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 / 口腔疾病防治

ABSTRACT

Objective@# 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.@*Methods@#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.@*Results @# 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)@*Conclusion@#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.