BMSCs-derived exosomes overexpressing TGM2 inhibit lactate-induced oxidative stress related apoptosis in nucleus pulposus cells

Abstract

Objective To explore the role and mechanism of exosomes derived from bone marrow mesenchymal stem cells (BMSCs) overexpressing transglutaminase 2 (TGM2) in lactate-mediated oxidative stress-induced apoptosis in nucleus pulposus cells. Methods Twenty 6-week-old male SD rats (350±50 g) were used to extract nucleus pulposus cells. Lactic acid was utilized to establish a model of oxidative stress-induced apoptosis in primary nucleus pulposus cells. Lentiviral vectors carrying TGM2 were employed to transfect BMSCs to overexpress the protein, and then exosomes were extracted from the BMSCs and identified. Subsequently, to investigate the potential mechanisms of TGM2-overexpressing exosomes against oxidative stress-induced apoptosis in nucleus pulposus cells, flow cytometry was used to detect the production of reactive oxygen species (ROS), and Western blotting and immunofluorescence assay were applied to measure the expression levels of Bax, Bcl-2, Cleaved-Caspase3, p-PI3K, p-Akt, n-Nrf2, c-Nrf2 and HO-1, and Nrf2 nuclear translocation. The rate of oxidative stress-induced apoptosis in nucleus pulposus cells and changes in the PI3K/Akt/Nrf2 pathway were assessed in 4 groups of cells (n=3): control group, lactate treatment group, lactate+control exosome group, and lactate+TGM2-overexpressing exosomes group. Results Lactic acid of 10 mmol/L was found to be more effective in inducing oxidative stress-induced apoptosis in nucleus pulposus cells (P < 0.05). Both control BMSCs and TGM2-overexpressing BMSCs could produce exosomes and stably overexpress TGM2 (P < 0.05, P < 0.01). Compared with the lactate treatment group, the nucleus pulposus cells in the lactate+TGM2-overexpressing exosome group and the lactate+control exosome group showed reduced apoptotic rates (P < 0.05), as well as decreased ROS level (P < 0.05). The lactate+TGM2-overexpressing exosome group exhibited better anti-apoptosis and ROS accumulation effects (P < 0.05). Additionally, the expression of Bax and Cleaved-Caspase3 was decreased (P < 0.05), that of Bcl-2 protein was increased (P < 0.05), the phosphorylation of PI3K/Akt was enhanced (P < 0.05), and both Nrf2 nuclear translocation and the expression of the antioxidant stress factor HO-1 were increased in nucleus pulposus cells. The lactic acid + TGM2-overexpressing exosome group showed stronger anti-apoptotic effects and activation of the PI3K/Akt/Nrf2 pathway (P < 0.05). Conclusion Exosomes derived from BMSCs overexpressing TGM2 can inhibit lactate-mediated oxidative stress-induced apoptosis in nucleus pulposus cells, and the mechanism may be related to the activation of the PI3K/Akt/Nrf2 pathway.

Keywords

• oxidative stress-induced apoptosis
• exosomes
• bone marrow mesenchymal stem cells
• transglutaminase 2
• nucleus pulposus cells