Análise de proteômica do efeito do peptídeo citoprotetor E8 na resposta inflamatória induzida pela IL-1β em condrócitos humanos
Data
2023-03-16
Autores
Pazelli, Gabriela Simões 
Orientadores
Chudzinski-Tavassi, Ana Marisa
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: A osteoartrite é uma doença crônica degenerativa que afeta as articulações, causando inflamação. Os tratamentos atualmente disponíveis proporcionam um alívio temporário, portanto ainda é necessário desenvolver tratamentos eficientes. O screening em modelo de resposta inflamatória em condrócito humano de peptídeos derivados do Lopap, os quais conservam a atividade citoprotetora da proteína, indicou o peptídeo E8 como um dos mais promissores quanto ao efeito anti-inflamatório, o qual reduz a liberação de citocinas inflamatórias no modelo de estudo. Portanto, o estudo acerca da ação do peptídeo citoprotetor E8 possibilitará o desenvolvimento de novos fármacos mais específicos e eficientes para o tratamento de doenças inflamatórias como a osteoartrite, sendo necessário entender como o peptídeo atua nesses potenciais alvos, bem como seus mecanismos de sinalização. Objetivo: Avaliar o efeito do peptídeo citoprotetor E8 em condrócitos humanos estimulados com a Interleucina-1β através da análise das vias de sinalização por meio do estudo de proteoma. Métodos: A partir da cultura de condrócitos primários humanos, indução da resposta inflamatória pela IL-1b e tratamento com o peptídeo E8, foi realizado o estudo de proteômica baseada em espectrometria de massas utilizando a quantificação relativa Label-free. Seguido da análise por enriquecimento de vias de sinalização, realizado partir das proteínas indicadas como diferencialmente abundantes entre as amostras. Resultados: Foram identificados, na condição sem resposta inflamatória, 1.048 grupos de proteínas nas amostras controle e 1.060 nas amostras tratadas com o peptídeo E8, das quais 27 apresentaram maior abundância nas células tratadas enquanto 47 apresentaram menor abundância. Nas amostras com resposta inflamatória induzida por IL-1β, foram identificadas 1.065 proteínas no controle e 1.190 no tratado com o E8, das quais 21 apresentaram maior abundância nas células tratadas com o E8 enquanto 18 apresentaram menor abundância. Das proteínas indicadas como diferencialmente abundantes, algumas recebem destaque por serem identificadas tanto na análise das amostras com resposta inflamatória induzida quanto nas amostras sem resposta inflamatória, as quais estão relacionadas a diferentes processos inflamatórios ou remodelagem tecidual. O enriquecimento de vias, realizado a partir das proteínas indicadas como diferencialmente abundantes em condrócitos sem resposta inflamatória, apontou 35 diferentes vias de atuação, enquanto o realizado a partir das proteínas indicadas como diferencialmente abundantes nas amostras com resposta inflamatória induzida apontou 31 vias. Em ambas as condições, a maioria das vias são de metabolismo de proteínas e metabolismo de DNA, com destaque para as vias NMD, de processamento de RNA ribossomal e de metabolismo de selenoaminoácido. Conclusões: O peptídeo é capaz de atuar alterando a abundância das proteínas, em especial, proteínas relacionadas a processos inflamatórios e de remodelagem tecidual, como: Proteína ribossômica 40S S23, Serina hidroximetiltransferase, Metaloproteinase de matriz-14, Cadeia alfa 1 do colágeno tipo III, e Heme oxigenase 1. O enriquecimento de vias realizado indica algumas vias interessantes para estudo do mecanismo de ação do peptídeo, a grande maioria envolvida em metabolismo de proteínas e metabolismo de RNA.
Introduction: Osteoarthritis is a chronic degenerative disease that affects the joints, causing inflammation. Currently available treatments provide temporary relief, thus there is still a need to develop efficient treatments. Screening for human chondrocyte inflammatory response model with peptides derived from Lopap, which retain the cytoprotective activity of the protein, indicated peptide E8 as one of the most promising in terms of anti-inflammatory effect, which reduces the release of inflammatory cytokines in the study model. Therefore, the study of the cytoprotective peptide E8 will enable the development of new, more specific and efficient drugs for the treatment of inflammatory diseases such as osteoarthritis, requiring an understanding of how the peptide acts on these potential targets, as well as its signaling mechanisms. Objective: To evaluate the effect of the cytoprotective peptide E8 on human chondrocytes stimulated with Interleukin-1β through the analysis of signaling pathways by means of proteome study. Methods: Based on the culture of primary human chondrocytes, induction of inflammatory response by IL-1β and treatment with peptide E8, a Label- free relative quantification-based proteomic study was conducted using mass spectrometry. This was followed by pathway enrichment analysis, performed on proteins indicated as differentially abundant between the samples. Results: Under the condition without inflammatory response, 1,048 protein groups were identified in the control samples and 1,060 in the samples treated with peptide E8, of which 27 showed higher abundance in treated cells while 47 showed lower abundance. In samples with IL-1β-induced inflammatory response, 1,065 proteins were identified in the control and 1,190 in the treated with E8, of which 21 showed higher abundance in cells treated with E8 while 18 showed lower abundance. Of the proteins indicated as differentially abundant, some stand out for being identified in both the analysis of induced inflammatory response samples and samples without inflammatory response, which are related to different inflammatory or tissue remodeling processes. Pathway enrichment analysis, performed on proteins indicated as differentially abundant in chondrocytes without inflammatory response, identified 35 different pathways of action, while that performed on proteins indicated as differentially abundant in samples with induced inflammatory response identified 31 pathways. In both conditions, most of the pathways are involved in protein metabolism and DNA metabolism, with emphasis on NMD pathways, ribosomal RNA processing, and selenoamino acid metabolism pathways. Conclusions: The peptide is able of acting by altering the abundance of proteins, especially those related to inflammatory and tissue remodeling processes, such as: 40S ribosomal protein S23, Serine hydroxy methyltransferase, Matrix metalloproteinase-14, Alpha 1 chain of type III collagen, and Heme oxygenase 1. Pathway enrichment analysis indicates some interesting pathways for studying the mechanism of action of the peptide, the vast majority of which are involved in protein metabolism and RNA metabolism.
Introduction: Osteoarthritis is a chronic degenerative disease that affects the joints, causing inflammation. Currently available treatments provide temporary relief, thus there is still a need to develop efficient treatments. Screening for human chondrocyte inflammatory response model with peptides derived from Lopap, which retain the cytoprotective activity of the protein, indicated peptide E8 as one of the most promising in terms of anti-inflammatory effect, which reduces the release of inflammatory cytokines in the study model. Therefore, the study of the cytoprotective peptide E8 will enable the development of new, more specific and efficient drugs for the treatment of inflammatory diseases such as osteoarthritis, requiring an understanding of how the peptide acts on these potential targets, as well as its signaling mechanisms. Objective: To evaluate the effect of the cytoprotective peptide E8 on human chondrocytes stimulated with Interleukin-1β through the analysis of signaling pathways by means of proteome study. Methods: Based on the culture of primary human chondrocytes, induction of inflammatory response by IL-1β and treatment with peptide E8, a Label- free relative quantification-based proteomic study was conducted using mass spectrometry. This was followed by pathway enrichment analysis, performed on proteins indicated as differentially abundant between the samples. Results: Under the condition without inflammatory response, 1,048 protein groups were identified in the control samples and 1,060 in the samples treated with peptide E8, of which 27 showed higher abundance in treated cells while 47 showed lower abundance. In samples with IL-1β-induced inflammatory response, 1,065 proteins were identified in the control and 1,190 in the treated with E8, of which 21 showed higher abundance in cells treated with E8 while 18 showed lower abundance. Of the proteins indicated as differentially abundant, some stand out for being identified in both the analysis of induced inflammatory response samples and samples without inflammatory response, which are related to different inflammatory or tissue remodeling processes. Pathway enrichment analysis, performed on proteins indicated as differentially abundant in chondrocytes without inflammatory response, identified 35 different pathways of action, while that performed on proteins indicated as differentially abundant in samples with induced inflammatory response identified 31 pathways. In both conditions, most of the pathways are involved in protein metabolism and DNA metabolism, with emphasis on NMD pathways, ribosomal RNA processing, and selenoamino acid metabolism pathways. Conclusions: The peptide is able of acting by altering the abundance of proteins, especially those related to inflammatory and tissue remodeling processes, such as: 40S ribosomal protein S23, Serine hydroxy methyltransferase, Matrix metalloproteinase-14, Alpha 1 chain of type III collagen, and Heme oxygenase 1. Pathway enrichment analysis indicates some interesting pathways for studying the mechanism of action of the peptide, the vast majority of which are involved in protein metabolism and RNA metabolism.