Inhibition of PI3K/AKT/mTOR signaling pathway promotes autophagy of articular chondrocytes and attenuates inflammatory response in rats with osteoarthritis.
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.
Xue JF
，Shi ZM
，Zou J
，Li XL
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Oroxin B alleviates osteoarthritis through anti-inflammation and inhibition of PI3K/AKT/mTOR signaling pathway and enhancement of autophagy.
Osteoarthritis (OA) is a common aging-related degenerative joint disease with chronic inflammation as its possible pathogenesis. Oroxin B (OB), a flavonoid isolated from traditional Chinese herbal medicine, possesses anti-inflammation properties which may be involved in regulating the pathogenesis of OA, but its mechanism has not been elucidated. Our study was the first to explore the potential chondroprotective effect and elucidate the underlying mechanism of OB in OA.
In vitro, primary mice chondrocytes were stimulated with IL-1β along with or without the administration of OB or autophagy inhibitor 3-methyladenine (3-MA). Cell viability assay was measured with a cell counting kit-8 (CCK-8). The phenotypes of anabolic-related (Aggrecan and Collagen II), catabolic-related (MMP3, MMP13, and ADAMTS5), inflammation-related (iNOS, COX-2, TNF-α, IL-6, and IL-1β), and markers of related signaling pathways in chondrocytes with different treatment were detected through western blot, RT-qPCR, and immunofluorescent staining. In vivo, the destabilized medial meniscus (DMM) operation was performed to establish the OA mice model. After knee intra-articular injection with OB for 8 weeks, the mice's knee joints were obtained for subsequent histological staining and analysis.
OB reversed the expression level of anabolic-related proteins (Aggrecan and Collagen II) and catabolic-related (MMP3, MMP13, and ADAMTS5) in IL-1β-induced chondrocytes. Mechanistically, OB suppressed the inflammatory response stimulated by IL-1β, as the inflammation-related (iNOS, COX-2, TNF-α, IL-6, and IL-1β) markers were downregulated after the administration of OB in IL-1β-induced chondrocytes. Besides, the activation of PI3K/AKT/mTOR signaling pathway induced by IL-1β could be inhibited by OB. Additionally, the autophagy process impaired by IL-1β could be rescued by OB. What's more, the introduction of 3-MA to specifically inhibit the autophagic process impairs the protective effect of OB on cartilage. In vivo, histological staining revealed that intra-articular injection of OB attenuated the cartilage degradation, as well as reversed the expression level of anabolic and catabolic-related proteins such as Aggrecan, Collagen II, and MMP13 induced in DMM-induced OA models.
The study verified that OB exhibited the chondroprotective effect by anti-inflammatory, inhibiting the PI3K/AKT/mTOR signaling pathway, and enhancing the autophagy process, indicating that OB might be a promising agent for the treatment of OA.
Lu R
，He Z
，Zhang W
，Wang Y
，Cheng P
，Lv Z
，Yuan X
，Guo F
，You H
，Chen AM
，Hu W
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《Frontiers in Endocrinology》
Silencing UHRF1 enhances cell autophagy to prevent articular chondrocytes from apoptosis in osteoarthritis through PI3K/AKT/mTOR signaling pathway.
Osteoarthritis (OA) is a common chronic degenerative joint disease, and chondrocyte apoptosis is one of most important pathological changes of OA pathogenesis. Growing studies have shown that Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is an important epigenetic regulatory factor that regulates cell proliferation and apoptosis of various tumors, but its role in OA remains ill-defined. In the present study, we found that UHRF1 expression was increased in human OA cartilage tissues, compared with normal cartilage tissues. Interleukin-1β (IL-1β), a major inflammatory cytokine that promotes cartilage degradation in OA, was used to stimulate primary human chondrocytes in vitro. The expression of UHRF1 was also enhanced in IL-1β-induced chondrocytes. Moreover, down-regulation of UHRF1 induced an increase on cell proliferation and autophagy, and a decrease on apoptosis of chondrocytes after IL-1β treatment. Further data indicated that silencing UHRF1 attenuated the up-regulation of IL-1β on phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway in chondrocytes. Then, an activator of PI3K weakened the effect of UHRF1 silencing on cell proliferation, autophagy, apoptosis of IL-1β-induced chondrocytes, and the cell autophagy special inhibitor 3-methyladenine (3-MA) also showed a same impact on UHRF1, hence suggesting that knockdown of UHRF1 enhances cell autophagy to protect chondrocytes from apoptosis in OA through PI3K/AKT/mTOR signaling pathway. In conclusion, our study suggests that UHRF1 may be a potential regulator of chondrocyte apoptosis in the pathogenesis of OA.
Shi X
，Han L
，Sun T
，Zhang F
，Ji S
，Zhang M
，Wang X
，Yang W
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ResearchGate
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