Red light-emitting diode light mediated by the KEAP1-NRF2/HO-1 pathway induces osteogenic differentiation and attenuates the oxidative stress damage of human periodontal ligament stem cells induced by high glucose

To explore the effects of red LED light mediated by the Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (KEAP1-NRF2/HO-1) pathway on osteogenic differentiation and oxidative stress damage of human periodontal ligament stem cells (hPDLSCs) induced by high glucose, which provides a basis for the application of red light-emitting diode (LED) light in cell antioxidative damage.

hPDLSCs were identified by flow cytometric analysis, alkaline phosphatase (ALP) staining and Alizarin red-S staining; hPDLSCs were pretreated in a high glucose environment for 48 hours and irradiated with 1, 3, or 5 J/cm² red LED light. A CCK-8 assay was performed to choose the radiant exposure that had the strongest effect on promoting the cell proliferation rate for subsequent experiments. hPDLSCs were divided into a control group, a high glucose group and a high glucose+light exposure group. ALP staining, ALP activity, Alizarin red-S staining and quantitative calcified nodules were used to detect the osteogenic differentiation of hPDLSCs; qRT-PCR and Western blot were used to detect the gene and protein expression levels of ALP, runt-related transcription factor 2 (RUNX2) and osterix (OSX); the relative mRNA expression levels of antioxidant enzyme-related genes superoxide dismutase 2 (SOD2) and catalase (CAT) in hPDLSCs were detected by qRT-PCR; reactive oxygen species (ROS) levels were detected by fluorescence microscopy and flow cytometry; the tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) levels in cell supernatants were detected by ELISA; the NRF2-specific inhibitor ML385 was used to inhibit the NRF2 pathway; ALP staining and ALP activity were used to detect the markers of early osteogenic differentiation; qRT-PCR was used to detect the gene expression of ALP, RUNX2 and OSX; and the protein expression levels of KEAP1, NRF2 and HO-1 were detected by Western blot.

Identified, and irradiant exposure of 5 J/cm² was chosen for subsequent experiments. Red LED light irradiation (5 J/cm²) improved the osteogenic differentiation of hPDLSCs induced by high glucose (P＜0.05), increased the mRNA and protein levels of ALP, RUNX2 and OSX (P＜0.05), upregulated the mRNA expression levels of SOD2 and CAT (P＜0.05), reduced the levels of ROS (P＜0.05), and reduced TNF-α and IL-1β levels in the cell supernatants (P＜0.05). When ML385 was added to inhibit the NRF2 pathway, the ALP activity of cells was decreased (P＜0.05); the gene expression levels of ALP, RUNX2 and OSX were downregulated (P＜0.05); the protein level of KEAP1 was upregulated (P＜0.05); and the protein levels of NRF2 and HO-1 were downregulated (P＜0.05).

Red LED light may promote the proliferation and osteoblastic differentiation of hPDLSCs induced by high glucose through the KEAP1-NRF2/HO-1 pathway and reduce the oxidative stress damage to hPDLSCs induced by high glucose.