Department of Orthopedics, Shanghai Jiaotong University Affiliated Sixth People's Hospital, Shanghai 200233, PR China.
Department of Orthopedics, Shanghai Jiaotong University Affiliated Sixth People's Hospital, Shanghai 200233, PR China.
Biomed Pharmacother. 2017 May;89:1252-1261. doi: 10.1016/j.biopha.2017.01.130. Epub 2017 Mar 17.
This study aims to explore the relationship between PI3K/AKT/mTOR signaling pathway and autophagy of articular chondrocytes in rats with osteoarthritis (OA).
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.
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.
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.
本研究旨在探讨 PI3K/AKT/mTOR 信号通路与骨关节炎（OA）大鼠关节软骨细胞自噬之间的关系。
分离并培养大鼠关节软骨细胞，然后用 PI3K/AKT/mTOR 信号通路蛋白抑制剂诱导。将软骨细胞分为空白组、IL-1β 诱导组（IL-1β 组）、PI3K 抑制剂+IL-1β 诱导组（PI3Ki+IL-1β 组）、AKT 抑制剂+IL-1β 诱导组（AKTi+IL-1β 组）和 mTOR 抑制剂+IL-1β 诱导组（mTORi+IL-1β 组）。通过 MTT 检测细胞增殖活性，流式细胞术检测细胞周期，单丹磺酰戊二胺（MDC）染色检测细胞自噬。通过 GFP-LC3 荧光显微镜评估自噬率。采用实时定量聚合酶链反应（qRT-PCR）检测自噬相关基因（Atg5 和 Atg7）的 mRNA 表达。Western blot 检测自噬标志物（LC3、Beclin1 和 p62）和 PI3K/AKT/mTOR 信号通路相关蛋白的表达。
细胞培养 24h 后，IL-1β 组细胞增殖率低于空白组，PI3Ki+IL-1β 组、AKTi+IL-1β 组和 mTORi+IL-1β 组细胞增殖率均高于 IL-1β 组。与空白组相比，IL-1β 组细胞停滞在 G1 期，S 期细胞减少，MDC 阳性染色细胞减少，染色强度减弱，自噬率降低，Atg5 和 Atg7 的 mRNA 表达及 LC3、Beclin1 和 p62 蛋白表达均显著下调。而在 PI3Ki+IL-1β、AKTi+IL-1β 和 mTORi+IL-1β 组中，二倍体细胞减少，S 期细胞比例增加，G1 期细胞比例减少，自噬率增加，Atg5 和 Atg7 的 mRNA 表达及 LC3、Beclin1 和 p62 蛋白表达均显著上调。与空白组相比，IL-1β 组中 PI3K、AKT 和 mTOR 的磷酸化蛋白水平升高，但 PI3K、AKT 和 mTOR 的总蛋白水平无明显变化。同时，在 PI3Ki+IL-1β、AKTi+IL-1β 和 mTORi+IL-1β 组中，PI3K、AKT 和 mTOR 的磷酸化蛋白水平相对较低。
炎症可抑制大鼠软骨细胞的增殖和细胞周期，降低自噬率。抑制 PI3K/AKT/mTOR 信号通路可促进 OA 大鼠关节软骨细胞自噬，减轻炎症反应。
