[1]侯敏杰,林淑璇,吕洋.苦参碱对食管癌Eca-109细胞自噬的影响及作用机制[J].新乡医学院学报,2023,40(10):901-908.[doi:10.7683/xxyxyxb.2023.10.001]
　HOU Minjie,LIN Shuxuan,LYU Yang.Effect and mechanism of matrine on autophagy of esophageal cancer Eca-109 cells[J].Journal of Xinxiang Medical University,2023,40(10):901-908.[doi:10.7683/xxyxyxb.2023.10.001]
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苦参碱对食管癌Eca-109细胞自噬的影响及作用机制
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
40卷
期数:
2023年10
页码:
901-908
栏目:
基础研究
出版日期:
2023-10-05

文章信息/Info

Title:
Effect and mechanism of matrine on autophagy of esophageal cancer Eca-109 cells
作者:
侯敏杰; 林淑璇; 吕洋
（河北北方学院病理学教研室，河北　张家口　075000）
Author(s):
HOU Minjie; LIN Shuxuan; LYU Yang
(Department of Pathology,Hebei North University,Zhangjiakou 075000,Hebei Province,China)
关键词:
苦参碱; 食管癌; 自噬; 肝激酶 B1/腺苷酸活化蛋白激酶/哺乳动物雷帕霉素靶蛋白通路; 多聚蛋白62
Keywords:
matrine; esophageal cancer; autophagy; liver kinase B1/adenylate-activated protein kinase/mammalian target of rapamycin pathway; polyprotein 62
分类号:
R285
DOI:
10.7683/xxyxyxb.2023.10.001
文献标志码:
A
摘要:
目的　探讨苦参碱对食管癌 Eca-109 细胞自噬的影响及其可能的作用机制。
方法　将传代培养的Eca-109细胞分为对照组、低浓度苦参碱组、中浓度苦参碱组和高浓度苦参碱组。对照组细胞加入含体积分数10%胎牛血清的RPMI 1640培养基，低、中、高浓度苦参碱组细胞分别加入终质量浓度为1.0、1.5、2.0 g·L-1的苦参碱溶液；采用四甲基偶氮唑盐法检测各组细胞的活性，免疫荧光细胞化学染色法检测各组细胞中自噬相关蛋白微管相关蛋白轻链3（LC3）的定位及表达，Western blot法检测各组细胞中多聚蛋白62（P62）、LC3 蛋白的相对表达量，实时荧光定量聚合酶链式反应法检测各组细胞中 Beclin1 mRNA的相对表达量，透射电子显微镜观察对照组和高浓度苦参碱组Eca-109细胞的超微结构。另取传代培养的Eca-109细胞，将细胞分为对照组、自噬抑制剂组、苦参碱组和自噬抑制剂+苦参碱组。对照组细胞加入5 mL含体积分数10%胎牛血清的RPMI 1640培养基，自噬抑制剂组细胞加入终浓度为7 μmol·L-1的3-甲基腺嘌呤（3-MA）溶液，苦参碱组细胞加入终质量浓度为2.0 g·L-1的苦参碱溶液，自噬抑制剂+苦参碱组细胞先加入终浓度为 7 μmol · L-1 3-MA溶液预孵育 3 h，再加入终质量浓度为2.0 g·L-1苦参碱溶液；培养 24 h后，采用Western blot 法检测各组细胞中Beclin1、LC3和肝激酶B1(LKB1)/腺苷酸活化蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路相关蛋白的表达。
结果　低、中、高浓度苦参碱组细胞增殖抑制率显著高于对照组（P<0.05）；中、高浓度苦参碱组细胞增殖抑制率显著高于低浓度苦参碱组（P<0.05）；高浓度苦参碱组细胞增殖抑制率显著高于中浓度苦参碱组（P<0.05）。对照组、低浓度苦参碱组、中浓度苦参碱组和高浓度苦参碱组Eca-109细胞质中均可见到红色荧光标记的LC3蛋白阳性表达。对照组细胞中LC3蛋白呈弥散状态；各浓度苦参碱组细胞中LC3蛋白呈斑点状态，且苦参碱浓度越高，细胞质中呈斑点状LC3蛋白越多。低、中、高浓度苦参碱组Eca-109细胞中P62蛋白的相对表达量显著低于对照组（P<0.05）；中、高浓度苦参碱组Eca-109细胞中P62蛋白的相对表达量显著低于低浓度苦参碱组（P<0.05）；中浓度苦参碱组与高浓度苦参碱组Eca-109细胞中P62蛋白的相对表达量比较差异无统计学意义（P>0.05）。低浓度苦参碱组与对照组Eca-109细胞中LC3-Ⅱ/LC3-Ⅰ比较差异无统计学意义（P>0.05）；中、高浓度苦参碱组Eca-109细胞中LC3-Ⅱ/LC3-Ⅰ显著高于对照组和低浓度苦参碱组（P<0.05）；高浓度苦参碱组Eca-109细胞中LC3-Ⅱ/LC3-Ⅰ显著高于中浓度苦参碱组（P<0.05）。低、中、高浓度苦参碱组Eca-109细胞中Beclin1 mRNA的相对表达量显著高于对照组（P<0.05）。中、高浓度苦参碱组Eca-109细胞中Beclin1 mRNA的相对表达量显著高于低浓度苦参碱组（P<0.05）；高浓度苦参碱组Eca-109细胞中Beclin1 mRNA的相对表达量显著高于中浓度苦参碱组（P<0.05）。对照组细胞中可见正常的细胞质、细胞器和细胞核，高浓度苦参碱组细胞质中可见大量大小不等的自噬泡，并可见包裹有细胞内容物的自噬体。自噬抑制剂组与对照组Eca-109细胞中Beclin1蛋白的相对表达量和LC3-Ⅱ/LC3-Ⅰ比较差异无统计学意义（P>0.05）；苦参碱组和自噬抑制剂+苦参碱组Eca-109细胞中Beclin1蛋白的相对表达量和LC3-Ⅱ/LC3-Ⅰ均显著高于对照组和自噬抑制剂组（P<0.05）；自噬抑制剂+苦参碱组Eca-109细胞中Beclin1蛋白的相对表达量和LC3-Ⅱ/LC3-Ⅰ均显著低于苦参碱组（P<0.05）。自噬抑制剂组Eca-109细胞中磷酸化LKB1（p-LKB1）/LKB1、磷酸化AMPK（p-AMPK）/AMPK显著低于对照组，磷酸化mTOR（p-mTOR）/mTOR显著高于对照组（P<0.05）；苦参碱组Eca-109细胞中p-LKB1/LKB1、p-AMPK/AMPK显著高于对照组，p-mTOR/mTOR显著低于对照组（P<0.05）；自噬抑制剂+苦参碱组Eca-109细胞中p-LKB1/LKB1、p-AMPK/AMPK显著高于对照组（P<0.05）；自噬抑制剂+苦参碱组与对照组Eca-109细胞中p-mTOR/mTOR比较差异无统计学意义（P>0.05）。苦参碱组和自噬抑制剂+苦参碱组Eca-109细胞中p-LKB1/LKB1、p-AMPK/AMPK显著高于自噬抑制剂组，p-mTOR/mTOR显著低于自噬抑制剂组（P<0.05）；自噬抑制剂+苦参碱组Eca-109细胞中p-LKB1/LKB1、p-AMPK/AMPK显著低于苦参碱组，p-mTOR/mTOR显著高于苦参碱组（P<0.05）。
结论　苦参碱可能通过调节LKB1/AMPK/mTOR信号通路相关蛋白的表达诱导食管癌 Eca-109 细胞发生自噬。
Abstract:
Objective　To investigate the effect of matrine on autophagy of esophageal cancer Eca-109 cells and its possible mechanism.
Methods　The subcultured Eca-109 cells were taken and divided into the control group,the low concentration matrine group,the medium concentration matrine group and the high concentration matrine group.The cells in the control group were added with the RPMI 1640 medium which containing volume fraction 10% fetal bovine serum,while the cells in the low,medium and high concentration matrine groups were added with matrine solutions with final concentrations of 1.0,1.5,2.0 g·L-1, respectively.The activity of cells in above each group was detected by 3-(4,5)-dimethylthahiazo (-z-y1)-3,5-di-phenytetrazolumromide method;the localization and expression of the autophagy related protein of microtubule-associated protein light chain 3(LC3) in each group was detected by immunofluorescence cytochemical staining;the expressions of polyprotein 62（P62）and LC3 protein were detected by Western blot;the expression of Beclin1 mRNA was detected by real-time quantitative polymerase chain reaction;the ultrastructure of Eca-109 cells in the control group and the high concentration matrine group was observed by transmission electron microscope.Another subcultured Eca-109 cells were taken and divided into control group,autophagy inhibitor group,matrine group and autophagy inhibitor + matrine group.The cells in the control group were added with 5 mL RPMI 1640 medium which containing volume fraction 10% fetal bovine serum;the cells in the autophagy inhibitor group were added with the final concentration of 7 μmol·L-1 3-methyladenine (3-MA) solution;the cells in the matrine group were added with the final concentration of 2.0 g·L-1 matrine solution;the cells in the autophagy inhibitor+matrine group were added with the final concentration of 7 μmol·L-1 3-MA solution for 3 hours,then they were added with the final concentration of 2.0 g·L-1 matrine solution;after 24 hours of cultivation,the expressions of Beclin1,LC3 and liver kinase B1(LKB1)/adenosine monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR) signaling pathway related proteins in cells in each group were detected by Western blot.
Results　The proliferation inhibition rate of cells in the low,medium and high concentration matrine groups was significantly higher than that in the control group(P<0.05);the proliferation inhibition rate of cells in the medium concentration matrine group,high concentration matrine group was significantly higher than that in the low concentration matrine group (P<0.05);the proliferation inhibition rate of cells in the high concentration matrine group was significantly higher than that in the medium concentration matrine group(P<0.05).The control group and the low,medium and high concentration matrine groups showed positive expression of LC3 protein labeled with red fluorescence in the cytoplasm of Eca-109 cells.The LC3 protein in cells in the control group showed a diffuse state;the LC3 protein in cells in different concentrations of matrine groups showed a spotted state,and the higher the concentration of matrine,the more the spotted LC3 protein in the cytoplasm.The relative expression of P62 protein in Eca-109 cells in low,medium and high concentration matrine groups was significantly lower than that in the control group(P<0.05);the relative expression of P62 protein in Eca-109 cells in the medium and high concentration matrine groups was significantly lower than that in the low concentration matrine group (P<0.05).The LC3-Ⅱ/LC3-Ⅰ in Eca-109 cells in the medium and high concentration matrine groups was significantly higher than that in the control group and low concentration matrine group (P<0.05);the LC3-Ⅱ/LC3-Ⅰ in Eca-109 cells in the high concentration matrine group was significantly higher than that in the medium concentration matrine group(P<0.05).The relative expression of Beclin1 mRNA in Eca-109 cells in the low,medium and high concentration matrine groups was significantly higher than that in the control group (P<0.05).The relative expression of Beclin1 mRNA in Eca-109 cells in the medium and high concentration matrine groups was significantly higher than that in the low concentration matrine group (P<0.05);the relative expression of Beclin1 mRNA in Eca-109 cells in the high concentration matrine group was significantly higher than that in the medium concentration matrine group(P<0.05).Normal cytoplasm,organelle and nuclei could be seen in the control group;a large number of autophagic vesicles with different sizes and the autophagosome wrapped with cell contents could be seen in the high concentration matrine group.There was no significant difference in the relative expression of Beclin1 protein and LC3-Ⅱ/LC3-Ⅰ in Eca-109 cells between the autophagy inhibitor group and the control group(P>0.05);the relative expressions of Beclin1 protein and LC3-Ⅱ/LC3-Ⅰ in Eca-109 cells in the matrine group and the autophagy inhibitor+matrine group were significantly higher than those in the control group and autophagy inhibitor group(P<0.05);the relative expressions of Beclin1 protein and LC3-Ⅱ/LC3-Ⅰ in Eca-109 cells in the autophagy inhibitor+matrine group were significantly lower than those in the matrine group(P<0.05).The levels of phosphorylated LKB1(p-LKB1)/LKB1 and phosphorylated AMPK(p-AMPK)/AMPK in Eca-109 cells in the autophagy inhibitor group were significantly lower than those in the control group,and the phosphorylated mTOR(p-mTOR)/mTOR was significantly higher than that in the control group(P<0.05);the levels of p-LKB1/LKB1 and p-AMPK/AMPK in Eca-109 cells in the matrine group were significantly higher than those in the control group,and the p-mTOR/mTOR was significantly lower than that in the control group (P<0.05).The levels of p-LKB1/LKB1 and p-AMPK/AMPK in Eca-109 cells in the matrine group and the autophagy inhibitor+matrine group were significantly higher than those in the autophagy inhibitor group,and the p-mTOR/mTOR was significantly lower than that in the autophagy inhibitor group(P<0.05);the levels of p-LKB1/LKB1 and p-AMPK/AMPK in Eca-109 cells in the autophagy inhibitor+matrine group were significantly lower than those in the matrine group,and the p-mTOR/mTOR was significantly higher than that in the matrine group (P<0.05).
Conclusion　Matrine may induce autophagy in esophageal cancer Eca-109 cells by regulating the expression of LKB1/AMPK/mTOR signaling pathway proteins.

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更新日期/Last Update: 2023-10-05