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Doctoral Thesis
DOI
https://doi.org/10.11606/T.25.2021.tde-07022022-111623
Document
Author
Full name
Tatiana Martini
Institute/School/College
Knowledge Area
Date of Defense
Published
Bauru, 2021
Supervisor
Committee
Buzalaf, Marilia Afonso Rabelo (President)
Cardoso, Cristiane de Almeida Baldini
Lima, Rafael Rodrigues
Silva, Thiago Cruvinel da
Title in English
Proteomics of acquired enamel pellicle and saliva in patients with gastroesophageal reflux disease with or without erosive tooth wear and in vitro evaluation of the protective potential of hemoglobin against initial erosion
Keywords in English
Acquired enamel pellicle
Gastroesophageal reflux
Hemoglobin
Proteomics
Tooth erosion
Abstract in English
This study compared the proteomic profile of the acquired enamel pellicle (AEP) of the palatal/lingual region and unstimulated saliva of patients (1) with gastroesophageal reflux disease (GERD) and erosive tooth wear (ETW) (BEWE, score 9; GE Group); (2) with GERD without ETW (BEWE = 0; GNE group); (3) without GERD without ETW (BEWE=0, Control group) using label-free shotgun quantitative proteomic analysis (nLC-ESI-MS / MS). Since in preliminary experiments we observed increased expression of different Hb subunits in patients with GERD without ETW when compared with patients with GERD and ETW, we also evaluated, in vitro, the potential of AEP modification with different concentrations of hemoglobin (Hb) to protect enamel against initial erosion. After prophylaxis, AEP was allowed to form for 120 min. The AEP was collected from the palatal/lingual surface with filter paper soaked in 3% citric acid. After extraction, proteins were identified by nLC-ESI-MS / MS. A total of 213 proteins were identified in the AEP. The GNE group had a high number of phosphorylated and calcium-binding proteins, in addition to Spectrin beta-chain isoforms found exclusively in this group. Regarding the proteomics of saliva, when the GNE group was compared with the GE group, a remarkable increase in distinct Hb subunits was observed (up to 22 times). Other increased proteins were Serum albumin (4.5-fold) and type II cytoskeletal keratin isoforms (more than 3-fold). Proteinase inhibitors, such as Alpha-1-antitrypsin and Alpha-2-macroglobulin, were identified exclusively in the GNE group. In the in vitro study, Hb at 1.0 and 4.0 mg/mL significantly reduced initial enamel erosion after 3 days. These results indicate that many proteins may be involved in the resistance of GERD patients to the ETW, with special emphasis on hemoglobin, in addition to intracellular proteins that have the potential to bind to hydroxyapatite or participate in the formation of supramolecular aggregates. These proteins may be candidates for inclusion in dental products for protection against ETW. Data from the in vitro study indicate that changing the composition of AEP by enriching with Hb at 1.0 mg/mL might be a good strategy for protection against intrinsic erosion.
Title in Portuguese
Proteoma da película adquirida do esmalte e saliva em pacientes com doença do refluxo gastroesofágico com ou sem desgaste dentário erosivo e avaliação in vitro do potencial protetor da hemoglobina contra a erosão inicial
Keywords in Portuguese
Erosão dentária
Hemoglobina
Película adquirida
Proteômica
Refluxo gastroesofágico
Abstract in Portuguese
Este estudo comparou o perfil proteômico da película adquirida do esmalte (PAE) da região palatina/lingual e saliva não estimulada de pacientes (1) com doença do refluxo gastresofágico (DRGE) e desgaste dentário erosivo (DDE) (BEWE, score 9; Grupo RE); (2) com DRGE e sem DDE (BEWE = 0; grupo RSE); c) sem DRGE e sem DDE (BEWE=0, grupo Controle) usando análise proteômica quantitativa livre de marcadores shotgun (nLC-ESI-MS / MS). Também foi avaliado, in vitro, o potencial da modificação do PAE com diferentes concentrações de hemoglobina (Hb) para proteger contra a erosão inicial do esmalte, uma vez que em experimentos preliminares observamos aumento da expressão de diferentes subunidades de Hb em pacientes com DRGE sem DDE, quando comparados com pacientes com DRGE e com DDE. A PAE foi formada 120 min após a profilaxia. A película foi coletada da superfície palatina/lingual com papel de filtro umedecido em ácido cítrico a 3%. Ao todo, foram identificadas 213 proteínas na PAE. O grupo RSE apresentou um alto número de proteínas fosforiladas e de ligação ao cálcio, além de isoformas de Spectrin beta-chain encontradas exclusivamente neste grupo. Em relação à análise proteômica da saliva, quando o grupo RSE foi comparado com o grupo RE, foi observado um aumento notável em subunidades distintas de Hb (até 22 vezes). Outras proteínas aumentadas foram Serum albumin (4,5 vezes) e isoformas de Cytoskeletal keratin II (mais de 3 vezes). Os inibidores de protease, como Alfa-1-antitripsin e Alfa-2-macroglobulin, foram identificados exclusivamente no grupo RSE. No estudo in vitro, a Hb nas concentrações de 1,0 e 4,0 mg / mL reduziu significativamente a erosão inicial do esmalte após 3 dias. Estes resultados indicam que muitas proteínas podem estar envolvidas na resistência de pacientes com DRGE à ocorrência do DDE, com especial destaque para a hemoglobina, além das proteínas intracelulares que têm potencial para se ligar à hidroxiapatita ou participar da formação de agregados supramoleculares. Dessa forma, essas proteínas podem ser candidatas a serem inclusas em produtos odontológicos para proteção contra o DDE. Os dados do estudo in vitro indicam que a alteração na composição da PAE, pelo enriquecimento com Hb a 1,0 mg/mL, pode ser uma boa estratégia para proteção contra a erosão intrínseca.
 
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Publishing Date
2022-02-09
 
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