Excessive reactive oxygen species (ROS) generation in degenerative intervertebral disc (IVD) indicates the contribution of oxidative stress to IVD degeneration (IDD), giving a novel insight into the pathogenesis of IDD. ROS are crucial intermediators in the signaling network of disc cells. They regulate the matrix metabolism, proinflammatory phenotype, apoptosis, autophagy, and senescence of disc cells. Oxidative stress not only reinforces matrix degradation and inflammation, but also promotes the decrease in the number of viable and functional cells in the microenvironment of IVDs. Moreover, ROS modify matrix proteins in IVDs to cause oxidative damage of disc extracellular matrix, impairing the mechanical function of IVDs. Consequently, the progression of IDD is accelerated. Therefore, a therapeutic strategy targeting oxidative stress would provide a novel perspective for IDD treatment. Various antioxidants have been proposed as effective drugs for IDD treatment. Antioxidant supplementation suppresses ROS production in disc cells to promote the matrix synthesis of disc cells and to prevent disc cells from death and senescence in vitro. However, there is not enough in vivo evidence to support the efficiency of antioxidant supplementation to retard the process of IDD. Further investigations based on in vivo and clinical studies will be required to develop effective antioxidative therapies for IDD.

The accumulation of senescent disc cells in degenerative intervertebral disc (IVD) suggests the detrimental roles of cell senescence in the pathogenesis of intervertebral disc degeneration (IDD). Disc cell senescence decreased the number of functional cells in IVD. Moreover, the senescent disc cells were supposed to accelerate the process of IDD via their aberrant paracrine effects by which senescent cells cause the senescence of neighboring cells and enhance the matrix catabolism and inflammation in IVD. Thus, anti-senescence has been proposed as a novel therapeutic target for IDD. However, the development of anti-senescence therapy is based on our understanding of the molecular mechanism of disc cell senescence. In this review, we focused on the molecular mechanism of disc cell senescence, including the causes and various molecular pathways. We found that, during the process of IDD, age-related damages together with degenerative external stimuli activated both p53-p21-Rb and p16-Rb pathways to induce disc cell senescence. Meanwhile, disc cell senescence was regulated by multiple signaling pathways, suggesting the complex regulating network of disc cell senescence. To understand the mechanism of disc cell senescence better contributes to developing the anti-senescence-based therapies for IDD.

Glutathione peroxidase-1 (GPx1) is a pivotal intracellular antioxidant enzyme that enzymatically reduces hydrogen peroxide to water to limit its harmful effects. This study aims to identify a microRNA (miRNA) that targets GPx1 to maintain redox homeostasis. Dual luciferase assays combined with mutational analysis and immunoblotting were used to validate the bioinformatically predicted miRNAs. We sought to select miRNAs that were responsive to oxidative stress induced by hydrogen peroxide (H2O2) in the H9c2 rat cardiomyocyte cell line. Quantitative real-time PCR (qPCR) demonstrated that the expression of miR-181a in H2O2-treated H9c2 cells was markedly upregulated. The downregulation of miR-181a significantly inhibited H2O2-induced cellular apoptosis, ROS production, the increase in malondialdehyde (MDA) levels, the disruption of mitochondrial structure, and the activation of key signaling proteins in the mitochondrial apoptotic pathway. Our results suggest that miR-181a plays an important role in regulating the mitochondrial apoptotic pathway in cardiomyocytes challenged with oxidative stress. MiR-181a may represent a potential therapeutic target for the treatment of oxidative stress-associated cardiovascular diseases.

Myeloid differentiation 1 (MD-1), a secreted protein interacting with radioprotective 105 (RP105), plays an important role in Toll-like receptor 4 (TLR4) signalling pathway. Previous studies showed that MD-1 may be restricted in the immune system. In this study, we demonstrated for the first time that MD-1 was highly expressed in both human and animal hearts. We also discovered that cardiac-specific overexpression of MD-1 significantly attenuated pressure overload-induced cardiac hypertrophy, fibrosis, and dysfunction, whereas loss of MD-1 had the opposite effects. Similar results were observed for in vitro angiotensin II-induced neonatal rat cardiomyocyte hypertrophy. The antihypertrophic effects of MD-1 under hypertrophic stimuli were associated with the blockage of MEK-ERK 1/2 and NF-κB signalling. Blocking MEK-ERK 1/2 signalling with a pharmacological inhibitor (U0126) greatly attenuated the detrimental effects observed in MD-1 knockout cardiomyocytes exposed to angiotensin II stimuli. Similar results were observed by blocking NF-κB signalling with a pharmacological inhibitor (BAY11–7082). Our data indicate that MD-1 inhibits cardiac hypertrophy and suppresses cardiac dysfunction during the remodelling process, which is dependent on its modulation of the MEK-ERK 1/2 and NF-κB signalling pathways. Thus, MD-1 might be a novel target for the treatment of pathological cardiac hypertrophy.

Senescence is a crucial driver of intervertebral disc degeneration (IDD). Disc cells are exposed to high oxygen tension due to neovascularization in degenerative discs. However, the effect of oxygen tension on disc cell senescence was unknown. Herein, rat nucleus pulposus (NP) cells were cultured under 20% O2 or 1% O2. Consequently, ROS induced by 20% O2 caused DNA damage and then activated p53-p21-Rb and p16-Rb pathways via ERK signaling to induce NP cell senescence. It also induced catabolic and proinflammatory phenotype of NP cells via MAPK and NF-κB pathways. Furthermore, 20% O2 was found to upregulate Nox4 in NP cells. Small interfering RNA against Nox4 reduced ROS production induced by 20% O2 and consequently suppressed premature senescence of NP cells. On the contrary, NP cells overexpressing Nox4 produced more ROS and rapidly developed senescent signs. In consistent with the in vitro studies, the expression of Nox4, p21, and Rb was upregulated in rat degenerative discs. This study, for the first time, demonstrates that Nox4 is an oxygen-sensing enzyme and a main ROS source in NP cells. Nox4-dependent ROS are genotoxic and a potent trigger of NP cell senescence. Nox4 is a potential therapeutic target for disc cell senescence and IDD.

Background Adult stem cells exist in a quiescent state (G0) within the in vivo niche; the loss of quiescence often leads to a decrease in the number and function of adult stem cells, impairing tissue regeneration and repair. Endogenous repair by nucleus pulposus-derived stem cells has recently shown promising regenerative potential for the treatment of intervertebral disc degeneration (IDD). However, the number and function of nucleus pulposus stem cells (NPSCs) declined throughout the process of IDD. This effect may have a specific relationship with quiescence. However, the biology of the quiescent NPSCs has not been reported. Methods First, we established an in vitro model for NPSC quiescence with serum starvation. The induction of G0 was confirmed by flow cytometry analyses of dual staining with Hoechst 33342 and Pyronin Y, immunofluorescent staining with Ki67 and Western blot analysis of P27 expression. NPSCs were cultured under serum starvation conditions for a long time period (21 days). To examine the functional phenotype of quiescent NPSCs, the cells were reactivated with 10% serum and differentiated into osteogenic and chondrogenic lineages in vitro. The number of colony-forming units was also estimated. To elucidate the role of autophagy in the quiescence of NPSCs, we activated and inhibited autophagy in starved cells with rapamycin and chloroquine, respectively. Then, the expression of P27 was evaluated by Western blot analysis, and the immunofluorescence of Ki67 was assessed. Finally, we assessed the role of P27 siRNA in NPSC quiescence by flow cytometry analyses and 5-ethynyl-20-deoxyuridine incorporation assays under normal and serum-starved conditions. Results NPSC quiescence was induced by 48 h of serum starvation, and they maintained quiescence for up to 21 days. Upon reactivation with serum, the quiescent NPSCs re-entered the cell cycle and exhibited enhanced clonogenic self-renewal, osteogenic differentiation and chondrogenic differentiation potentials compared to control NPSCs under normal culture conditions. We also found that autophagy underlay serum starvation-induced NPSC quiescence. Further study demonstrated that autophagy mediated the quiescence of NPSCs by regulating P27. Conclusions Serum starvation efficiently induces quiescence in NPSCs. Quiescent NPSCs maintain stem cell properties. Our study reveals that autophagy plays a role in maintaining NPSC quiescence and that autophagy mediates the quiescence of NPSCs by regulating P27. We conclude that the induction of quiescence in cultured NPSCs provides a useful model for the analysis of mechanisms that might be relevant to the biology of NPSCs in vivo.

Myeloid differentiation protein 1 (MD1) has been implicated in numerous pathophysiological processes, including immune regulation, obesity, insulin resistance, and inflammation. However, the role of MD1 in cardiac remodelling remains incompletely understood. We used MD1-knockout (KO) mice and their wild-type littermates to determine the functional significance of MD1 in the regulation of aortic banding (AB)-induced left ventricular (LV) structural and electrical remodelling and its underlying mechanisms. After 4 weeks of AB, MD1-KO hearts showed substantial aggravation of LV hypertrophy, fibrosis, LV dilation and dysfunction, and electrical remodelling, which resulted in overt heart failure and increased electrophysiological instability. Moreover, MD1-KO-AB cardiomyocytes showed increased diastolic sarcoplasmic reticulum (SR) Ca2+ leak, reduced Ca2+ transient amplitude and SR Ca2+ content, decreased SR Ca2+-ATPase2 expression, and increased phospholamban and Na+/Ca2+-exchanger 1 protein expression. Mechanistically, the adverse effects of MD1 deletion on LV remodelling were related to hyperactivated CaMKII signalling and increased impairment of intracellular Ca2+ homeostasis, whereas the increased electrophysiological instability was partly attributed to exaggerated prolongation of cardiac repolarisation, decreased action potential duration alternans threshold, and increased diastolic SR Ca2+ leak. Therefore, our study on MD1 could provide new therapeutic strategies for preventing/treating heart failure.

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