小檗碱对永生化结肠细胞系生长的影响及其机制

The effects of Berberine on growth of Immorto-Min colonic epithelial cell line

目的 观察小檗碱对永生化结肠细胞系（IMCE）生长的影响,探讨其可能机制.方法 IMCE细胞在存在及不存在表皮生长因子（EGF）或TNFα的情况下加入小檗碱,以Ki-67免疫细胞化学染色对有增殖活性的细胞进行标记,采用原位末端转移酶标记技术分析细胞凋亡情况,以Western blot方法检测EGF受体（EGFR）和蛋白激酶B（Akt）及其磷酸化的水平.结果 （1）EGF组的IMCE细胞增殖活性较空白对照组增强,增殖细胞比例为（10.64±1.41）%,EGF＋小檗碱组比例为（1.81±0.85）%,显著低于EGF组（P＜0.01）,小檗碱组比例最低[（0.49±0.42）%].（2）小檗碱组与TNFα组的细胞凋亡比例分别为（8.47±2.52）%和（9.39±2.13）%,明显高于空白对照组[（0.27±0.30）%],P＜0.01.（3）EGF组的磷酸化EGFR（p-EGFR）明显增加,EGF＋不同浓度小檗碱组的p-EGFR呈浓度依赖性下降.（4）TNFα组的磷酸化Akt（p-Akt）增加,TNF＋不同浓度小檗碱组的p-Akt表达较TNFα组下降.结论 小檗碱具有抑制IMCE细胞增殖和促进凋亡的作用,这种作用可能与抑制EGFR和Akt磷酸化有关.

Objective To investigate the effects of Berberine on growth of Immorto-Min colonic epithelial cell line （IMCE） and explore its possible mechanisms. Methods IMCE cells were treated with Berberine in the absence or presence of epidermal growth factor （EGF） and TNFα. Ki-67 staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling （TUNEL） assay were used to identify the cell proliferation and apoptosis respectively. Furthermore, Western blot analysis was performed to detect the epidermal growth factor receptor （ EGFR）, protein kinase B （Akt） and their phosphorylation.Results （ 1 ） Proliferating activity of IMCE cells was increased after adding EGF and the proportion of cell proliferation was （ 10. 64 ± 1.41 ） %. The proportion was significantly lowed in EGF plus Berberine group [（1.81 ±0. 85）%] compared to the EGF group（P 〈0. 01 ）, while the lowest was the Berberine group [（0.49 ± 0.42） %]. （2） The proportions of cell apoptosis were （ 8.47 ± 2. 52 ） % and （9. 39 ± 2. 13 ） %in the Berberine group and TNFt group respectively which were significantly higher compared to the normal control [（0. 27 ± 0. 30）%], both P 〈 0. 01. （3） The phosphorylation of EGFR was significantly increased after adding EGF and p-EGFR was decreased in EGF plus Berberine group at a concentration-dependent manner. （4） Moreover, the phosphorylation of Akt was enhanced after addition of TNFα , while the phosphorylation in the TNFα and Berberine group was inhibited compared to the TNFα group. Conclusions Berberine may suppress the proliferation and promote the apoptosis of IMCE cells. The mechanisms may relate to the inhibition of the phosphorylation of EGFR and Akt.