·L-1LA group and 60 μmol·L-1LA group (P>0.05).The relative expression levels of Bax and caspase-3 protein in U251 cells in the 60 μmol·L-1 LA group were significantly higher than those in the 30 μmol·L-1 LA group(P<0.05).There was no significant difference in the relative expression levels of p53 and Bcl-2 proteins between the 60 μmol·L-1 LA group and 30 μmol·L-1 LA group (P>0.05).There was no significant difference in the relative expression of LC3-Ⅰ protein in U251 cells among the four groups (P>0.05).The relative expression level of LC3-Ⅱ protein and LC3-Ⅱ/LC3-Ⅰ ratio in U251 cells in the 60 μmol·L-1 LA group and 90 μmol·L-1 LA group were significantly higher than those in the blank group and 30 μmol·L-1 LA group (P<0.05).There was no significant difference in the relative expression level of LC3-Ⅱ protein and LC3-Ⅱ/LC3-Ⅰ ratio of U251 cells between the 30 μmol· L-1LA group and the blank group (P>0.05).There was no significant difference in the relative expression level of LC3-Ⅱ protein and LC3-Ⅱ/LC3-Ⅰ ratio between the 60 μmol·L-1LA group and 90 μmol·L-1LA group (P>0.05).Conclusion　LA can inhibit the proliferation of U251 cells and promote the apoptosis and autophagy of U251 cells by increasing the expression of apoptotic proteins including p53,Bax,caspase-3 and decreasing the expression of Bcl-2 protein,as well as increasing the expression of LC3-Ⅱ protein.

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更新日期/Last Update: 2022-03-05