盐酸小檗碱抑制PI3K/AKT/mTOR通路的活化对结肠癌SW480细胞生长、凋亡和自噬的调节作用

Berberine Hydrochloride Regulates the Growth,Apoptosis and Auto-phagy of Colon Cancer SW480 Cells via PI3K/AKT/mTOR Pathway

目的探究盐酸小檗碱抑制PI3K/AKT/mTOR通路的活化对结肠癌SW480细胞生长、调亡和自噬的调节作用的机制。方法体外培养结肠癌SW480细胞,并分为空白组(Control)、低剂量盐酸小檗碱组(BH 25μmol/L)、中剂量盐酸小檗碱组(BH 50μmol/L)、高剂量盐酸小檗碱组(BH 100μmol/L)分别使用对应剂量的盐酸小檗碱预处理。使用1~500μmol/L浓度梯度的盐酸小檗碱处理48 h后,采用MTT检测细胞存活率;采用克隆形成实验检测细胞生长情况;采用Hoechst 33258染色检测细胞凋亡情况;采用免疫荧光检测LC3含量;采用Westerm印迹检测Ki67、Caspase-3、Beclinl,P62、LC3Ⅱ/LC3Ⅰ、PI3K、AKT、mTOR表达情况。结果MTT检测结果显示,随着盐酸小檗碱处理浓度增大细胞存活率降低,当剂量超过200μmol/L后差异有统计学意义(P<0.05);克隆形成实验显示,随着盐酸小檗碱处理浓度增大SW480细胞形成率降低(P<0.05);Hoechst 33258染色检测结果显示,随着盐酸小檗碱处理浓度增大SW480细胞凋亡率升高(P<0.05),免疫荧光检测结果显示,随着盐酸小檗碱处理浓度增大SW480细胞细胞内荧光点数显著增加(P<0.05);Western印迹检测结果显示,相比空白对照组,使用盐酸小檗碱处理各组Ki67、P62、p-PI3K、p-AKT、p-mTOR蛋白表达显著降低(P<0.05)且呈计量依赖性,Caspase-3、Beclin1、LC3Ⅱ蛋白表达显著升高(P<0.05)且呈计量依赖性,LC3Ⅰ、PI3K、AKT、mTOR蛋白表达无显著差异(P>0.05)。结论盐酸小檗碱可以通过抑制PI3K/AKT/mTOR通路的活化实现抑制结肠癌SW480细胞的生长、促进结肠癌SW480细胞的凋亡和自噬。

Objective To investigate the regulating mechanism of berberine hydrochloride(BH)on the growth,apoptosis and autophagy of colon cancer SW480 cells by inhibiting the PI3KV AKT/mTOR pathway.Methods Colon cancer SW480 cells were culturedin vitroand divided into blank control group,low-dose BH group(BH 25μmol/L),medium-dose BH group(BH 50μmol/L)and high-dose BH group)(BH 100μmol/L).After treatment with BH at a concentration of 1-500μumol/L for 48 h,cell survival rates were measured by MTT,cell growth by colony forma-tion assay,apoptosis by Hoechst 33258 staining,LC3 content by immunofluorescence and the ex-pression of Ki67,caspase-3,Beclin-1,P62,LC3Ⅱ/LC3Ⅰ,PI3K,AKT and mTOR by West-ern blotting.Results MTT assay showed that the cell survival rates were decreased with the in-crease of BH concentration.The differences were statistically significant when the dose was more than 200μmol/L(P<0.05).The colony formation assay showed that the SW480 cell formation rates decreased with BH concentration increasing(P<0.05).Hoechst 33258 staining revealed that the apoptosis rates of SW480 cells increased with the increase of BH concentration(P<0.05)Immunofluorescence assay showed that number of fluorescent points in SW480 cells significantly in-creased with the increase of BH concentration(P<0.05).Western blotting showed that the expres-sion of Ki67,P62,p-PI3K,p-AKT and p-mTOR protein was significantly decreased in the BH group compared with those in blank control group(P<0.05),and the decreases were in a dose-dependent manner,while the expression of caspase-3,Beclin1 and LC3Ⅱprotein was dose-de-pendently significantly increased(P<0.05).There was no significant difference in expression of LC3Ⅰ,PI3K,AKT or mTOR protein between the BH groups and blank control group(P>0.05).Conclusion BH can inhibit the growth of colon cancer SW480 cells,and promote the apopto-sis and autophagy of colon cancer SW480 cells by inhibiting the activation of PI3K/AKT/mTOR path-way.