中华皮肤科杂志 ›› 2020, Vol. 53 ›› Issue (9): 685-691.doi: 10.35541/cjd.20191201

• 论著 • 上一篇    下一篇

小檗碱对皮肤鳞状细胞癌裸鼠模型的抑制作用及其机制研究

邢天娇1    李东霞1    张娟2    贾敏鑫1   

  1. 1内蒙古医科大学附属医院皮肤科，呼和浩特  010050；2昆明医科大学第一附属医院皮肤科  650045
  • 收稿日期:2019-12-30 修回日期:2020-06-18 发布日期:2020-08-31
  • 通讯作者: 李东霞 E-mail:ldx828@sina.com
  • 基金资助:
    2016年“草原英才”高层次培养人才项目（CYYC6027）

Inhibitory effect of berberine on a nude mouse model of cutaneous squamous cell carcinoma and its mechanisms

Xing Tianjiao1, Li Dongxia1, Zhang Juan2, Jia Minxin1   

  1. 1Department of Dermatology, The Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010050, China; 2Department of Dermatology, First Affiliated Hospital of Kunming Medical University, Kunming 650045, China
  • Received:2019-12-30 Revised:2020-06-18 Published:2020-08-31
  • Contact: Li Dongxia E-mail:ldx828@sina.com
  • Supported by:
    2016 "Grassland Talents" High?Level Talent Training Project （CYYC6027）

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摘要： 【摘要】 目的 研究小檗碱对皮肤鳞状细胞癌（cSCC）-A431裸鼠移植瘤的体积、重量、细胞增殖及凋亡的影响，探索小檗碱抑制cSCC的可能机制。方法 培养A431细胞，于20只裸鼠背部皮下注射A431细胞悬液建立cSCC移植瘤裸鼠模型。接种第15天，将荷瘤裸鼠随机平均分为4组：低、中、高剂量组裸鼠腹腔分别注射10、20、25 mg/kg小檗碱溶液，对照组腹腔注射生理氯化钠溶液，每日1次，连续给药35 d。分别于给药前、给药第7、14、21、28、35天检测移植瘤体积。实验结束后，处死裸鼠并随即剥离瘤块称重，计算肿瘤生长抑制率。移植瘤行组织病理检查，免疫组化检测Ki67表达水平，TUNNEL染色检测移植瘤组织中凋亡细胞数，荧光定量PCR和Western印迹法分别检测凋亡相关蛋白Bax、Bcl-2、caspase-3和Ezrin mRNA和蛋白的表达。多组间比较采用单因素方差分析，各组与对照组比较采用Dunnett-t检验。结果 随小檗碱剂量升高和作用时间延长，肿瘤生长曲线逐渐变平缓，各小檗碱组肿瘤生长受到不同程度的抑制，其中高剂量组肿瘤生长抑制作用最明显。小檗碱低、中、高剂量组肿瘤生长抑制率分别为31.05%、66.68%、76.49%，瘤重均显著低于对照组（t = 4.07、6.33、7.26，均P < 0.01）。移植瘤组织病理检查显示，随小檗碱剂量的增加，肿瘤细胞坏死程度和范围增加。免疫组化显示，随小檗碱剂量的增加，Ki67阳性细胞逐渐减少。此外，低、中、高剂量组Ki67阳性细胞数均显著低于对照组（均P < 0.01），而凋亡细胞数均显著高于对照组（P < 0.05或 < 0.01）。4组Bax、Bcl-2、caspase-3、Ezrin mRNA及蛋白表达差异均有统计学意义（均P < 0.01）。其中，小檗碱低、中、高剂量组Bcl-2 mRNA的表达均显著低于对照组（均P < 0.01），中、高剂量组Bcl-2蛋白表达显著低于对照组（均P < 0.01）；高剂量组Bax mRNA及中、高剂量组caspase-3 mRNA的表达均显著高于对照组（P < 0.05或 < 0.01），而低、中、高剂量组Bax、caspase-3蛋白表达量均显著高于对照组（均P < 0.01）；仅高剂量组Ezrin mRNA表达显著高于对照组（均P < 0.01），而低、中、高剂量组Ezrin蛋白表达均显著低于对照组（均P < 0.01）。结论 小檗碱可抑制cSCC-A431裸鼠移植瘤细胞的增殖并促进凋亡，从而一定程度抑制cSCC-A431裸鼠移植瘤的生长，其机制可能与小檗碱上调Bax、caspase-3的表达，同时下调Bcl-2、Ezrin的表达有关。

关键词: 癌, 鳞状细胞, 小檗碱, 疾病模型, 动物, 细胞凋亡, A431细胞

Abstract: 【Abstract】 Objective To evaluate the effect of berberine on the volume and weight of, cell proliferation and apoptosis in transplanted cutaneous squamous cell carcinoma （cSCC） in nude mice inoculated with A431 cells, and to explore the possible mechanism underlying the inhibitory effect of berberine on cSCC. Methods A431 cells were cultured, and A431 cell suspension was subcutaneously injected into the back of 20 nude mice to establish a nude mouse model of transplanted cSCC. On day 15 after inoculation, these tumor-bearing mice were randomly and equally divided into 4 groups: low-, medium- and high-dose berberine groups intraperitoneally injected with 10, 20 and 25 mg/kg berberine solution respectively, and control group intraperitoneally injected with sodium chloride physiological solution. The treatment was performed once a day for 35 consecutive days. The tumor volume was measured before, and on days 7, 14, 21, 28 and 35 after the start of treatment. After the end of the experiment, the nude mice were sacrificed, and the tumors were removed and weighed to calculate the tumor growth inhibition rate. Histopathological examination was performed in these transplanted tumors, immunohistochemical study was conducted to determine the expression of Ki67, TUNNEL staining was conducted to determine the number of apoptotic cells in the transplanted tumor tissues, fluorescence-based quantitative PCR and Western blot analysis were employed to determine the mRNA and protein expression of apoptosis-related proteins Bax, Bcl-2, caspase-3 and Ezrin respectively. One-way analysis of variance was used for comparisons among multiple groups, and Dunnett-t test for comparisons of each berberine group with the control group. Results Along with the increase in the dose of berberine and treatment duration, the tumor growth curve gradually became flat, the tumor growth was inhibited to different degrees in the berberine groups, and the high-dose berberine showed the strongest inhibitory effect on the tumor growth. The tumor growth inhibition rate was 31.05%, 66.68%, 76.49% in the low-, medium-, and high-dose berberine groups respectively, and the tumor weight was significantly lower in the 3 berberine groups than in the control group （t = 4.07, 6.33, 7.26, respectively, all P < 0.01）. Along with the increase in the dose of berberine, histopathological examination of the transplanted tumors showed that the extent and area of tumor cell necrosis increased, while immunohistochemical study showed that the number of Ki67-positive cells gradually decreased. Moreover, the number of Ki67-positive cells was significantly lower in the low-, medium- and high-dose berberine groups than in the control group （all P < 0.01）, but the number of apoptotic cells was significantly higher in the berberine groups than in the control group （P < 0.05 or < 0.01）. The mRNA and protein expression of Bax, Bcl-2, caspase-3 and Ezrin significantly differed among the 4 groups （all P < 0.01）. In addition, the mRNA expression of Bcl-2 was significantly lower in the low-, medium- and high-dose berberine groups than in the control group （all P < 0.01）, and the protein expression of Bcl-2 was significantly lower in the medium- and high-dose berberine groups than in the control group （both P < 0.01）; Bax mRNA expression in the high-dose berberine group and caspase-3 mRNA expression in the medium- and high-dose berberine groups were significantly higher than the corresponding mRNAs in the control group respectively （P < 0.05 or < 0.01）, and the protein expression of Bax and caspase-3 was significantly higher in the low-, medium- and high-dose berberine groups than in the control group （all P < 0.01）; Ezrin mRNA expression was significantly higher in the high-dose berberine group than in the control group （P < 0.01）, but its protein expression was significantly lower in the low-, medium-, and high-dose berberine groups than in the control group （all P < 0.01）. Conclusion Berberine can inhibit the proliferation of A431 cells and promote their apoptosis in the transplanted cSCC of the nude mice, and then suppress the growth of transplanted cSCC in the nude mice to a certain extent, which may be related to the upregulation of Bax and caspase-3 and downregulation of Bcl-2 and Ezrin.

Key words: Carcinoma, squamous cell, Berberine, Disease models, animal, Apoptosis, A431 cells