吴茱萸碱联合小檗碱对CCD-18Co诱导的结肠上皮细胞表型转化的调节效应

doi:10.24920/003670

摘要/Abstract

摘要：

目的基质细胞之间或基质细胞与癌细胞之间存在着细胞转分化过程。吴茱萸碱和小檗碱是中药方剂左金丸的主要药理成分,二者在肿瘤微环境重塑中起着至关重要的作用。本研究旨在探讨吴茱萸碱联合小檗碱（cBerEvo）对肿瘤相关成纤维细胞诱导的结肠上皮细胞表型转化的影响及其相关机制。
方法采用制备好的人结肠肌成纤维细胞系CCD-18Co条件化培养基诱导正常人结肠上皮细胞系HCoEpiCs向间质细胞转化,同时观察TGF-β受体抑制剂LY364947或cBerEvo对HCoEpiCs细胞转化是否有抑制作用。用相差显微镜观察HCoEpiCs形态学变化。免疫荧光显微镜观察及检测上皮细胞-间充质转化标记物包括E-钙粘蛋白（E-cadherin）,波形蛋白（vimentin）以及α-平滑肌肌动蛋白（α-SMA）的表达。创伤愈合试验评估细胞迁移情况。Western blotting检测E-cadherin、vimentin、α-SMA、Snail、ZEB1及Smads蛋白表达。
结果与对照组细胞相比,经肿瘤相关成纤维细胞样CCD-18Co细胞诱导的HCoEpiCs细胞表达E-cadherin下调、表达vimentin、α-SMA、Snail、ZEB1上调（P<0.05）,同时HCoEpiCs细胞迁移能力提高（P<0.05）,表达Smads蛋白包括Smad2、Smad3、p-Smad2、p-Smad3和Smad4上调。TGF-B抑制剂LY364947或吴茱萸碱联合小檗碱能够以浓度依赖的方式部分逆转上述上述CCD-18Co细胞的诱导作用。此外,cBerEvo能以浓度依赖性的方式抑制p-Smad2/Smad2及p-Smad3/Smad3的比例。
结论本研究结果表明cBerEvo通过抑制TGF-β/Smads信号通路的激活调节由CCD-18Co细胞诱导的结肠上皮细胞转分化过程。

关键词: 吴茱萸碱, 小檗碱, 肿瘤微环境, 上皮-间质转化, TGF-β信号

Abstract:

Objective Transdifferentiation exists between stromal cells or between stromal cells and cancer cells. Evodiamine and berberine are predominant pharmacological components of Zuojin pill, a prescription of Traditional Chinese Medicine, playing crucial functions in remolding of tumor microenvironment. This study aimed to explore the effect of combination of evodiamine with berberine (cBerEvo) on the phenotypic transition of colon epithelial cells induced by tumor-associated fibroblasts, as well as the involved mechanisms.
Methods Human normal colon epithelial cell line HCoEpiC cells were treated with the prepared conditioned medium of CCD-18Co, a human colon myofibroblast line, to induce epithelial-mesenchymal transition. Phase contrast microscope was used to observe the morphological changes. Epithelial-mesenchymal transition markers including E-cadherin, vimentin and alpha-smooth muscle actin (α-SMA) were observed with immunofluorescence microscopy. Migration was assessed by wound healing assay. Western blotting was used to detect the expressions of E-cadherin, vimentin, α-SMA, Snail, ZEB1 and Smads.
Results In contrast to the control, the tumor-associated fibroblasts-like CCD-18Co cells induced down-regulation of E-cadherin and up-regulation of vimentin, α-SMA, Snail and ZEB1 (P<0.05), and promoted migration of HCoEpiCs (P<0.05), with over expression of Smads including Smad2, p-Smad2, Smad3, p-Smad3 and Smad4 (P<0.05), which were abolished by a transforming growth factor-β (TGF-β) receptor inhibitor LY364947 and by cBerEvo in a concentration dependent manner. In addition, cBerEvo-inhibited ratios of p-Smad2/Smad2 and p-Smad3/Smad3 were also dose dependent.
Conclusion The above results suggest that cBerEvo can regulate the differentiation of colon epithelial cells induced by CCD-18Co through suppressing activity of TGF-β/Smads signaling pathway.

Key words: evodiamine, berberine, tumor microenvironment, epithelial-mesenchymal transition, TGF-β signaling

Huang Chao, Xiang Keming, Liang Bingjun, Huang Weixuan, Zhang Fanjun, Shao Yuwan, Wang Xiulian, Liu Haosheng, Shen Weizeng. Combination of Evodiamine with Berberine Reveals a Regulatory Effect on the Phenotypic Transition of Colon Epithelial Cells Induced by CCD-18Co[J].Chinese Medical Sciences Journal, 2020, 35(3): 195-206.

图/表 6

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