PHLDA2 overexpression facilitates senescence and apoptosis via the mitochondrial route in human nucleus pulposus cells by regulating Wnt/ß-catenin signalling pathway.

Low back pain is a common clinical symptom of intervertebral disc degeneration (IVDD), which seriously affects the quality of life of the patients. The abnormal apoptosis and senescence of nucleus pulposus cells (NPCs) play important roles in the pathogenesis of IVDD. PHLDA2 is an imprinted gene related to cell apoptosis and tumour progression. However, its role in NPC degeneration is not yet clear. Therefore, this study was set to explore the effects of PHLDA2 on NPC senescence and apoptosis and the underlying mechanisms. The expression of PHLDA2 was examined in human nucleus pulposus (NP) tissues and NPCs. Immunohistochemical staining, magnetic resonance imaging imaging and western blot were performed to evaluate the phenotypes of intervertebral discs. Senescence and apoptosis of NPCs were assessed by SA-ß-galactosidase, flow cytometry and western blot. Mitochondrial function was investigated by JC-1 staining and transmission electron microscopy. It was found that the expression level of PHLDA2 was abnormally elevated in degenerated human NP tissues and NPCs. Furthermore, knockdown of PHLDA2 can significantly inhibit senescence and apoptosis of NPCs, whereas overexpression of PHLDA2 can reverse senescence and apoptosis of NPCs in vitro. In vivo experiment further confirmed that PHLDA2 knockdown could alleviate IVDD in rats. Knockdown of PHLDA2 could also reverse senescence and apoptosis in IL-1ß-treated NPCs. JC-1 staining indicated PHLDA2's knockdown impaired disruption of the mitochondrial membrane potential and also ameliorated superstructural destruction of NPCs as showed by transmission electron microscopy. Finally, we found the PHLDA2 knockdown promoted Collagen-II expression and suppressed MMP3 expression in NPCs by repressing wnt/ß-catenin pathway. In conclusion, the results of the present study showed that PHLDA2 promotes IL-1ß-induced apoptosis and senescence of NP cells via mitochondrial route by activating the Wnt/ß-catenin pathway, and suggested that therapy targeting PHLDA2 may provide valuable insights into possible IVDD therapies.

Pretreatment of nucleus pulposus mesenchymal stem cells with appropriate concentration of H2O2 enhances their ability to treat intervertebral disc degeneration.

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.

The biological effect of cobalt chloride mimetic-hypoxia on nucleus pulposus cells and the comparability with physical hypoxia in vitro.

Objective: Nucleus pulposus cells (NPCs) are cells extracted from the intervertebral disc and are important for research into intervertebral disc degeneration (IVDD). NPCs live in an avascular and relatively hypoxic environment. Cobalt chloride (CoCl2) has been used in many cell studies to mimic hypoxia. The objective of this study was to explore the possibility of using CoCl2 to induce mimetic-hypoxia for NPCs and the comparison with hypoxia (1% O2) in vitro. Materials and methods: Rat nucleus pulposus cells of Passage 3-5 were used in this research. Cell viability, rate of cell apoptosis, ROS (reactive oxygen species) generation, cell migration, extracellular pH and extracellular matrix metabolism were determined to compare the influence of hypoxia (1% O2) and CoCl2 on NPCs. Results: We found that the effects of CoCl2 on NPCs was dose-dependent. At the proper concentration, CoCl2 could be used to elicit chemical hypoxia for nucleus pulposus cells in vitro and many biological effects, analogous to physical hypoxia (1% O2), could be achieved such as enhanced cell viability, decreased apoptosis and activated extracellular matrix metabolism. On the other hand, CoCl2 mimetic-hypoxia did not affect NPCs glycolysis and migration compared to physical hypoxia. In addition, high concentration of CoCl2 (>200 µM) is harmful to NPCs with high rates of apoptosis and ECM (extracellular matrix) degradation. Conclusions: It is feasible and convenient to use CoCl2 to induce chemical mimetic hypoxia for culturing NPCs on the premise of appropriate concentration. But in aspects of cell migration and glycolysis, CoCl2 could not achieve similar results with physical hypoxia. This study may provide a convenient method and enlightenment to induce mimetic-hypoxia for researchers studying NPCs and IVVD.

Exosomes derived from stem cells as an emerging therapeutic strategy for intervertebral disc degeneration.

Intervertebral disc (IVD) degenerative diseases are a common problem in the world, and they cause substantial social and economic burdens for people. The current methods for treating IVD degenerative diseases mainly include surgery and conservative treatment, which cannot fundamentally restore the normal structure of the disc. With continuous research on the mechanism of degeneration and the development of regenerative medicine, rapid progress has been made in the field of regenerative medicine regarding the use of stem cell-derived exosomes, which are active biological substances used in intercellular communication, because they show a strong effect in promoting tissue regeneration. The study of exosomes in the field of IVD degeneration has just begun, and many surprising achievements have been made. This paper mainly reviews the biological characteristics of exosomes and highlights the current status of exosomes in the field of IVD degeneration, as well as future developments regarding exosomes.