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目的:探讨PI3K/Akt/m TOR双靶点抑制剂NVP-BEZ235诱导多囊肾(polycystic kidney,PCK)大鼠胆管上皮细胞自噬的作用。方法:免疫组化法检测p-m TOR和p-Akt在PCK大鼠胆管上皮细胞中的水平。WST-1比色法检测NVP-BEZ235对胆管细胞活力的抑制作用以及LC3、Beclin 1基因沉默和自噬特异性抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)对细胞活力的影响。蛋白免疫印迹法检测NVP-BEZ235对PI3K/Akt/m TOR信号通路相关蛋白及自噬标志蛋白LC3和Beclin 1的变化。结果:p-m TOR和p-Akt在PCK大鼠胆管上皮细胞中显著升高,NVP-BEZ235可明显抑制胆管上皮细胞的活力,且呈浓度和时间依赖性改变(P<0.05);NVP-BEZ235明显抑制PI3K/Akt/m TOR信号通路相关蛋白的水平;并上调自噬标志蛋白LC3 II/LC3 I比值和Beclin 1蛋白的表达水平。LC3、Beclin 1基因沉默和3-MA均可明显减弱NVP-BEZ235对细胞活力的抑制作用(P<0.01)。结论:NVPBEZ235可抑制PCK大鼠胆管上皮细胞的活力,其机制与自噬密切相关。
AIM: To investigate the role of PI3K / Akt / m TOR dual target inhibitor NVP-BEZ235 in inducing biliary epithelial cell autophagy in polycystic kidney (PCK) rats. Methods: The levels of p-m TOR and p-Akt in bile duct epithelial cells of PCK rats were detected by immunohistochemistry. The inhibitory effect of NVP-BEZ235 on the viability of cholangiocytes and the effect of LC3, Beclin 1 gene silencing and 3-methyladenine (3-MA), a specific inhibitor of autophagy, on cell viability influences. Western blotting was used to detect the changes of PI3K / Akt / m TOR signaling pathway related proteins and autophagy markers LC3 and Beclin 1 by NVP-BEZ235. Results: pm TOR and p-Akt were significantly increased in bile duct epithelial cells of PCK rats. NVP-BEZ235 significantly inhibited the viability of bile duct epithelial cells in a concentration- and time-dependent manner (P <0.05). NVP-BEZ235 was significantly Inhibited the protein level of PI3K / Akt / m TOR signaling pathway and upregulated the expression of autophagy marker protein LC3 II / LC3 I and Beclin 1 protein. LC3, Beclin 1 gene silencing and 3-MA significantly attenuated the inhibitory effect of NVP-BEZ235 on cell viability (P <0.01). Conclusion: NVPBEZ235 can inhibit the viability of bile duct epithelial cells in PCK rats. The mechanism is closely related to autophagy.