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

Abstract

摘要 目的 探讨Kelch样ECH相关蛋白1-核因子E2相关因子2/血红素加氧酶-1（Kelch-like ECH associated protein 1-nuclear factor erythroid 2-related factor 2/heme oxygenase-1，KEAP1-NRF2/HO-1）通路介导发光二极管（light-emitting diode，LED）红光对高糖诱导下人牙周膜干细胞（human periodontal ligament stem cells，hPDLSCs）成骨分化和氧化损伤的影响，为LED红光在细胞抗氧化损伤中的应用提供依据。方法 流式细胞术、碱性磷酸酶（alkaline phosphatase，ALP）染色和茜素红染色鉴定hPDLSCs；高糖预处理hPDLSCs 48 h，用1、3、5 J/cm2 LED红光照射细胞，CCK-8实验选择促细胞增殖率高的辐射曝光量进行后续实验。将hPDLSCs分为对照组、高糖组、高糖+光照组；ALP染色、ALP活性检测、茜素红染色和半定量分析检测成骨分化能力，qRT-PCR和Western blot检测细胞成骨相关基因ALP、Runt相关转录因子2（runt-related transcription factor 2，RUNX2）、成骨细胞特异性转录因子（osterix，OSX）基因和蛋白表达；qRT-PCR检测相关抗氧化酶基因超氧化物歧化酶2（superoxide dismutase 2，SOD2）、过氧化氢酶（catalase，CAT）表达；荧光显微镜观察和流式细胞术测定细胞内活性氧簇（reactive oxygen species，ROS）水平；ELISA检测细胞上清液中肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素-1β（interleukin-1β，IL-1β）水平。以NRF2特异性抑制剂ML385抑制NRF2通路，ALP染色、ALP活性检测细胞早期成骨分化能力，qRT-PCR检测早期成骨分化标志物ALP、RUNX2、OSX基因表达，Western blot检测细胞KEAP1、NRF2、HO-1蛋白表达水平。结果 选择促高糖诱导下hPDLSCs增殖率最高的5 J/cm2辐射曝光量进行后续实验（P<0.05）。5 J/cm2 LED红光促进高糖诱导下hPDLSCs的成骨分化（P<0.05），上调ALP、RUNX2、OSX的基因与蛋白表达（P<0.05），上调SOD2、CAT基因表达（P<0.05），降低细胞ROS水平（P<0.05），减少细胞上清液中TNF-α、IL-1β水平（P<0.05）。ML385抑制NRF2通路，细胞ALP活性降低（P<0.05），ALP、RUNX2、OSX基因表达下降（P<0.05），KEAP1蛋白表达上升（P<0.05），NRF2、HO-1蛋白表达下降（P<0.05）。结论 LED红光可能通过KEAP1-NRF2/HO-1通路促进高糖诱导下hPDLSCs增殖和成骨分化，减轻氧化损伤。 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.

Keywords

• light-emitting diode
• human periodontal ligament stem cells
• high glucose
• osteogenic differentiation
• reactive oxygen species
• antioxidant
• kelch-like ech associated protein 1
• nuclear factor erythroid 2-related factor 2
• heme oxygenase-1