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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
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          谢金金 ，陈 燕，杜 鑫，李雨珂，赵梦楠，石三军（成都中医药大学药学院，成都 611137）
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          中图分类号  R285；R965      文献标志码  A      文章编号  1001-0408（2024）12-1443-08
          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
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          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
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          例和NANOG、OCT4荧光强度均显著升高（P＜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，
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          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（P＜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（P＜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，P＜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。
          关键词  小檗碱；肿瘤干细胞；焦亡；焦孔素E；安全性

          Study on mechanism of berberine inhibiting tumor stem cells proliferation and its in vivo safety evaluation
          XIE Jinjin，CHEN Yan，DU Xin，LI Yuke，ZHAO Mengnan，SHI Sanjun（College  of  Pharmacy，  Chengdu

          University of Traditional Chinese Medicine， Chengdu 611137， China）

          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
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          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
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          fluorescence  intensity  of  NANOG  and  OCT4  were  significantly  increased （P＜0.000  1）.  The  half-inhibitory  concentration  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
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          significantly （P＜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
             Δ  基金项目 国 家 自 然 科 学 基 金 青 年 科 学 基 金 项 目（No.
          82003689）；四川省科技计划项目（No.2022JDJQ0052）               release rate of LDH of cells was significantly increased and the
             ＊第一作者 硕士研究生。研究方向：中药活性小分子体外药效评                   release  rate  of  LDH  increased  with  increasing  dose；  the
          价。E-mail：xjj18402807749@163.com
                                                             protein  expressions  of  GSDME-N  and  cleaved-caspase-3  of
             # 通信作者 教授，博士生导师，博士。研究方向：中药活性成分发
          掘与肿瘤干细胞。E-mail：shisanjuns@163.com                  cells  in  berberine  20，  40  μmol/L  groups  were  significantly


          中国药房  2024年第35卷第12期                                              China Pharmacy  2024 Vol. 35  No. 12    · 1443 ·
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