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.