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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 |

REPOSITORIES: biostudies-literature

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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:Objective In periodontitis, excessive oxidative stress combined with subsequent apoptosis and cell death further exacerbated periodontium destruction. TRPA1, an important transient receptor potential (TRP) cation channel, may participate in the process. This study is aimed at exploring the role and the novel therapeutic function of TRPA1 in periodontitis. Methods Periodontal ligament cells or tissues derived from healthy and periodontitis (PDLCs/Ts and P-PDLCs/Ts) were used to analyze the oxidative and apoptotic levels and TRPA1 expression. TRPA1 inhibitor (HC030031) was administrated in inflammation induced by P. gingivalis lipopolysaccharide (P.g.LPS) to investigate the oxidative and apoptotic levels of PDLCs. The morphology of the endoplasmic reticulum (ER) and mitochondria was identified by transmission electron microscope, and the PERK/eIF2α/ATF-4/CHOP signal pathways were detected. Finally, HC030031 was administered to periodontitis mice to evaluate its effect on apoptotic and oxidative levels in the periodontium and the relieving of periodontitis. Results The oxidative, apoptotic levels and TRPA1 expression were higher in P-PDLC/Ts from periodontitis patients and in P.g.LPS-induced inflammatory PDLCs. TRPA1 inhibitor significantly decreased the intracellular calcium, oxidative stress, and apoptosis of inflammatory PDLCs and decreased ER stress by downregulating PERK/eIF2α/ATF-4/CHOP pathways. Meanwhile, the overall calcium ion decrease induced by EGTA also exerted similar antiapoptosis and antioxidative stress functions. In vivo, HC030031 significantly reduced oxidative stress and apoptosis in the gingiva and periodontal ligament, and less periodontium destruction was observed. Conclusion TRPA1 was highly related to periodontitis, and TRPA1 inhibitor significantly reduced oxidative and apoptotic levels in inflammatory PDLCs via inhibiting ER stress by downregulating PERK/eIF2α/ATF-4/CHOP pathways. It also reduced the oxidative stress and apoptosis in periodontitis mice thus ameliorating the development of periodontitis.

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

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