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Doctoral Thesis
DOI
https://doi.org/10.11606/T.17.2022.tde-13072022-152534
Document
Author
Full name
Bruna Amanda da Cruz Rattis
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2022
Supervisor
Committee
Celes, Mara Rubia Nunes (President)
Zinni, Cibele Maria Prado
Pereira, Jonathas Xavier
Silva, João Santana da
Title in Portuguese
Efeito da curcumina na expressão dos componentes da via mTOR no coração de camundongos submetidos a sepse experimental
Keywords in Portuguese
Cardiomiopatia séptica
Curcumina
mTOR
Nanocurcumina
Sepse
Abstract in Portuguese
Introdução: A disfunção cardíaca induzida pela sepse (DCIS) é um fator determinante no prognóstico do paciente, e apesar do conhecimento considerável de sua fisiopatologia, pouco avanço terapêutico foi feito, mantendo a sepse como uma das principais causas de morte pelo mundo. A etiologia da DCIS vem sendo elucidada ao longo dos anos, as alterações estruturais e ultraestruturais nas células cardíacas já estão bem estabelecidas na literatura. Entretanto, ainda há um longo caminho a ser percorrido a nível molecular, afim de compreender pontualmente o inicio da sinalização celular mediante ao estresse séptico. Dessa forma, a mTOR, via de sinalização envolvida na sobrevivência celular, na resposta da célula ao estresse e na síntese proteica pode ser ponto chave. A curcumina surgiu como um possível inibidor da via da mTOR. Objetivo: Dessa forma, o objetivo deste estudo é avaliar a ação da curcumina sobre a via mTOR no coração de camundongos submetidos a sepse experimental por ligadura e perfuração do ceco. Metodologia: Foram utilizadas duas formulações de curcumina, sendo uma composta pela curcumina livre (CL) e outra de nanocurcumina (NC). A toxicidade da NC foi avaliada in vitro a partir de células H9c2. Posteriormente, foram utilizados camundongos da linhagem C57BL/6 para indução de sepse através da técnica de ligadura e perfuração do ceco distribuídos em seis grupos: falso-operados (SHAM), falso-operados tratados com CL (SH+CL); falso-operados tratados com NC (SH+NC); séptico (CLP); séptico tratado com CL (CLP+CL); séptico tratado com NC (CLP+NC). O tratamento foi realizado logo após o procedimento cirúrgico. As coletas foram feitas após 24 e 120 horas após a sepse. As análises histopatológicas foram analisadas por metacrilato. A expressão gênica e proteica da via mTOR foi avaliada por Western Blotting e real-time PCR. Resultados: O tratamento com nanocurcumina em células H9c2 foi bem tolerado, não apresentando toxicidade celular. A análise da sobrevida, temperatura, peso e escore clínico revelou que o tratamento não interferiu na progressão da sepse. Do ponto de vista histopatológico e ultraestrutural, os tratamentos com CL e NC reduziram as lesões cardíacas ocasionadas pela sepse. Ademais, observamos que a curcumina atuou de maneira pontual na via mTOR, onde o tratamento com CL e NC reduziu os níveis de mRNA nos grupos (CLP+CL e CLP+NC) em 24 horas. A análise dos níveis proteicos em 120 horas mostrou aumento significativo no grupo CLP em relação ao controle, além disso, foi observado aumento no grupo séptico tratado (CLP+NC) quando comparado ao séptico sem tratamento (CLP). Conclusão: Nossos resultados indicam que a curcumina, mesmo que na sua forma livre, apresenta efeito cardioprotetor na sepse murina. Além disso, mostramos que a sepse atua sobre a via da mTOR e a curcumina teve ação sobre a expressão gênica de mTOR, se destacando como um alvo terapêutico promissor para futuras pesquisas pré-clinicas e clínicas.
Title in English
Effect of curcumin on the expression of components of the mTOR pathway in the heart of mice subjected to experimental sepsis
Keywords in English
Cardiac dysfunction
Curcumin
mTOR
Nanocurcumin
Sepsis
Abstract in English
Introduction: Sepsis-induced myocardial dysfunction (SIMD) is a determining factor in patient prognosis, and despite considerable knowledge of its pathophysiology, little therapeutic progress has been made, maintaining sepsis as one of the leading causes of death worldwide. The etiology of SIMD has been elucidated over the years, structural and ultrastructural changes in cardiac cells are already well established in the literature. However, there is still a long way to go at the molecular level to understand the initiation of cell signaling through septic stress punctually. In this way, mTOR, a signaling pathway involved in cell survival in the cell's response to stress and protein synthesis, may be a critical point. Curcumin has emerged as a possible inhibitor of the mTOR pathway. Objective: Thus, the objective of this study is to evaluate the action of curcumin on the mTOR pathway in the heart of mice submitted to experimental sepsis by ligation and perforation of the cecum. Methodology: Two curcumin formulations were used, one composed of free curcumin (CL) and the other of nanocurcumin (NC). NC toxicity was evaluated in vitro from H9c2 cells. Subsequently, C57BL/6 mice were used to induce sepsis through the technique of ligation and perforation of the cecum, divided into six groups: sham-operated (SHAM), sham-operated treated with CL (SH+CL); false-operated treated with NC (SH+NC); septic (CLP); CL-treated septic (CLP+CL); septic treated with NC (CLP+NC). Treatment was performed shortly after the surgical procedure. Collections were made after 24 and 120 hours after sepsis. Histopathological analyzes were analyzed by methacrylate. The mTOR pathway gene and protein expression was evaluated by Western Blotting and real-time PCR. Results: Nanocurcumin treatment in H9c2 cells was well tolerated, showing no cellular toxicity. Analysis of survival, temperature, weight, and clinical score revealed that the treatment did not interfere with the progression of sepsis. From the histopathological and ultrastructural point of view, treatments with CL and NC reduced cardiac lesions caused by sepsis. Furthermore, we observed that curcumin acted punctually in the mTOR pathway, where treatment with CL and NC reduced mRNA levels in the groups (CLP+CL and CLP+NC) within 24 hours. The analysis of protein levels in 120 hours showed a significant increase in the CLP group compared to the control, and in addition, an increase was observed in the treated septic group (CLP+NC) compared to the untreated septic group (CLP). Conclusion: Our results indicate that curcumin has a cardioprotective effect in murine sepsis, even in its free form. In addition, we showed that sepsis acts on the mTOR pathway and curcumin had action on mTOR gene expression, standing out as a promising therapeutic target for future preclinical and clinical research.
 
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Publishing Date
2022-08-04
 
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