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 a low ability to resist the harsh microenvironment of the degenerated intervertebral disc. There is an urgent need to explore 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 (range from 25 to 300 μM) of H₂O₂. The proliferation, reactive oxygen species (ROS) level, and apoptosis of NPMSCs were detected by cell counting kit-8 (CCK-8) assay, 5-ethynyl-2′-deoxyuridine (EdU) staining, and flow cytometry in vitro. The underlying signalling pathways were explored utilizing Western blotting. A rat needle puncture-stimulated IVDD model was established. X-ray, histological staining, and a multimode small animal live imaging system were used to evaluate the therapeutic effect of H₂O₂-pretreated NPMSCs in vivo. Results: NPMSCs pretreated with 75 μM H₂O₂ demonstrated the strongest elevated cell proliferation by inhibiting the Hippo pathway (P < 0.01). Meanwhile, 75 μM H₂O₂-pretreated NPMSCs exhibited significantly enhanced antioxidative stress ability (P < 0.01), which is related to downregulated Brd4 and Keap1 and upregulated Nrf2. NPMSCs pretreated with 75 μM H₂O₂ also exhibited distinctly decreased apoptosis (P < 0.01). In vivo experiments verified that 75 μM H₂O₂-pretreated NPMSCs-transplanted rats exhibited an enhanced disc height index (DHI% = 90.00 ± 4.55, P < 0.01) and better histological morphology (histological score = 13.5 ± 0.5, P < 0.01), 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 proliferation, antioxidative stress, and antiapoptotic ability of transplanted NPMSCs, which is expected to provide a new feasible method to improve the stem cell therapy efficacy of IVDD.

Intervertebral disc degeneration (IDD) is the most common chronic disease. Oxidative stress and apoptosis of nucleus pulposus (NP) cells disrupt intervertebral disc (IVD) homeostasis, which is the main cause of IDD. Glioma-associated oncogene 1 (Gli1) is an important transcription factor in the Hedgehog (Hh) pathway. Depletion of Gli1 accelerates the occurrence and development of degenerative diseases. This study aimed to explore the role of aging related Gli1 depletion in the progression of IDD. The relationship between aging related Gli1 depletion and IDD was studied in the NP tissues of human and rats of different ages, and the levels of oxidative stress and NP cell apoptosis during IDD were explored. Gli1 depletion of NP cells were established by targeting inhibitor GANT61 or lentivirus-coated Gli1 sh-RNA (sh-Gli1) to explore the role of Gli1 in NP cells and underlying mechanism. Exogenous Gli1 depletion induced IDD of rats was established by intraperitoneal injection of GANT61. Also, the roles of Fos in the Gli1 depletion induced NP cell oxidative stress, apoptosis and IDD were investigated. Gli1 was down-regulated in the tissues of degenerative NP, and the level of Gli1 was negatively correlated with the severity of aging related IDD in human and rats. Furthermore, we found enhanced oxidative stress and apoptosis in degenerative NP tissues. Gli1 depletion promoted oxidative stress and apoptosis of NP cells and resulted in the degradation of extracellular matrix (ECM) and decreased ECM synthesis. Transcriptome sequencing showed that Gli1 depletion caused Fos activation in NP cells. the effect of Gli1 depletion on the oxidative stress and apoptosis of NP cells were retarded by Fos inhibitor. In vivo, Fos inhibition alleviated the IDD induced by exogenous Gli1 depletion. This study revealed for the first time that Gli1 is gradually depleted in NP with IDD progression. Exogenous Gli1 depletion causes oxidative stress and apoptosis of NP cells both in vivo and in vitro. Fos suppression effectively retards the destructive effects of Gli1 depletion on IVD homoeostasis. The translational potential of this article: This study may provide new potential targets for preventing and reversing IDD. Maintaining Gli1 expression in NP and suppressing Fos activation may be an effective treatment strategy for IDD.

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.