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Nrf2 Inhibits Periodontal Ligament Stem Cell Apoptosis under Excessive Oxidative Stress.

ABSTRACT: The present study aimed to analyze novel mechanisms underlying Nrf2-mediated anti-apoptosis in periodontal ligament stem cells (PDLSCs) in the periodontitis oxidative microenvironment. We created an oxidative stress model with H₂O₂-treated PDLSCs. We used real-time PCR, Western blotting, TUNEL staining, fluorogenic assay and transfer genetics to confirm the degree of oxidative stress and apoptosis as well as the function of nuclear factor-erythroid 2-related factor 2 (Nrf2). We demonstrated that with upregulated levels of reactive oxygen species (ROS) and malondialdehyde (MDA), the effect of oxidative stress was obvious under H₂O₂ treatment. Oxidative molecules were altered after the H₂O₂ exposure, whereby the signaling of Nrf2 was activated with an increase in its downstream effectors, heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1) and γ-glutamyl cysteine synthetase (γ-GCS). Additionally, the apoptosis levels gradually increased with oxidative stress by the upregulation of caspase-9, caspase-3, Bax and c-Fos levels in addition to the downregulation of Bcl-2. However, there was no alterations in levels of caspase-8. The enhanced antioxidant effect could not mitigate the occurrence of apoptosis. Furthermore, Nrf2 overexpression effectively improved the anti-oxidative levels and increased cell proliferation. At the same time, overexpression effectively restrained TUNEL staining and decreased the molecular levels of caspase-9, caspase-3, Bax and c-Fos, but not that of caspase-8. In contrast, silencing the expression of Nrf2 levels had the opposite effect. Collectively, Nrf2 alleviates PDLSCs via its effects on regulating oxidative stress and anti-intrinsic apoptosis by the activation of oxidative enzymes.

SUBMITTER: Liu Y

PROVIDER: S-EPMC5454985 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Nrf2 Inhibits Periodontal Ligament Stem Cell Apoptosis under Excessive Oxidative Stress.

Liu Yanli Y Yang Hongxu H Wen Yi Y Li Bingyi B Zhao Yinhua Y Xing Jing J Zhang Min M Chen Yongjin Y

International journal of molecular sciences 20170517 5

The present study aimed to analyze novel mechanisms underlying Nrf2-mediated anti-apoptosis in periodontal ligament stem cells (PDLSCs) in the periodontitis oxidative microenvironment. We created an oxidative stress model with H₂O₂-treated PDLSCs. We used real-time PCR, Western blotting, TUNEL staining, fluorogenic assay and transfer genetics to confirm the degree of oxidative stress and apoptosis as well as the function of nuclear factor-erythroid 2-related factor 2 (Nrf2). We demonstrated that ...[more]

PMID: 28513573

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Project description:The periodontal ligament (PDL) is a specialized connective tissue that provides structural support to the tooth and is crucial for oral functions. The mechanical properties of the PDL are mainly derived from the tissue-specific composition and structural characteristics of the extracellular matrix (ECM). The ECM also plays key roles in determining cell fate in the cellular microenvironment thus crucial in the PDL tissue homeostasis. In the present study, we determined the comprehensive ECM profile of mouse molar PDL using laser microdissection and mass spectrometry-based proteomic analysis with ECM-oriented data curation. Additionally, we evaluated changes in the ECM proteome under mechanical loading using a mouse orthodontic tooth movement (OTM) model and analyzed potential regulatory networks using a bioinformatics approach. Proteomic changes were evaluated in reference to the novel second harmonic generation (SHG)-based fiber characterization. Our ECM-oriented proteomics approach succeeded in illustrating the comprehensive ECM profile of the mouse molar PDL. We revealed the presence of type II collagen in PDL, possibly associated with the load-bearing function upon occlusal force. Mechanical loading induced unique architectural changes in collagen fibers along with dynamic compositional changes in the matrisome profile, particularly involving ECM glycoproteins and matrisome-associated proteins. We identified several unique matrisome proteins which responded to the different modes of mechanical loading in PDL. Notably, the proportion of type VI collagen significantly increased at the mesial side, contributing to collagen fibrogenesis. On the other hand, type XII collagen increased at the PDL-cementum boundary of the distal side. Furthermore, a multifaceted bioinformatics approach illustrated the potential molecular cues, including PDGF signaling, that maintain ECM homeostasis under mechanical loading. Our findings provide fundamental insights into the molecular network underlying ECM homeostasis in PDL, which is vital for clinical diagnosis and development of biomimetic tissue-regeneration strategies.

Project description:BackgroundPeriodontitis is a highly prevalent dental disease which is associated with diabetes and is challenging to cure in diabetic patients. However, the mechanism of comorbid diabetes and periodontitis is still unclear. This study aimed to uncover the role of endoplasmic reticulum (ER) stress in high glucose-associated periodontitis.MethodsPeriodontal tissues were obtained from diabetic patients with periodontitis, periodontitis patients without systemic disease, and healthy teeth. The expressions of ER stress-related factors GRP78, ATF6, PERK and XBP1 were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immumohistochemical staining. Periodontal ligament stem cells (PDLSCs) from three states of periodontal tissues were isolated and cultured as diabetic PDLSCs (dPDLSCs), inflamed PDLSCs (iPDLSCs) and healthy PDLSCs (hPDLSCs), and the cell stemness was assayed. Different concentrations (8, 11, and 25 mmol/L) of D-glucose were used on hPDLSCs to simulate high glucose microenvironment. The changes of osteogenic ability of PDLSCs were observed, and the expressions of ER stress-related factors in different time point (6, 12, 24, and 72 h) were detected. Finally, GRP78 shRNA lentivirus was used to block ER stress on PDLSCs in the 25 mmol/L D-glucose microenvironment, and the osteogenic ability of PDLSCs was observed.ResultsThe results showed that the expressions of GRP78, ATF6, PERK, and XBP1 were highest in the diabetic periodontitis group and lowest in the healthy periodontal tissue group (P<0.05). The clone formation, osteogenic and lipogenic differentiation abilities were lowest in dPDLSCs and highest in hPDLSCs. With the increase of glucose concentration, the osteogensis ability of PDLSCs decreased. After 6 hours of stimulation with D-glucose 25 mmol/L, the ER stress pathways in PDLSCs were effectively activated, and the peak value was reached at 12 hours. The decrease in the osteogensis ability of PDLSCs in a high glucose microenvironment reversed when ER stress was blocked.ConclusionsThe osteogenic differentiation ability of PDLSCs cells is inhibited in a high glucose microenvironment, and this effect is realized by ER stress activation. Blocking ER stress can partially restore the reduced osteogenic ability of PDLSCs. These results suggest that high glucose inhibits the osteogenic differentiation ability of PDLSCs by activating ER stress, which ultimately exacerbates periodontitis.

Dataset Information

Nrf2 Inhibits Periodontal Ligament Stem Cell Apoptosis under Excessive Oxidative Stress.

Publications

Nrf2 Inhibits Periodontal Ligament Stem Cell Apoptosis under Excessive Oxidative Stress.

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