Objective: This study aims to explore the relationship between PI3K/AKT/mTOR signaling pathway and autophagy of articular chondrocytes in rats with osteoarthritis (OA).
Methods: Rat articular chondrocytes were isolated and cultured, and then induced by protein inhibitors of PI3K/AKT/mTOR signaling pathway. Chondrocytes were assigned into blank group, IL-1β induction group (IL-1β group), PI3K inhibitor+IL-1β induction group (PI3Ki+IL-1β group), AKT inhibitor+IL-1β induction group (AKTi+IL-1β group) and mTOR inhibitor+IL-1β induction group (mTORi+IL-1β group). Cell proliferation activity was detected by MTT assay, cell cycle by flow cytometry and cell autophagy by monodansylcadaverine (MDC) staining. Autophagy rates were evaluated by GFP-LC3 fluorescence microscopy. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect mRNA expressions of autophagy-related genes (Atg5 and Atg7). Western blotting was utilized to detect expressions of autophagy markers (LC3, Beclin1 and p62) and of relevant proteins in the PI3K/AKT/mTOR signaling pathway.
Results: The cell proliferation rate of the IL-1β group was lower than that of the blank group after cells were cultured for 24h, and the cell proliferation rates of the PI3Ki+IL-1β group, the AKTi+IL-1β group and the mTORi+IL-1β group were higher than those of the IL-1β group. In comparison with the blank group, cells in the IL-1β group were arrested at the G1 phase and decreased in the S phase, MDC positive staining cells were decreased with attenuated staining intensity, the autophagy rate was decreased, the mRNA expressions of Atg5 and Atg7 and the protein expressions of LC3, Beclin1 and p62 were significantly down-regulated. While in the groups of PI3Ki+IL-1β, AKTi+IL-1β and mTORi+IL-1β, haploid cells were reduced, coupled with an increased proportion of cells in the S phase and decreased proportion of cells in the G1 phase, the autophagy rate was increased, the mRNA expressions of Atg5 and Atg7 and the protein expressions of LC3, Beclin1 and p62 were significantly up-regulated. Compared with the blank group, the protein phosphorylation levels of PI3K, AKT and mTOR were elevated, while there were no significant difference observed in the total amount of PI3K, AKT and mTOR in the IL-1β group. Meanwhile, there were relatively low protein phosphorylation levels of PI3K, AKT and mTOR in the groups of PI3Ki+IL-1β, AKTi+IL-1β and mTORi+IL-1β.
Conclusions: Inflammation could inhibit the proliferation and cell cycle of rat chondrocytes and reduce the autophagy rate. Inhibition of PI3K/AKT/mTOR signaling pathway could promote the autophagy of articular chondrocytes and attenuate inflammation response in rats with OA.
Keywords: Autophagy; IL-1β; Induction; Osteoarthritis; PI3K/AKT/mTORi; Rat articular chondrocytes.
Copyright © 2017. Published by Elsevier Masson SAS.