Study on mechanism of berberine inhibiting tumor stem cells proliferation and its <italic style="font-style: italic">in vivo </italic>safety evaluation

XIE Jinjin; CHEN Yan; DU Xin; LI Yuke; ZHAO Mengnan; SHI Sanjun

doi:10.6039/j.issn.1001-0408.2024.12.06

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Study on mechanism of berberine inhibiting tumor stem cells proliferation and its in vivo safety evaluation

更新时间：2024-06-25

- Study on mechanism of berberine inhibiting tumor stem cells proliferation and its in vivo safety evaluation
- ZHONGGUO YAOFANG Vol. 35, Issue 12, Pages: 1443-1450(2024)
- 作者机构：
  
  成都中医药大学药学院，成都　611137
- 作者简介：
- 基金信息：
- DOI：10.6039/j.issn.1001-0408.2024.12.06
  CLC： R285;R965
- Published：30 June 2024，
  
  Received：04 January 2024，
  
  Revised：19 March 2024，
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谢金金,陈燕,杜鑫等.小檗碱抑制肿瘤干细胞增殖的机制研究及体内安全性评价 ^Δ[J].中国药房,2024,35(12):1443-1450.

XIE Jinjin,CHEN Yan,DU Xin,et al.Study on mechanism of berberine inhibiting tumor stem cells proliferation and its in vivo safety evaluation[J].ZHONGGUO YAOFANG,2024,35(12):1443-1450.
谢金金,陈燕,杜鑫等.小檗碱抑制肿瘤干细胞增殖的机制研究及体内安全性评价 ^Δ[J].中国药房,2024,35(12):1443-1450. DOI： 10.6039/j.issn.1001-0408.2024.12.06.

XIE Jinjin,CHEN Yan,DU Xin,et al.Study on mechanism of berberine inhibiting tumor stem cells proliferation and its in vivo safety evaluation[J].ZHONGGUO YAOFANG,2024,35(12):1443-1450. DOI： 10.6039/j.issn.1001-0408.2024.12.06.

摘要

目的

研究小檗碱对肿瘤干细胞增殖的体外抑制作用机制，并评价其体内安全性。

方法

采用流式细胞术从小鼠皮肤黑色素瘤B16F10细胞中分选肿瘤干细胞；以CD44、CD133、Nanog同源盒蛋白（NANOG）、八聚体结合转录因子4（OCT4）为指标对肿瘤干细胞进行表征。将肿瘤干细胞分为对照组、全反式维甲酸（ATRA）组和小檗碱组，采用CCK-8法检测小檗碱对肿瘤干细胞活力的影响；采用流式细胞仪检测细胞凋亡率、CD44

/CD133

细胞比例和性别决定区Y框蛋白2（SOX2）阳性细胞率；记录小檗碱处理后肿瘤球的形态变化；记录各组细胞焦亡形态图并检测乳酸脱氢酶（LDH）释放率，采用Western blot法检测焦亡相关蛋白消皮素E（GSDME）、消皮素E氮端（GSDME-N）、胱天蛋白酶3（caspase-3）和裂解胱天蛋白酶3（cleaved-caspase-3）的蛋白表达水平。采用斑马鱼胚胎毒性实验对小檗碱的体内安全性进行初步评价。

结果

与B16F10细胞比较，肿瘤干细胞中CD44

/CD133

细胞比例和NANOG、OCT4荧光强度均显著升高（

＜0.000 1）。小檗碱对肿瘤干细胞的半数抑制浓度为50.98 μmol/L；与对照组比较，小檗碱组细胞凋亡率显著升高，CD44

/CD133

细胞比例和SOX2阳性细胞率均显著降低（

＜0.000 1），肿瘤干细胞球皱缩，部分细胞死亡。小檗碱处理后的肿瘤干细胞，其最外

层细胞膜肿胀，LDH释放率显著增加，且随剂量增大而升高（

＜0.05）；小檗碱20、40 μmol/L组的GSDME-N、cleaved-caspase-3蛋白表达水平显著上升，GSDME、caspase-3蛋白表达水平显著下降（小檗碱20 μmol/L组除外，

＜0.05）。经小檗碱处理的斑马鱼胚胎发育几乎未受影响，胚胎存活率达到100%，未观察到明显畸形。

结论

小檗碱具有较好的抗肿瘤干细胞增殖活性，其作用机制可能与激活GSDME、促进细胞焦亡有关；且小檗碱体内安全性良好。

Abstract

OBJECTIVE

To investigate the

in vitro

inhibitory mechanism of berberine on the proliferation of tumor stem cells and evaluate its

in vivo

safety.

METHODS

Flow cytometry was used to select tumor stem cells from mouse skin melanoma B16F10 cells； CD44， CD133， Nanog homologous box protein （NANOG） and octamer-binding transcription factor 4 （OCT4） were used as indicators to characterize tumor stem cells. Tumor stem cells were divided into control group， all-trans retinoic acid （ATRA） group， and berberine group， and the CCK-8 method was used to detect the effects of berberine on the viability of tumor stem cells； flow cytometry was adopted to detect cell apoptotic rate， the proportion of CD44

/CD133

and the positive cell rate of sex determining region Y box protein 2 （SOX2）； the morphological changes of tumor balls were recorded after treatment with berberine； the morphology of cell pyroptosis in each group was recorded， and the release rate of lactate dehydrogenase （LDH） was detected； Western blot assay was adopted to detect the expressions of pyroptosis-related protein gasdermin E （GSDME）， GSDME-N， caspase-3 and cleaved caspase-3. Preliminary evaluation of

in vivo

safety of berberine was conducted by using zebrafish embryo toxicity experiments.

RESULTS

Compared with B16F10 cells， the proportion of CD44

/CD133

cells in tumor stem cells and the fluorescence intensity of NANOG and OCT4 were significantly increased （

＜0.000 1）. The half-inhibitory con

centration of berberine to tumor stem cells was 50.98 μmol/L. Compared with the control group， the apoptotic rate of cells in the berberine group was significantly increased， while the proportion of CD44

/CD133

cells and the rate of SOX2 positive cells were reduced significantly （

＜0.000 1）； tumor stem cell spheroids were atrophied， with partial cell death. After treatment with berberine， tumor stem cells exhibited swelling in their outermost layer， the release rate of LDH of cells was significantly increased and the release rate of LDH increased with increasing dose； the protein expressions of GSDME-N and cleaved-caspase-3 of cells in berberine 20， 40 μmol/L groups were significantly increased， and the protein expressions of GSDME and caspase-3 were significantly reduced （except for berberine 20 μmol/L group，

＜0.05）. The embryonic development of zebrafish treated with berberine was almost unaffected， and the survival rate of embryo reached 100%， with no obvious abnormalities observed.

CONCLUSIONS

Berberine has good activity against the proliferation of tumor stem cells， and its mechanism of action may be related to activating GSDME and promoting cell pyroptosis； berberine has good

in vivo

safety.

关键词

小檗碱肿瘤干细胞焦亡焦孔素E安全性

Keywords

tumor stem cellspyroptosisgasdermin Esafety

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Study on mechanism of berberine inhibiting tumor stem cells proliferation and its in vivo safety evaluation

DOI：10.6039/j.issn.1001-0408.2024.12.06

摘要

Abstract

关键词

Keywords

references