Research of dihydroartemisinin on anti‑colorectal cancer by regulating MAPK/PI3K/Akt signaling pathway

XU Jiaruo; JIA Fengjing; CHEN Jialiang; YAO Guangtao

doi:10.16306/j.1008-861x.2024.02.012

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Research of dihydroartemisinin on anti‑colorectal cancer by regulating MAPK/PI3K/Akt signaling pathway

Experiment Research | 更新时间：2024-04-15

- Research of dihydroartemisinin on anti‑colorectal cancer by regulating MAPK/PI3K/Akt signaling pathway
- Academic Journal of Shanghai University of Traditional Chinese Medicine Vol. 38, Issue 2, Pages: 83-92(2024)
- 作者机构：
  
  1.上海中医药大学（上海　201203）
  2.四川省药品检验研究院，四川省医疗器械检测中心（四川　成都　611731）
  3.国家药品监督管理局药物制剂体内外相关性技术研究重点实验室（四川　成都　611731）
- 作者简介：
- 基金信息：
- DOI：10.16306/j.1008-861x.2024.02.012
  CLC：
- Published：25 March 2024，
  
  Received：02 July 2023，
  
  Revised：19 September 2023，
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徐嘉若,贾丰菁,陈佳靓等.双氢青蒿素通过调控MAPK/PI3K/Akt信号通路抗结直肠癌作用研究[J].上海中医药大学学报,2024,38(02):83-92.

XU Jiaruo,JIA Fengjing,CHEN Jialiang,et al.Research of dihydroartemisinin on anti‑colorectal cancer by regulating MAPK/PI3K/Akt signaling pathway[J].Academic Journal of Shanghai University of Traditional Chinese Medicine,2024,38(02):83-92.
徐嘉若,贾丰菁,陈佳靓等.双氢青蒿素通过调控MAPK/PI3K/Akt信号通路抗结直肠癌作用研究[J].上海中医药大学学报,2024,38(02):83-92. DOI： 10.16306/j.1008-861x.2024.02.012.

XU Jiaruo,JIA Fengjing,CHEN Jialiang,et al.Research of dihydroartemisinin on anti‑colorectal cancer by regulating MAPK/PI3K/Akt signaling pathway[J].Academic Journal of Shanghai University of Traditional Chinese Medicine,2024,38(02):83-92. DOI： 10.16306/j.1008-861x.2024.02.012.

摘要

目的

探讨双氢青蒿素（DHA）在体内对人结直肠癌（RKO）细胞的抗癌活性及体外机制。

方法

将40只SPF级雄性裸鼠建立RKO荷瘤小鼠模型，随机分为模型组、顺铂组及DHA高剂量（200 mg/kg）、低剂量（100 mg/kg）组，每组10只；对比各组裸鼠体质量、肿瘤体积、肿瘤质量、抑瘤率、血清肿瘤坏死因子（TNF-α）水平等；苏木素-伊红（HE）染色观察各组裸鼠肿瘤组织病理学变化；噻唑蓝（MTT）比色法检测不同浓度（25、50、100 μmol/L）DHA干预0、24、48、72 h内对RKO细胞增殖的影响；流式细胞仪、Hoechst 33258检测（0、50、95、150 μmol/L）DHA干预48 h后对RKO细胞周期和细胞凋亡的影响；划痕实验观察95 μmol/L DHA干预对细胞迁移能力的影响；实时荧光定量PCR（RT-qPCR）检测DHA对细胞内半胱氨酸天冬氨酸蛋白酶-3（

Caspase

）、

Caspase

、B细胞淋巴瘤-2（

Bcl

）、Bcl-2相关X蛋白（

Bax

） mRNA表达水平的影响；蛋白质印迹法（Western blot）检测DHA对细胞内Caspase-3、Caspase-9、Bcl-2、Bax以及p38-丝裂原活化蛋白激酶（p38-MAPK）、p-p38-MAPK、磷脂酰肌醇3-激酶（PI3K）、p-PI3K、蛋白激酶B（Akt）、p-Akt、基质金属蛋白酶（MMP-9）等表达水平的影响。

结果

200 mg/kg DHA显著降低肿瘤体积和质量，降低血清TNF-α水平，抑瘤率为41.45%；DHA可随浓度的增加而增强对RKO细胞增殖的抑制作用；（50、95、150 μmol/L）DHA阻滞RKO细胞周期在G

/M期，且促进RKO细胞凋亡作用随DHA浓度增加而增强； 95 μmol/L DHA干预12、24 h后可显著降低RKO细胞迁移能力（

0.01）；95 μmol/L DHA可显著上调RKO细胞内

Caspase

、

Caspase

mRNA表达水平（

0.01）以及

Bax

Bcl

mRNA表达比值（

0.05）；95 μmol/L DHA可增加RKO细胞内剪切型Caspase-9（cleaved-Caspase-9）/Caspase-9、Bax/Bcl-2蛋白表达（

0.01），同时降低MMP-9、p-P38 MAPK、p-PI3K、p-Akt及Akt蛋白表达水平（

0.01）。

结论

DHA有较好的抑制结直肠癌作用，其机制可能为抑制MAPK/PI3K/Akt信号通路，触发RKO细胞内源性凋亡，阻止其增殖与迁移。

Abstract

Objective： To investigate the anticancer activity of dihydroartemisinin（DHA）on RKO cells

in vivo

and its mechanism

in vitro

Methods

40 RKO tumor-bearing mouse models were established in specific pathogen-free male nude mice and randomly divided into model group，cisplatin group，high dose DHA（200 mg/kg）group，and low dose DHA（100 mg/kg）group，with 10 mice in each group. The body weight，tumor volume，tumor mass，tumor inhibition rate and serum，tumor necrosis factor-α（TNF-α）level of each group were compared. Hematoxylin-eosin （H

E） staining was used to observe tumor histopathological changes in nude mice of each group. MTT assay was used to detect the effects of DHA at different concentrations（25，50，100 μmol/L）on the proliferation of RKO cells within 0，24，48，and 72 h. Flow cytometry and Hoechst 33258 were used to detect the effects of DHA （0，50，95，150 μmol/L）on the cell cycle and apoptosis of RKO cells after 48 h intervention. Wound healing assay was used to observe the effect of 95 μmol/L DHA on cell migration. Real Time Quantitative PCR （RT-qPCR） was used to detect the effect of DHA on the mRNA expression levels of

Caspase

，

Caspase

，

Bcl

and

Bax

. Western blot was used to detect the effect of DHA on the expression levels of Caspase-3， Caspase-9， Bcl-2， Bax， p38-MAPK， p-p38-MAPK， PI3K， p-PI3K， Akt， p-Akt and MMP-9.

Results

Tumor volume and mass were significantly reduced and the serum level of TNF-α was lowered by 200 mg/kg DHA， and the tumor inhibition rate was 41.45%. The inhibitory effect on RKO cell proliferation was enhanced with increasing concentration of DHA. RKO cell cycle was arrested in the G2/M phase by （50，95，150 μmol/L）DHA，and the effect of promoting RKO cell apoptosis was enhanced with the increase of DHA concentration. The migration ability of RKO cells was significantly reduced after intervention with 95 μmol/L DHA for 12 and 24 h （

0.01）. The mRNA expression levels of

Caspase

and

Caspase

in RKO cells were significantly up-regulated by 95 μmol/L DHA（

0.01）， and

Bax

Bcl

mRNA expression ratio was also significantly up-regulated（

0.05）. The protein expressions of cleaved-Caspase-9/Caspase-9 and Bax/Bcl-2 in RKO cells were increased by 95 μmol/L DHA（

0.01）， while the protein expression levels of MMP-9， p-P38 MAPK， p-PI3K， p-Akt and Akt were decreased（P

0.01）.

Conclusion

DHA has a good inhibitory effect on colorectal cancer，and its mechanism may be to trigger the endogenous apoptosis of RKO cells to prevent their proliferation and migration through inhibiting the MAPK/PI3K/Akt signaling pathway.

关键词

双氢青蒿素结直肠癌RKO细胞细胞凋亡MAPK/PI3K/Akt信号通路

Keywords

dihydroartemisinincolorectal cancerRKO cellscell apoptosisMAPK/PI3K/Akt signaling pathway

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