<italic style="font-style: italic">Celastrus orbiculatus </italic>Extract Promotes Apoptosis of Gastric Cancer Cells by Destroying Mitochondrial Structure

Haibo WANG; Yuanyuan LUO; Xinyi FENG; Tengyang NI; Zewen CHU; Miao ZHU; Yanqing LIU

doi:10.13422/j.cnki.syfjx.20221823

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Celastrus orbiculatus Extract Promotes Apoptosis of Gastric Cancer Cells by Destroying Mitochondrial Structure

更新时间：2022-08-11

- Celastrus orbiculatus Extract Promotes Apoptosis of Gastric Cancer Cells by Destroying Mitochondrial Structure
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 28, Issue 18, Pages: 46-53(2022)
- 作者机构：
  
  扬州大学医学院，国家中医药管理局胃癌毒邪论治重点研究室，扬州大学-扬州市肿瘤研究所，江苏扬州 225001
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20221823
  CLC： R22;R242;R2-031;R285.5
- Published：20 September 2022，
  
  Published Online：30 June 2022，
  
  Received：04 April 2022，
- 稿件说明：
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王海波,罗园园,冯心怡等.南蛇藤提取物通过破坏线粒体结构促进胃癌AGS细胞凋亡的机制[J].中国实验方剂学杂志,2022,28(18):46-53.

WANG Haibo,LUO Yuanyuan,FENG Xinyi,et al.Celastrus orbiculatus Extract Promotes Apoptosis of Gastric Cancer Cells by Destroying Mitochondrial Structure[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(18):46-53.
王海波,罗园园,冯心怡等.南蛇藤提取物通过破坏线粒体结构促进胃癌AGS细胞凋亡的机制[J].中国实验方剂学杂志,2022,28(18):46-53. DOI： 10.13422/j.cnki.syfjx.20221823.

WANG Haibo,LUO Yuanyuan,FENG Xinyi,et al.Celastrus orbiculatus Extract Promotes Apoptosis of Gastric Cancer Cells by Destroying Mitochondrial Structure[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(18):46-53. DOI： 10.13422/j.cnki.syfjx.20221823.

摘要

目的

观察南蛇藤提取物（COE）对胃癌细胞的抑制作用，探讨COE通过影响线粒体结构和功能促进胃癌凋亡的一种全新的作用机制，为南蛇藤的进一步开发和临床应用提供实验依据。

方法

5-溴脱氧尿嘧啶核苷（Brdu）染色联合流式细胞实验用来检测不同质量浓度COE （20、40、80 mg·L

-1

）对胃癌细胞增殖的影响。通过膜联蛋白V-异硫氰酸荧光素（AnnexinV-FITC）染色实验联合流式细胞术检测COE对胃癌细胞凋亡的影响，用JC-1线粒体膜电位检测试剂盒检测线粒体膜电位的变化，通过蛋白免疫印迹法（Western blot）检测COE对胃癌细胞凋亡相关蛋白B细胞淋巴瘤-2（Bcl-2）、B细胞淋巴瘤-xL（Bcl-xL）、Bcl-2相关X蛋白（Bax）和胱天蛋白酶-3（Caspase-3）表达的影响，用透射电镜检测COE处理后的胃癌细胞线粒体微观结构的变化，包括线粒体膜完整性和内部嵴的变化。COE处理后，Western blot检测胃癌细胞线粒体标志蛋白超氧化物歧化酶1（SOD1）、电压依赖性阴离子通道蛋白（VDAC）、抗增殖蛋白1（PHB1）和热休克蛋白60（HSP60）水平的变化。

结果

Brdu染色显示，与空白组比较，COE组（40、80 mg·L

-1

）胃癌细胞的增殖比例明显下降（

0.05）。AnnexinV-FITC染色显示，与空白组比较，COE组（40、80 mg·L

-1

）胃癌细胞的凋亡数量明显增加（

0.05）。JC-1线粒体膜电位检测显示，COE处理后组胃癌细胞的线粒体膜电位水平明显降低。Western blot结果显示，与空白组比较，COE组（20、40、80 mg·L

-1

）促进胃癌凋亡的相关蛋白Bax和Caspase-3表达明显增加（

0.05，

0.01），COE组（40、80 mg·L

-1

）胃癌细胞凋亡抵抗相关蛋白Bcl-2和Bcl-xL表达显著降低（

0.01）。透射电镜结果显示，与空白组比较，COE组胃癌细胞的微观结构发生了明显变化，细胞膜内出现了很多空泡，线粒体结构出现破坏，线粒体内出现空泡。Western blot结果显示，与空白组比较，COE组（20、40、80 mg·L

-1

）胃癌细胞中与应激反应有关的蛋白SOD1表达增高（

0.05，

0.01），COE组（80 mg·L

-1

）与线粒体稳定和通透性相关的蛋白VDAC、PHB1和HSP60表达均显著下降（

0.01）。

结论

COE能够显著抑制胃癌细胞的增殖，促进胃癌细胞的凋亡，其机制可能与COE破坏胃癌细胞线粒体结构和功能从而激活细胞线粒体凋亡途径有关。

Abstract

Objective

To study the inhibitory effect of

Celastrus orbiculatus

extract （COE） on gastric cancer cells， to clarify the specific mechanism of COE promoting the apoptosis of gastric cancer cells by affecting the mitochondrial structure and function， and to provide an experimental basis for the further development and clinical application of

C. orbiculatus

Method

Brdu staining combined with flow cytometry and Annexin V-fluorescein isothiocyanate （AnnexinV-FITC） staining combined with flow cytometry were employed to detect the effects of COE （20， 40， 80 mg·L

-1

） on the proliferation and apoptosis of gastric cancer cells， respectively. The changes in mitochondrial membrane potential were detected with JC-1 mitochondrial membrane potential assay kit. The expression of apoptosis-associated proteins including B-cell lymphoma-2 （Bcl-2）， B-cell lymphoma-xL （Bcl-xL）， Bcl-2-associated X （Bax）， and cysteine aspartutespecific protease-3 （Caspase-3） in gastric cancer cells was determined by Western blot. Transmission electron microscopy was employed to detect changes in the mitochondrial microstructure of gastric cancer cells exposed to COE. Western blot was employed to measure the expression of mitochondrial marker proteins ［superoxide dismutase 1 （SOD1）， voltage-dependent anion channel （VDAC）， prohibitin 1 （PHB1）， and heat shock protein 60 （HSP60）］ in gastric cancer cells.

Result

Compared with the control group， COE （40， 80 mg·L

-1

） inhibited the proliferation and promoted the apoptosis of gastric cancer cells （

0.05）. Furthermore， COE reduced the mitochondrial membrane potential of gastric cancer cells. Compared with the control group， COE （20， 40， 80 mg·L

-1

） up-regulated the expression of Bax and Caspase-3 which promoted apoptosis of gastric cells （

0.05，

0.01）， and COE at 40 and 80 mg·L

-1

down-regulated the expression of Bcl-2 and Bcl-xL which inhibited the apoptosis of gastric cancer cells （

0.01）. The results of transmission electron microscopy showed that COE changed the microstructure of gastric cancer cells， which led to the appearance of vacuoles in the cell membrane and mitochondria and damaged the mitochondrial structure. Compared with the control group， COE （20， 40， 80 mg·L

-1

） changed the expression of mitochondrial marker proteins. Specifically， it up-regulated the expression of SOD1 involved in stress response （

0.05，

0.01） and down-regulated that of VDAC， PHB1， and HSP60 associated with mitochondrial stability and permeability （

0.01）.

Conclusion

COE can significantly inhibit the proliferation and promote the apoptosis of gastric cancer cells. It may activate the mitochondrial apoptosis pathway by destroying the mitochondrial structure and function of gastric cancer cells.

关键词

南蛇藤提取物线粒体胃癌凋亡

Keywords

Celastrus orbiculatus extractmitochondriagastric cancerapoptosis

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Celastrus orbiculatus Extract Promotes Apoptosis of Gastric Cancer Cells by Destroying Mitochondrial Structure

DOI：10.13422/j.cnki.syfjx.20221823

摘要

Abstract

关键词

Keywords

references