Alterações macromoleculares da matriz extracelular em córneas com ceratocone e após crosslinking com extrato de açaí (Euterpe oleracea): um estudo ex vivo e in vitro
Arquivos
Data
2024-09-18
Autores
Peres, Murilo Bertazzo 
Orientadores
Campos, Mauro Silveira de Queiroz
Tipo
Tese de doutorado
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Título de Volume
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Objetivo: Analisar os proteoglicanos e outras macromoléculas de matriz extracelular em córneas humanas normais (NL) e com ceratocone (KC), antes e após tratamento com crosslinking (CXL) e avaliar a ação e toxicidade do extrato de açaí como um potencial agente de CXL. Métodos: Córneas descartadas de banco de olhos (grupo NL) e provenientes de transplante óptico em paciente com ceratocone (grupo KC) foram obtidas para estudo. Para análise ex vivo, parte das córneas foram fixadas e preparadas para avalição da imunofluorescência em microscopia confocal. Outra parte foi submetida à extração das macromoléculas com hidrocloreto de guanidina (GuHCl) para dosagem de proteínas, identificação por eletroforese em gel de agarose, em gel de poliacrilamida com sodium dodecyl sulfate (SDS-PAGE) e western blot, e quantificação por enzyme-linked immunosorbent assay (ELISA). Para a identificação da ação do extrato de açaí como agente indutor de CXL, foram preparados modelos experimentais de discos de colágeno feitos de gelatina, tratados com diferentes concentrações do extrato, para avaliar a resistência desses discos à digestão pela colagenase. Para análise in vitro, foram realizadas culturas primárias de ceratócitos do grupo NL e KC em meio Dulbecco's Modified Eagle Medium (DMEM)/Ham's F-12 nutrient mixture (F-12) com soro fetal bovino 2%. Os ceratócitos foram submetidos a tratamento com extrato de açaí em diferentes concentrações (2%, 4% e 8%) e tempos de exposição (30 min , 1 h, 2 h) e, após 24 h, a viabilidade celular foi avaliada (MTT e azul tripan) e comparada com os controles e com o tratamento padrão com riboflavina 0,1% e luz ultravioleta 3 mW/cm2 por 30 min (RBUV). Macromoléculas dos ceratócitos de ambos os grupos foram avaliadas por imunofluorescência antes e após 24 h do tratamento com CXL. Resultados: Na análise ex vivo das córneas extraídas (N=8 para NL e N=12 para KC), não houve diferença em relação ao sexo, lateralidade, peso úmido, somente em relação à idade (NL – 68 anos, KC – 30 anos). No western blot, detectou-se marcação para queratam sulfato (KS), decorim e lumicam em ambos os grupos, com diferença estrutural no decorim no grupo KC, que apresentou maior peso molecular. A média da dosagem total de proteínas extraídas por peso úmido de córnea foi estatisticamente menor no grupo KC. As médias das dosagens por ELISA do decorim, lumicam e KS não apresentaram diferença entre os grupos quando normalizadas por peso úmido, mas apresentaram uma maior concentração significativa no grupo KC, quando normalizadas por μg de proteína extraída. Na imunofluorescência, observou-se distribuição semelhante dessas macromoléculas no estroma de ambos os grupos. Nos modelos experimentais de colágeno, observou-se uma maior resistência dos discos à digestão da colagenase, quanto maior foi a concentração do tratamento com extrato de açaí. No estudo in vitro de viabilidade pelo MTT (NL N=3 e KC N=2), observou-se que, em ambos os grupos, apenas houve menor viabilidade média significativa em relação ao controle no tratamento 8% por 2 h. Quando comparada a viabilidade celular por MTT (N=3 para NL e KC) para o tratamento com extrato de açaí 4% por 2 h com o tratamento padrão (RBUV), observou-se menor viabilidade média significativa em ambos os grupos no tratamento RBUV em relação ao açaí e em relação ao controle. O teste azul tripan mostrou resultados semelhantes. Na avaliação por imunofluorescências dos ceratócitos de ambos os grupos, antes e após tratamento com extrato de açaí, observou-se que os filamentos de actina do citoesqueleto aparecem com um padrão organizado em rede e mais cortical no grupo NL, e um padrão em feixe (fibras de estresse) no grupo KC, especialmente após o tratamento com extrato do açaí. Conclusões: Nas córneas com KC, ocorre diminuição de proteínas não colágenas, mas não às custas do decorim, lumicam e KS, que estão preservados, e pode haver uma maior glicosilação no decorim. O tratamento com extrato de açaí diminui a ação da colagenase em discos de colágeno, indicando capacidade de realizar CXL. In vitro, o extrato de açaí tem menor toxicidade aos ceratócitos, em comparação com o tratamento com RBUV. Os ceratócitos de córnea com ceratocone in vitro apresentam uma organização de citoesqueleto mais típica de ceratócitos ativados, principalmente após o CXL.
Objective: To analyze proteoglycans and other extracellular matrix macromolecules in normal (NL) and keratoconus (KC) human corneas, before and after crosslinking treatment (CXL) and to evaluate açaí extract action and toxicity as a potential CXL agent. Methods: Discarded corneas from eye banks (NL group) and cornea from penetrating keratoplasty in a patient with keratoconus (KC group) were obtained. For ex vivo analysis, parts of the corneas were fixed and prepared for immunofluorescence in confocal microscopy, other parts were subjected to macromolecules extraction with GuHCl for protein dosage, identification by agarose gel electrophoresis, polyacrylamide gel with sodium dodecyl sulfate (SDS-PAGE) and western blot, and quantification by enzyme-linked immunosorbent assay (ELISA). To evaluate the açaí extract as a CXL agent, experimental models of collagen discs made of gelatin treated with different concentrations of the extract were prepared to assess the resistance of these discs against collagenase digestion. For in vitro analysis, primary cultures of NL and KC keratocytes were performed in DMEM/F12 medium with 2% fetal bovine serum. The keratocytes were submitted to treatment with açaí extract in different concentrations (2%, 4% and 8%) and exposure times (30 min, 1 h, 2 h) and after 24 h the cell viability was evaluated with MMT and trypan blue assay comparing with the untreated controls and with the standard treatment with 0.1% riboflavin and ultraviolet light 3 mW/cm2 for 30 min (RBUV). Keratocyte macromolecules from both groups were evaluated by immunofluorescence before and after 24 h of CXL treatment. Results: In the ex vivo analysis of the extracted corneas (N=8 for NL and N=12 for KC) there was no difference in sex, laterality, or wet weight, only in age (NL 68 years, KC 30 years). In the western blot, staining for keratan sulfate (KS), decorin, and lumican was detected in both groups, with structural differences in decorin in the KC group, presenting higher molecular weight. The mean total dosage of extracted proteins per corneal wet weight was statistically lower in the KC group. The mean ELISA dosage of decorin, lumicam, and KS did not show any difference between the groups when normalized by wet weight, but it showed a higher concentration in the KC group when normalized by μg of extracted protein. Immunofluorescence showed a similar distribution of these macromolecules in the stroma of both groups. In the experimental models of collagen, it was observed that the discs had greater resistance to collagenase digestion with higher concentrations of açaí extract treatment. In the in vitro viability assay by MMT (NL N=3 and KC N=2) it was observed that in both groups there was only a significantly lower mean viability compared to the control in the 8% 2 h treatment. In the MMT assay comparing cell viability in both groups (N=3 for NL and KC) between açaí (4%, 2 h) with the standard treatment (RBUV), a significant mean lower viability was observed in both groups in the RBUV treatment than the açaí and the control. The blue trypan test showed similar results. In the immunofluorescence evaluation of the keratocytes of both groups, before and after treatment with açaí extract, it was observed that the actin filaments of the cytoskeleton showed a more cortical and network pattern in the NL group and a bundle pattern (stress fibers) in the KC group, especially after treatment with açaí extract. Conclusions: In KC corneas there is a decrease in non-collagen proteins without affecting the levels of decorin, lumican, and KS, which are preserved and decorin may be more glycosylated. Treatment with açaí extract decreases the action of collagenase on collagen discs, indicating the ability to perform CXL. In vitro, acai extract has lower keratocyte toxicity compared to RBUV treatment. Corneal keratocytes with keratoconus in vitro have a cytoskeletal organization more typical of activated keratocytes, especially after CXL.
Objective: To analyze proteoglycans and other extracellular matrix macromolecules in normal (NL) and keratoconus (KC) human corneas, before and after crosslinking treatment (CXL) and to evaluate açaí extract action and toxicity as a potential CXL agent. Methods: Discarded corneas from eye banks (NL group) and cornea from penetrating keratoplasty in a patient with keratoconus (KC group) were obtained. For ex vivo analysis, parts of the corneas were fixed and prepared for immunofluorescence in confocal microscopy, other parts were subjected to macromolecules extraction with GuHCl for protein dosage, identification by agarose gel electrophoresis, polyacrylamide gel with sodium dodecyl sulfate (SDS-PAGE) and western blot, and quantification by enzyme-linked immunosorbent assay (ELISA). To evaluate the açaí extract as a CXL agent, experimental models of collagen discs made of gelatin treated with different concentrations of the extract were prepared to assess the resistance of these discs against collagenase digestion. For in vitro analysis, primary cultures of NL and KC keratocytes were performed in DMEM/F12 medium with 2% fetal bovine serum. The keratocytes were submitted to treatment with açaí extract in different concentrations (2%, 4% and 8%) and exposure times (30 min, 1 h, 2 h) and after 24 h the cell viability was evaluated with MMT and trypan blue assay comparing with the untreated controls and with the standard treatment with 0.1% riboflavin and ultraviolet light 3 mW/cm2 for 30 min (RBUV). Keratocyte macromolecules from both groups were evaluated by immunofluorescence before and after 24 h of CXL treatment. Results: In the ex vivo analysis of the extracted corneas (N=8 for NL and N=12 for KC) there was no difference in sex, laterality, or wet weight, only in age (NL 68 years, KC 30 years). In the western blot, staining for keratan sulfate (KS), decorin, and lumican was detected in both groups, with structural differences in decorin in the KC group, presenting higher molecular weight. The mean total dosage of extracted proteins per corneal wet weight was statistically lower in the KC group. The mean ELISA dosage of decorin, lumicam, and KS did not show any difference between the groups when normalized by wet weight, but it showed a higher concentration in the KC group when normalized by μg of extracted protein. Immunofluorescence showed a similar distribution of these macromolecules in the stroma of both groups. In the experimental models of collagen, it was observed that the discs had greater resistance to collagenase digestion with higher concentrations of açaí extract treatment. In the in vitro viability assay by MMT (NL N=3 and KC N=2) it was observed that in both groups there was only a significantly lower mean viability compared to the control in the 8% 2 h treatment. In the MMT assay comparing cell viability in both groups (N=3 for NL and KC) between açaí (4%, 2 h) with the standard treatment (RBUV), a significant mean lower viability was observed in both groups in the RBUV treatment than the açaí and the control. The blue trypan test showed similar results. In the immunofluorescence evaluation of the keratocytes of both groups, before and after treatment with açaí extract, it was observed that the actin filaments of the cytoskeleton showed a more cortical and network pattern in the NL group and a bundle pattern (stress fibers) in the KC group, especially after treatment with açaí extract. Conclusions: In KC corneas there is a decrease in non-collagen proteins without affecting the levels of decorin, lumican, and KS, which are preserved and decorin may be more glycosylated. Treatment with açaí extract decreases the action of collagenase on collagen discs, indicating the ability to perform CXL. In vitro, acai extract has lower keratocyte toxicity compared to RBUV treatment. Corneal keratocytes with keratoconus in vitro have a cytoskeletal organization more typical of activated keratocytes, especially after CXL.
Descrição
Citação
PERES, Murilo Bertazzo. Alterações macromoleculares da matriz extracelular em córneas com ceratocone e após crosslinking com extrato de açaí (Euterpe oleracea): um estudo ex vivo e in vitro. 2024. 108 f. Tese (Doutor em Oftalmologia) – Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP). São Paulo, 2024.