Recombinant Klotho Protects Human Periodontal Ligament Stem Cells by Regulating Mitochondrial Function and the Antioxidant System during H₂O₂-Induced Oxidative Stress

Abstract

Human periodontal ligament stem cells (hPDLSCs) are a favourable source for tissue engineering, but oxidative stress conditions during cell culture and transplantation could affect stem cell viability and stemness, leading to failed regeneration. The aim of this study was to evaluate the antioxidant and protective effects of Klotho, an antiageing protein, against cell damage and the loss of osteogenesis in hPDLSCs in H 2 O 2 -induced oxidative environments. H 2 O 2 was used as an exogenous reactive oxygen species (ROS) to induce oxidative stress. Recombinant human Klotho protein was administered before H 2 O 2 treatment. Multitechniques were used to assess antioxidant activity, cell damage, and osteogenic ability of hPDLSCs in oxidative stress and the effects of Klotho on hPDLSCs. Mitochondrial function was analyzed by an electron microscopy scan of cellular structure, mitochondrial DNA copy number, and cellular oxygen consumption rate (OCR). Furthermore, we explored the pathway by which Klotho may function to regulate the antioxidant system. We found that pretreatment with recombinant human Klotho protein could enhance SOD activity and reduce intracellular oxidative stress levels. Klotho reduced H 2 O 2 -induced cellular damage and eventually maintained the osteogenic differentiation potential of hPDLSCs. Notably, Klotho promoted mitochondrial function and activated antioxidants by negatively regulating the PI3K/AKT/FoxO1 pathway. The findings suggest that Klotho protein enhanced the antioxidative ability of hPDLSCs and protected stem cell viability and stemness from H 2 O 2 -induced oxidative stress by restoring mitochondrial functions and the antioxidant system.