N-cadherin Alleviates Apoptosis and Senescence of Nucleus Pulposus Cells via Suppressing ROS-dependent ERS in the Hyper-osmolarity Microenvironment.

The in-situ osmolarity is an important physicochemical factor that regulates cell fate of nucleus pulposus cells (NPCs). Our previous studies demonstrated that reduced N-cadherin (NCDH) expression in nucleus pulposus cells is associated with cellular damage under hyper-osmolarity microenvironment. This study was aimed at exploring the impacts of NCDH on senescence and apoptosis of NPCs, as well as the potential molecular mechanism. By comparing NPCs from patients with lumbar fractures and lumbar disc herniation, we identified a correlation between decreased NCDH expression and increased endoplasmic reticulum stress (ERS), resulting in undesirable cell fate (senescence and apoptosis). After blocking Reactive oxygen species (ROS) or ERS, it was indicated that hyper-osmolarity microenvironment induced ERS was ROS-dependent. Further results demonstrated the correlation in rat NPCs. Upregulation of NCDH expression reduced ROS-dependent ERS, thus limiting undesirable cell fates in vitro. This was further confirmed through the rat tail acupuncture injection model. NCDH overexpression successfully mitigated ERS, preserved extracellular matrix production and alleviating intervertebral disc degeneration in vivo. Together, NCDH can alleviate senescence and apoptosis of NPCs by suppressing ROS-dependent ERS via the ATF4-CHOP signaling axis in the hyper-osmolarity microenvironment, thus highlighting the therapeutic potential of NCDH in combating degenerative disc diseases.

Spheroid Formation Enhances the Regenerative Capacity of Nucleus Pulposus Cells via Regulating N-CDH and ITGß1 Interaction.

Background: Nucleus pulposus (NP) degeneration is the core pathological change of intervertebral disc (IVD) degenerative diseases, but currently, no effective therapy is available. With the rapid development of biomaterials and tissue engineering in recent years, biomaterial-assisted cell transplantation becomes a promising therapy for IVD degeneration. However, the application is severely limited by the weak biological characteristics of NP cells (NPCs), such as a moderate proliferation ability, weak self-renewal capacity, and minimal extracellular matrix (ECM) synthesis capacity, caused by the current inappropriate cell seeding or grafting methods. Methods: Here, we developed a three-dimensional (3D) spheroidizing culture method to construct NPC spheroids and investigated repair and regeneration potential of these spheroids in vitro and in vivo. The in vitro biological characteristics (including cell viability and proliferation), and in vivo functions (including anti-degeneration potential and ability to induce tissue repair) of NPC spheroids and monolayer-cultured NPCs were compared. Furthermore, an RNA-seq-based transcriptome analysis and a series of function experiments were performed to elucidate the potential mechanisms of their differences that were involved in the tissue regeneration process. Results: NPC spheroids exhibited obviously superior self-renewal and ECM synthesis capacities compared to monolayers of NPCs in vitro. In vivo, NPC spheroids generated more functional ECM components, primarily aggrecan (ACAN) and collagen type II (Col2), and markedly promoted NP regeneration in the disc degeneration model induced by partial NP excision. Additionally, the biological characteristics and functions of NPC spheroids were to some extent regulated by the interaction of N-cadherin (N-CDH) and Integrinß1 (ITGß1), two key mechanosensing ECM-receptors expressed on NPCs. Conclusions: The NPC spheroidizing culture method is beneficial for cell renewal and the generation of functional ECM in NP tissue. The molecular mechanism involved in this regeneration process is closely associated with the regulation of the N-CDH and ITGß1 interaction-mediated ECM homeostesis. Moreover, the strategy of hydrogel-assisted NPC spheroids transplantation may potentially be used in the future treatment of IVD degeneration.

Bavachin Induces Ferroptosis through the STAT3/P53/SLC7A11 Axis in Osteosarcoma Cells.

Ferroptosis is a new form of regulated cell death, which is mediated by intracellular iron. Although it is reported that bavachin has antitumour effects on several tumour cells and prompts the reactive oxygen species (ROS) generation, it is unclear whether ferroptosis can be induced by bavachin in osteosarcoma (OS) cells. In this study, we found that bavachin inhibits the viability of MG63 and HOS OS cell lines along with an increase in the ferrous iron level, ROS accumulation, malondialdehyde overexpression, and glutathione depletion. Moreover, iron chelators (deferoxamine), antioxidants (Vit E), and ferroptosis inhibitors (ferrostatin-1 and liproxstatin-1) reverse bavachin-induced cell death. Bavachin also altered the mitochondrial morphology of OS cells, leading to smaller mitochondria, higher density of the mitochondrial membrane, and reduced mitochondrial cristae. Further investigation showed that bavachin upregulated the expression of transferrin receptor, divalent metal transporter-1, and P53, along with downregulating the expression of ferritin light chain, ferritin heavy chain, p-STAT3 (705), SLC7A11, and glutathione peroxidase-4 in OS cells. More importantly, STAT3 overexpression, SLC7A11 overexpression, and pretreatment with pifithrin-α (P53 inhibitor) rescued OS cell ferroptosis induced by bavachin. The results show that bavachin induces ferroptosis via the STAT3/P53/SLC7A11 axis in OS cells.

SIRT1 Attenuates Apoptosis of Nucleus Pulposus Cells by Targeting Interactions between LC3B and Fas under High-Magnitude Compression.

Mechanical overloading-induced nucleus pulposus cell (NPC) apoptosis plays a core role in the pathogenesis of intervertebral disc degeneration. In this study, we investigated the involvement of mammalian silent information regulator 2 homolog (SIRT1) in NPC apoptosis under high-magnitude compression. Our results showed that high-magnitude compression aggravated cellular apoptosis and attenuated the expression levels of SIRT1 and microtubule-associated protein-1 light chain-3B (LC3B) in rat NPCs in a three-dimensional (3D) cell culture model and an in vivo rat tail compression model, whereas SIRT1 overexpression in NPCs partially reversed these indicators. Moreover, SIRT1 overexpression increased the formation of the LC3B/Fas complex, alleviated activation of the NF-κB pathway, and reduced NPC apoptosis. Finally, downregulation of LC3B partially activated the NF-κB pathway and aggravated NPC apoptosis. Overall, upregulation of SIRT1 increases formation of the LC3B/Fas complex, which contributes to suppression of NPC apoptosis by inhibiting the NF-κB pathway under high compressive stress.

HO-1 induced autophagy protects against IL-1 ß-mediated apoptosis in human nucleus pulposus cells by inhibiting NF-κB.

In this study, we investigated the role of heme oxygenase-1 (HO-1) in intervertebral disc degeneration (IDD) by assessing the effects of HO-1 overexpression on IL-1ß-induced apoptosis in nucleus pulposus cells (NPCs). Immunohistochemical staining showed HO-1 expression to be lower in NPCs from IDD patients than from patients with lumbar vertebral fractures (LVF). Western blot analysis showed HO-1 and LC3-II/I levels to be lower in NP tissues from IDD patients than from LVF patients, suggesting suppression of autophagy in degenerative intervertebral disc. Consistent with that idea, autophagy was increased in HO-1-overexpressing NPCs while IL-1ß-induced apoptosis was reduced. These effects were reversed by treatment with the early autophagy inhibitor 3-methyl adenine, which suggests HO-1-induced autophagy suppresses IL-1ß-induced apoptosis in NPCs. HO-1 overexpression promoted autophagy by increasing levels of Beclin-1/PI3KC3 complex. Phospho-P65 levels were lower in HO-1-overexpressing NPCs, suggesting inhibition of NF-κB-mediated apoptosis. Our study thus demonstrates that HO-1 promotes autophagy by enhancing formation of Beclin-1/PI3KC3 complex and suppresses IL-1ß-induced apoptosis by inhibiting NF-κB. We suggest that HO-1 is a potential therapeutic target to alleviate IDD.