Peptídeos catiônicos e flavonoides como alternativas biológicas para aplicação em odontologia: efeito antimicrobiano, citotóxico e na indução de mineralização / Cationic peptides and flavonoids as biological alternatives in dentistry: antimicrobial, cytotoxic and mineralization induction effects

O presente estudo avaliou o efeito antimicrobiano, citotóxico e na indução de mineralização de peptídeos catiônicos e flavonoides para aplicação em Odontologia. Para isso, foram realizados 7 estudos que neste trabalho foram divididos em capítulos. Os capítulos 1 e 2 tiveram por objetivo avaliar a toxicidade em células epiteliais (linhagens HaCat e OBA-9) e o efeito de fragmentos de peptídeos catiônicos sobre bactérias cariogênicas em condições planctônicas e sobre biofilme e posteriormente inserir o fragmento peptídico com melhor efeito antimicrobiano em um sistema líquido cristalino (SLC), previamente caracterizado por microscopia de luz polarizada, reologia e ensaio de bioadesão, e avaliar seu efeito sobre biofilme de Streptococcus mutans. Os capítulos 3 e 4 avaliaram a toxicidade em fibroblastos (linhagem 1929) e a atividade antimicrobiana/antibiofilme de análogos de peptídeos catiônicos sobre microrganismos de interesse endodôntico e seu efeito sobre os marcadores de mineralização dentinária: atividade da fosfatase alcalina (ALP), formação de nódulos de mineralização (NM) e expressão dos genes DSPP ­ sialofosfoproteína dentinária e DMP-I ­ fosfoproteína da matriz dentinária) em células semelhantes a odontoblastos da linhagem MDPC-23. O capítulo 5 teve por objetivo avaliar a atividade antimicrobiana/antibiofilme dos flavonoides: taxifolina, galangina, pinocembrina e crisina e sua toxicidade sobre fibroblastos L929. Na sequência, os capítulos 6 e 7 avaliaram o potencial da taxifolina sobre a indução de marcadores de mineralização dentinária (ALP, NM, expressão dos genes DSPP e DMP-I) nas células MDPC-23 e óssea (ALP, NM e expressão dos genes ALP e COL-I ­ colagenase 1) nas células semelhantes a osteoblastos da linhagem Saos-2, utilizando tratamentos de 24, 72h e contínuo. Os resultados dos capítulos 1 e 2 mostraram que, dentre os peptídeos testados, DI-23, fragmento da porção N-teminal do Defb14-1CV (ortólogo do hBD-3), apresentou o melhor efeito sobre cultura de S. mutans, S. mitis and S. salivarius e potente ação contra biofilme de cepas padrão e clínicas de S. mutans, além de baixa toxicidade em células epiteliais. Na presença de saliva, o SLC demonstrou alta viscosidade e altos valores de bioadesão. O SLC contendo DI-23 apresentou melhor efeito antibiofilme após 24h quando comparado à 4h de tratamento e não apresentou citoxicidade, nas condições testadas. Os resultados dos capítulos 3 e 4 mostraram que os peptídeos afetaram o metabolismo de L929 em concentrações acima de 500 gg/mL. KR-12-a5 inibiu o crescimento de todos os microrganismos testados, em baixas concentrações, e reduziu significativamente os biofilmes de S. mutans e Enterococcus faecalis formados nos túbulos dentinários. KR-12-a5 também estimulou a deposição inicial de NM, similar aos outros peptídeos, entretanto, em concentrações mais baixas e minimamente tóxicas. Nenhum peptídeo influenciou a expressão de DMP-I e DSPP nas células MDPC-23, nas concentrações testadas. No capítulo 5, a taxifolina não causou toxicidade sobre 1929 em nenhuma concentração testada e apresentou o melhor efeito inibitório sobre cultura de E. faecalis, S. mutans e Actinomyces israelii. Esse flavonoide também foi capaz de eliminar biofilmes de E. faecalis e S. mutans e reduzir significativamente biofilme de A. israelii em placas de poliestireno, além de reduzir substanciamente biofilme de E. faecalis no interior dos túbulos dentinários. No capítulo 6, o tratamento com taxifolina a 10gM por 72h estimulou a atividade de ALP, a formação de NM e a expressão de DMP-I. Taxifolina a 5gM por 24h estimulou a expressão de DSPP nas células MDPC-23. No capítulo 7, taxifolina a 10gM por 24h estimulou a atividade e a expressão de ALP e COL-I e taxifolina a 10gM a por 72h estimulou a formação de NM e também a expressão de COL-I. Conclui-se que os peptídeos DI-23 e KR-12-a5 são potentes agentes antimicrobianos e antibiofilme. A taxifolina, além da ação antimicrobiana/antibiofilme, também demonstrou capacidade de indução de marcadores fenotípicos e genotípicos de mineralização dentinária e óssea, indicando seu potencial uso em Odontologia(AU)

The present study evaluated the antimicrobial, cytotoxic and mineralization induction effects of cationic peptides and flavonoids for application in Dentistry. Seven studies were carried out and divided into chapters for this work. Chapters 1 and 2 aimed to evaluate the toxicity in epithelial cells (HaCat and OBA-9 lines) and the effect of cationic peptide fragments on cariogenic bacteria under planktonic and biofilm conditions and the peptide fragment with the best effect was incorporated in a crystalline liquid system (SLC) previously characterized by polarized light microscopy, rheology and bioadhesion assay, and to evaluate its effect on biofilm of clinical and standard strains of S. mutans. Chapters 3 and 4 evaluated the toxicity on fibroblasts (line L929) and the antimicrobial/antibiofilm activity of analogs of cationic peptides on microorganisms of endodontic interest and their effect on the markers of dentin mineralization: alkaline phosphatase activity (ALP), formation of mineralization nodules (MN) and expression of DSPP dentin sialophosphoprotein and DMP-I - dentin matrix phosphoprotein genes) in MDPC-23 odontoblasts-like cells. The objective of chapter 5 was to evaluate the antimicrobial/antibiofilm activity of the flavonoids: taxifolin, galangin, pinocembrin and chrysin and their toxicity on L929 fibroblasts. In addition, chapters 6 and 7 evaluated the potential of taxifolin on the induction of dentin mineralization markers (ALP, MN, DSPP and DMP-I gene expression) in MDPC-23 and bone mineralization markers (ALP, MN, ALP and COL-1-collagenase 1 gene expression) in the osteoblast-like cells of Saos-2 line, using 24h, 72h and continuous treatments. The results of chapters 1 and 2 showed that, among the peptides tested, DI-23, fragment of the N-terminal portion of Defb14-1CV (hBD-3 ortholog), showed the best effect on culture of S. mutans, S. mitis and S. salivarius and potent biofilm action on standard and clinical strains of S. mutans, as well as low toxicity in epithelial cells. In the presence of saliva, SLC demonstrated high viscosity and high bioadhesion values. SLC containing DI-23 presented better antibiofilm effect after 24h when compared to 4h of treatment and did not present cytotoxicity under the conditions tested. The results of chapters 3 and 4 showed that peptides affected the metabolism of L929 at concentrations above 500 gg/mL. KR-12-a5 inhibited the growth of all tested microorganisms at low concentrations and significantly reduced the biofilms of S. mutans and E. faecalis formed in dentin tubules. KR-12-a5 also stimulated the initial deposition of MN, similar to the other peptides, however, in lower and minimally toxic concentrations. No peptide influenced the expression of DMP-I and DSPP in MDPC-23 cells. In Chapter 5, taxifolin no cause toxicity on L929 at any tested concentration and it had the best effect against culture of Actinomyces israelii, S. mutans and E. faecalis. This flavonoid was also able to eliminate biofilms of E. faecalis and S. mutans and significantly reduce A. israelii biofilm on polystyrene plates, besides substantially reducing E. faecalis biofilms within dentinal tubules. In chapter 6, treatment with taxifolin at IOgM for 72h stimulated ALP activity, MN formation and DMP-I expression. Taxifolin at 51.1Mfo r 24h stimulated DSPP expression in MDPC-23 cells. In chapter 7, taxifolin at 10gM for 24h stimulated the activity and expression of ALP and COL-I and taxifolin at 10gM for 72h stimulated MN formation and also expression of COL-I. It is concluded that peptides DI-23 and KR-12-a5 are potent antimicrobial and antibiofilm agents. Taxifolin, besides the antimicrobial/antibiofilm action, also demonstrated the capacity for induction of phenotypic and genotypic markers of dentin and bone mineralization, indicating their potential use in Dentistry(AU)

Determination of disulfide bridges of two spider toxins: hainantoxin-III and hainantoxin-IV

Peptide toxins are usually highly bridged proteins with multipairs of intrachain disulfide bonds. Analysis of disulfide connectivity is an important facet of protein structure determination. In this paper, we successfully assigned the disulfide linkage of two novel peptide toxins, called HNTX-III and HNTX-IV, isolated from the venom of Ornithoctonus hainana spider. Both peptides are useful inhibitors of TTX-sensitive voltage-gated sodium channels and are composed of six cysteine residues that form three disulfide bonds, respectively. Firstly, the peptides were partially reduced by tris(2-carboxyethyl)-phosphine (TCEP) in 0.1 M citrate buffer containing 6 M guanidine-HCl at 40° C for ten minutes. Subsequently, the partially reduced intermediates containing free thiols were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and alkylated by rapid carboxamidomethylation. Then, the disulfide bonds of the intermediates were analyzed by Edman degradation. By using the strategy above, disulfide linkages of HNTX-III and HNTX-IV were determined as I-IV, II-V and III-VI pattern. In addition, this study also showed that this method may have a great potential for determining the disulfide bonds of spider peptide toxins.(AU)

Behavioral and electroencephalographic analysis of seizures induced by intrahippocampal injection of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera

In this study, the behavioral and electroencephalographic (EEG) analysis of seizures induced by the intrahippocampal injection in rats of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera, was determined. The first alterations occurred during microinjection of granulitoxin (8 µg) into the dorsal hippocampus and consisted of seizure activity that began in the hippocampus and spread rapidly to the occipital cortex. This activity lasted 20-30 s, and during this period the rats presented immobility. During the first 40-50 min after its administration, three to four other similar short EEG seizure periods occurred and the rats presented the following behavioral alterations: akinesia, facial automatisms, head tremor, salivation, rearing, jumping, barrel-rolling, wet dog shakes and forelimb clonic movements. Within 40-50 min, the status epilepticus was established and lasted 8-12 h. These results are similar to those observed in the acute phase of the pilocarpine model of temporal lobe epilepsy and suggest that granulitoxin may be a useful tool not only to study the sodium channels, but also to develop a new experimental model of status epilepticus.

Partial sequence and toxic effects of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera

A neurotoxic peptide, granulitoxin (GRX), was isolated from the sea anemone Bunodosoma granulifera. The N-terminal amino acid sequence of GRX is AKTGILDSDGPTVAGNSLSGT and its molecular mass is 4958 Da by electrospray mass spectrometry. This sequence presents a partial degree of homology with other toxins from sea anemones such as Bunodosoma caissarum, Anthopleura fuscoviridis and Anemonia sulcata. However, important differences were found: the first six amino acids of the sequence are different, Arg-14 was replaced by Ala and no cysteine residues were present in the partial sequence, while two cysteine residues were present in the first 21 amino acids of other toxins described above. Purified GRX injected ip (800 µg/kg) into mice produced severe neurotoxic effects such as circular movements, aggressive behavior, dyspnea, tonic-clonic convulsion and death. The 2-h LD50 of GRX was 400 ñ 83 µg/kg