The Effects And Mechanism Of PP242 Induced Autophagy On Polycystic Kidney Rat Cholangiocytes

Background:Caroli’s disease is characterized by the progressive, multiple cystic dilatations of intrahepatic bile ducts, and is frequently associated with portal fibrosis corresponding to congenital hepatic fibrosis. It belongs to a group of congenital hepatorenal fibrocystic syndrome, and is a hepatic manifestation of autosomal recessive polycystic kidney disease (ARPKD). PP242 is a novel mTORC inhibitor, acts on ATP binding site of mTORC, and targets mTOR complex 2 (mTORC2). It is function as two distinct multiprotein complexes, mTORC1 and mTORC2.Compare with rapamycin, it is easy to combine with mTOR site, therefore, it has the effect of inhibiting protein synthesis and cell proliferation. Using the PCK rat, potential therapeutic strategies for Caroli’s disease and ARPKD have been analyzed; however, no effective therapy applicable to human disease has been established.Objectives:To explore the mechanisms of dual PI3K/Akt/mTOR inhibitor PP242 induced proliferation and autophagy in PCK rat cholangiocytes.Materials and methods:Expression of p-mTOR and p-Akt protein in the bile duct epithelial was examined by immunohistochemistry. The effect of PP242 on proliferation activity of cholangiocytes was detected using the WST-1 assay. The expression of PI3K/Akt/mTOR signaling pathway and autophagy-related proteins with PP242 treatment were detected by using western blotting. The effect of LC3silencing and Beclin1 silencing on the cell proliferation activity were determined using the WST-1 assay.Results:1.Theexpressions of p-Akt and p-mTOR protein were highly expressed inthe bile duct epithelium of the PCK rat. PP242 significantly inhibited cholangiocytes cell proliferation, which showed adose and time-dependent manner (p<0.05).2. PP242 significantly reduced the expression of PI3K/Akt/mTOR signaling pathway related proteins with dose-dependent manner in PCK cholangiocytes.3. PP242 upregulated the autophagy-specific protein LC3Ⅱ and Beclinl. 4. The inhibitory effect of PP242 on cell proliferation was weakened by treatment with knockdown of LC3 and Beclinl (p<0.01).Conclusions:The p-mTOR and p-Akt proteins were over expressed in PCK cholangiocytes. The PI3K/Akt/mTOR inhibitor PP242 suppressed the PCK cholangiocyte proliferation, and which mechanism is closely related with autophagy.