Low back pain is thought to be mainly caused by intervertebral disc degeneration (IVDD), and there is a lack of effective treatments. Cellular senescence and matrix degradation are important factors that cause disc degeneration. Mitochondrial dysfunction induced by oxidative stress is an important mechanism of cellular senescence and matrix degradation in the nucleus pulposus (NP), and mitophagy can effectively remove damaged mitochondria, restore mitochondrial homeostasis, and mitigate the damage caused by oxidative stress. Optineurin (OPTN) is a selective mitophagy receptor, and its role in intervertebral disc degeneration remains unclear. Here, we aimed to explore the effect of OPTN on H2O2-induced nucleus pulposus cell (NPCs) senescence and matrix degradation in a rat model of disc degeneration. Western blot analysis showed that OPTN expression was reduced in degenerative human and rat nucleus pulposus tissues and increased in H2O2-induced senescent NPCs. OPTN overexpression significantly inhibited H2O2-induced senescence and increased matrix-associated protein expression in NPCs, but OPTN knockdown showed the opposite effect. As previous reports have suggested that mitophagy significantly reduces mitochondrial damage and reactive oxygen species (ROS) caused by oxidative stress, and we used the mitophagy agonist CCCP, the mitophagy inhibitor cyclosporin A (CsA), and the mitochondrial ROS (mtROS) scavenger mitoTEMPO and confirmed that OPTN attenuated NPCs senescence and matrix degeneration caused by oxidative stress by promoting mitophagy to scavenge damaged mitochondria and excess reactive oxygen species, thereby slowing the progression of IVDD. In conclusion, our research suggests that OPTN is involved in IVDD and exerts beneficial effects against IVDD.
Background Intervertebral disc degeneration (IVDD) is closely associated with the structural damage in the annulus fibrosus (AF). Aberrant mechanical loading is an important inducement of annulus fibrosus cells (AFCs) apoptosis, which contributes to the AF structural damage and aggravates IVDD, but the underlying mechanism is still unclear. This study aims to investigate the mechanism of a mechanosensitive ion channel protein Piezo1 in aberrant mechanical loading-induced AFCs apoptosis and IVDD. Methods Rats were subjected to lumbar instability surgery to induce the unbalanced dynamic and static forces to establish the lumbar instability model. MRI and histological staining were used to evaluate the IVDD degree. A cyclic mechanical stretch (CMS)-stimulated AFCs apoptosis model was established by a Flexcell system in vitro. Tunel staining, mitochondrial membrane potential (MMP) detection, and flow cytometry were used to evaluate the apoptosis level. The activation of Piezo1 was detected using western blot and calcium fluorescent probes. Chemical activator Yoda1, chemical inhibitor GSMTx4, and a lentiviral shRNA-Piezo1 system (Lv-Piezo1) were utilized to regulate the function of Piezo1. High-throughput RNA sequencing (RNA-seq) was used to explore the mechanism of Piezo1-induced AFCs apoptosis. The Calpain activity and the activation of Calpain2/Bax/Caspase3 axis were evaluated by the Calpain activity kit and western blot with the siRNA-mediated Calapin1 or Calpain2 knockdown. Intradiscal administration of Lv-Piezo1 was utilized to evaluate the therapeutic effect of Piezo1 silencing in IVDD rats. Results Lumbar instability surgery promoted the expression of Piezo1 in AFCs and stimulated IVDD in rats 4 weeks after surgery. CMS elicited distinct apoptosis of AFCs, with enhanced Piezo1 activation. Yoda1 further promoted CMS-induced apoptosis of AFCs, while GSMTx4 and Lv-Piezo1 exhibited opposite effects. RNA-seq showed that knocking down Piezo1 inhibited the calcium signaling pathway. CMS enhanced Calpain activity and elevated the expression of BAX and cleaved-Caspase3. Calpain2, but not Calpain1 knockdown, inhibited the expression of BAX and cleaved-Caspase3 and alleviated AFCs apoptosis. Lv-Piezo1 significantly alleviated the progress of IVDD in rats after lumbar instability surgery. Conclusions Aberrant mechanical loading induces AFCs apoptosis to promote IVDD by activating Piezo1 and downstream Calpain2/BAX/Caspase3 pathway. Piezo1 is expected to be a potential therapeutic target in treating IVDD.
Background: Nucleus pulposus mesenchymal stem cells (NPMSCs) transplantation is a promising treatment for intervertebral disc degeneration (IVDD). However, the transplanted NPMSCs exhibited weak cell proliferation, high cell apoptosis, and low ability to resist the harsh microenvironment of the degenerated intervertebral disc. There is an urgent need in exploring feasible methods to enhance the therapeutic efficacy of NPMSCs transplantation.Objective: To identify the optimal concentration for NPMSCs pretreatment with hydrogen peroxide (H2O2) and explore the therapeutic efficacy of NPMSCs transplantation using H2O2 pretreatment in IVDD.Methods: Rat NPMSCs were pretreated with different concentrations of H2O2. The proliferation, ROS level, and apoptosis of NPMSCs were detected by CCK-8 test, EdU staining, and flow cytometry in vitro. The underlying signaling pathways were explored utilizing western blot. A rat needle-puncture stimulated IVDD model was established. X-ray, histological staining, and multi-mode small animal live imaging system were performed to evaluate the therapeutic effect of H2O2-pretreated NPMSCs in vivo.Results: 75 μM H2O2 pretreated NPMSCs demonstrated the strongest elevated cell proliferation by inhibiting the Hippo pathway. Meanwhile, 75 μM H2O2 pretreated NPMSCs exhibited significantly enhanced anti-oxidative stress ability, which is related to downregulated Brd4, Keap1, and upregulated Nrf2. 75 μM H2O2 pretreated NPMSCs also exhibited distinctly declined apoptosis. In vivo experiments results verified that 75 μM H2O2 pretreated NPMSCs-transplanted rats exhibited enhanced disc height index (DHI%) and better histological morphology, which means 75 μM H2O2 pretreated NPMSCs can better adapt to the environment of degenerative intervertebral discs and promote the repair of IVDD.Conclusions: Pretreatment with 75 μM H2O2 was the optimal concentration to improve the ability of proliferation, anti-oxidative stress, and anti-apoptosis of transplanted NPMSCs, which is expected to provide a new feasible method to improve the stem cell therapy efficacy of IVDD.
The accumulation of metabolites in the intervertebral disc is considered to be an important cause of intervertebral disc degeneration (IVDD). Lactate, which is a metabolite that is produced by cellular anaerobic glycolysis, has been proven to be closely associated with IVDD. However, little is known about the role of lactate in nucleus pulposus cell (NPC) senescence and oxidative stress. This study attempted to investigate the effect of lactate on NPC senescence and oxidative stress as well as the underlying mechanism. A puncture-induced disc degeneration (PIDD) model was established in rats. Metabolomics analysis proved that lactate levels were significantly increased in the degenerated intervertebral discs. Elimination of excessive lactate levels using lactate oxidase (LOx)-overexpressing lentivirus alleviated the progression of IVDD. In vitro experiments showed that high concentrations of lactate could induce senescence and oxidative stress in NPC. High-throughput RNA sequencing results and bioinformatic analysis demonstrated that the induction of NPC senescence and oxidative stress by lactate may be related to the PI3K/Akt signalling pathway. Further study verified that high concentrations of lactate could induce NPC senescence and oxidative stress by inhibiting PI3K/Akt signalling and the downstream Akt/p21/p27/cyclin D1 and Akt/Nrf2/HO-1 pathways. Utilizing molecular docking and microscale thermophoresis assay, we found that lactate could suppress Akt phosphoactivation by binding to the Lys39 and Leu52 residues in the PH domain of Akt. These results highlight the involvement of lactate in NPC senescence and oxidative stress, and lactate may become a novel potential therapeutic target for the treatment of IVDD.
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