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/cm^(2)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,q RT-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/cm^(2)辐射曝光量进行后续实验(P<0.05)。5 J/cm^(2)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。
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/cm^(2)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;q RT-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;q RT-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/cm^(2)was chosen for subsequent experiments.Red LED light irradiation(5 J/cm^(2))improved the osteogenic differentiation of h PDLSCs induced by high glucose(P<0.05),increased the mRNA and protein levels of ALP,RUNX2 and OSX(P<0.05),upregulated the m RNA 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 h PDLSCs induced by high glucose through the KEAP1-NRF2/HO-1pathway and reduce the oxidative stress damage to hPDLSCs induced by high glucose.
作者
姜冰
冯茂耕
郑艮子
刘源
李昊
王瑶
JIANG Bing;FENG Maogeng;ZHENG Genzi;LIU Yuan;LI Hao;WANG Yao(Department of Preventive Health Care,The Affiliated Stomatological Hospital of Southwest Medical University,Luzhou 646000,China;Oral&Maxillofacial Reconstruction and Regeneration of Luzhou Key Laboratory,Luzhou 646000,China;Insti-tute of Stomatology,Southwest Medical University,Luzhou 646000,China;Department of Oral and Maxillofacial Sur-gery,The Affiliated Stomatological Hospital of Southwest Medical University,Luzhou 646000,China)
出处
《口腔疾病防治》
2023年第6期389-399,共11页
Journal of Prevention and Treatment for Stomatological Diseases
基金
四川省自然科学基金面上项目(2022NSFSC0599)
四川省科技计划联合创新专项项目(2022YFS0634)
四川省医学科研课题计划(S21015)
西南医科大学校级科研项目(2021ZKMS013)。
关键词
发光二极管
人牙周膜干细胞
高糖
成骨分化
活性氧簇
抗氧化
Kelch样ECH相关蛋白1
核因子E2相关因子2
血红素加氧酶-1
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