Inhibition of Rac1 activity by NSC23766 prevents cartilage endplate degeneration via Wnt/β-catenin pathway.

Cartilage endplate (CEP) degeneration has been considered as one of important factors related to intervertebral disc degeneration (IVDD). Previous researches have showed that Rac1 played a pivotal role in chondrocyte differentiation. However, the effect of Rac1 during the process of CEP degeneration remains unclear. Herein, we explored the effect of Rac1 on CEP degeneration and elucidated the underlying molecular mechanism. We found expression of Rac1-GTP increased in human-degenerated CEP tissue and IL-1β-stimulated rat endplate chondrocytes (EPCs). Our study revealed that Rac1 inhibitor NSC23766 treatment promoted the expression of collagen II, aggrecan and Sox-9, and decreased the expression of ADTAMTS5 and MMP13 in IL-1β-stimulated rat EPCs. Moreover, we also found that NSC23766 could suppress the activation of Wnt/β-catenin pathway, suggesting that the beneficial effects of Rac1 inhibition in EPCs are mediated through the Wnt/β-catenin signalling. Besides, puncture-induced rats models showed that NSC23766 played a protective role on CEP and disc degeneration. Collectively, these findings demonstrated that Rac1 inhibition delayed the EPCs degeneration and its potential mechanism may be associated with Wnt/β-catenin pathway regulation, which may help us better understand the association between Rac1 and CEP degeneration and provide a promising strategy for delaying the progression of IVDD.

Jiang C ，Sun ZM ，Zhu DC ，Guo Q ，Xu JJ ，Lin JH ，Chen ZX ，Wu YS ... -  《-》

Inhibition of EZH2 ameliorates cartilage endplate degeneration and attenuates the progression of intervertebral disc degeneration via demethylation of Sox-9.

Cartilaginous endplate (CEP) degeneration is considered as one of the major causes of intervertebral disc degeneration (IVDD) which causes low back pain. Recent studies have proved that epigenetic alteration is involved in a variety of diseases. This work explored the role of histone methyltransferase enhancer of zeste homologue 2 (EZH2) in CEP degeneration, as well as its underlying epigenetic mechanisms, and confirmed the effect of EZH2 knockdown on delaying IVDD development. Western blotting, immunofluorescence staining, and ChIP assay were applied to demonstrate the molecular mechanism of EZH2 in CEP tissue. The therapeutic potential of EZH2 was investigated using puncture-induced rat models. The EZH2 expression was upregulated in human and rat CEP tissue. It was also found that the overexpression of EZH2 suppressed the expression of Collagen II, aggrecan and Sox-9, and promoted the expression of ADTAMTS5 and MMP13 in rat endplate chondrocytes (EPCs), which could be reversed by EZH2 silencing. The correlation between EZH2 and Sox-9 was further explored, while overexpression of Sox-9 could reverse the effect of EZH2 in rat EPCs. Moreover, inhibition of EZH2 upregulated the level of Sox-9 by demethylating H3K27me3 at Sox-9 promoter sites, revealing the regulatory mechanism of EZH2 on Sox-9. Meanwhile, puncture-induced rat models showed that EZH2 knockdown exerted a protective effect on CEP and disc degeneration. This study reveals that EZH2 inhibition is a promising strategy for mitigating the symptoms and progression of IVDD. This study was funded by the Natural Science Foundation of Zhejiang Province (Y16H060034). Authors declare that the funders had no involvement in the study design, data analysis and interpretation of the results.

Jiang C ，Guo Q ，Jin Y ，Xu JJ ，Sun ZM ，Zhu DC ，Lin JH ，Tian NF ，Sun LJ ，Zhang XL ，Wu YS ... -  《EBioMedicine》

Rac1 regulates nucleus pulposus cell degeneration by activating the Wnt/β-catenin signaling pathway and promotes the progression of intervertebral disc degeneration.

Yang X ，Sun Y ，Li X ，Zhang W ... -  《-》

Lycorine Suppresses Endplate-Chondrocyte Degeneration and Prevents Intervertebral Disc Degeneration by Inhibiting NF-κB Signalling Pathway.

Cartilaginous endplate (CEP) degeneration is an important cause for intervertebral disc (IVD) degeneration that leads to low-back pain. The identification of compounds that may prevent CEP degeneration is of interest for the prevention of IVD degeneration. Catabolic protease expression in the CEP of disc degeneration patients was first assessed. The toxicity, function and underlying mechanism of lycorine (LY) on CEP-derived chondrocytes degeneration were assessed in vitro by flow cytometry analysis and western blotting. The concentration and function of LY in rat-tail disc-degeneration models were also assessed by HPLC (High Performance Liquid Chromatography) quantification and histological analysis. In CEP cells, Interleukin (IL)-1β upregulated the expression of matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 that is critical for the degradation of cartilage extracellular matrix. Interestingly, LY suppressed the expression of these enzymes via the inhibition of nuclear factor-κB (NFκB) signalling and thus prevented IL-1β-induced endplate cell degeneration in vitro. More importantly, LY also reduced the expression of MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 in CEP and exerted a protective effect on both CEP and nucleus pulposus (NP) degeneration. In addition to its inhibitory effect on matrix-degrading protease expression, LY treatment also reduced positive regulators of proinflammatory cytokines, such as MIF, which can be secreted by CEP cells and subsequently target NP cells. LY could serve as a potential drug for treating IVD disease.

Wang G ，Huang K ，Dong Y ，Chen S ，Zhang J ，Wang J ，Xie Z ，Lin X ，Fang X ，Fan S ... -  《-》

Attenuation of the degenerative effects of endothelin-1 on cartilaginous end plate cells by the endothelin receptor antagonist BQ-123 via the Wnt/β-catenin signaling pathway.

Endothelin-1 (ET-1) is an inflammatory mediator associated with cartilage end plate (CEP) degeneration in the intervertebral disc (IVD). SOX9 is downregulated during CEP degeneration, along with its targets, collagen II and aggrecan. Wnt/β-catenin signaling is associated with CEP degeneration and a downstream target of SOX9; however, the precise mechanism of CEP degeneration and the role of ET-1 are largely unknown. The purpose of the study was to evaluate the influence of the endothelin-A receptor antagonist, BQ-123, on ET-1-induced effects on cartilaginous end plate cells (CECs) associated with CEP degeneration via the Wnt/β-catenin signaling pathway. The influence of ET-1 on the expression levels of collagen II, aggrecan, and SOX9 in CECs and the effect of BQ-123 in this context were investigated. To establish a model for CEP degeneration, three lumbar discs (L3-L4, L4-L5, and L5-L6 levels) in New Zealand white rabbits were punctured close to the vertebral end plate using a 14G needle. Intervertebral disc degeneration was evaluated by magnetic resonance imaging 4 weeks after vertebral end plate injury. CECs were then isolated from the degenerated CEPs to allow evaluation of the role of ET-1 and BQ-123 and to investigate their effects on the Wnt/β-catenin signaling pathway. The expression of ET-1 in CECs from degenerated CEPs was analyzed by immunofluorescent staining. Changes in the levels of collagen II, aggrecan, and SOX9 were evaluated in CECs by real-time polymerase chain reaction and by Western blotting. The Wnt/β-catenin signaling pathway was also investigated by Western blotting. After 4 weeks, IVDs with vertebral end plate injury exhibited clear signs of disc degeneration. Immunofluorescent staining showed that ET-1 was expressed in the cytoplasm of CECs. Endothelin-1 stimulation significantly inhibited the expression of collagen II, aggrecan, and SOX9 in CECs, whereas BQ-123 increased the levels of these three molecules. In addition, ET-1 stimulation increased the expression of β-catenin, cyclin D1, and Dvl1 in the Wnt/β-catenin signaling pathway of CECs from degenerated discs and reduced the expression of GSK-3β, whereas BQ-123 had the opposite effect. Endothelin-1 can reduce levels of collagen II, aggrecan, and SOX9 in CECs through activation of the Wnt/β-catenin signaling pathway, whereas BQ-123 attenuates these negative effects, highlighting a new molecular mechanism with potential for exploitation for treatment of CEP degeneration.

Yuan W ，Li ZX ，Zhao CL ，Hou TH ，Hu SW ，Liu WB ，Yuan FL ，Xiao JR ... -  《-》