Avaliação da etapa final do sinal insulínico, via inflamatória, estresse oxidativo, mecanismo epigenético por metilação do DNA e expressão de GLUT4 no músculo gastrocnêmio de ratos adultos, proles de ratas com periodontite apical
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Data
2023-04-05
Autores
Tsosura, Thais Veronica Saori [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A programação fetal sugere que estímulos adversos quando aplicados durante o início do desenvolvimento fetal podem alterar o metabolismo da prole, aumentando o risco de doenças na sua vida adulta. A metilação do DNA é um dos mecanismos epigenéticos envolvida nesta programação e regula a expressão gênica. Estudos anteriores verificaram que a periodontite apical (AP) materna em ratas promove em sua prole adulta: resistência insulínica (RI), alteração na etapa inicial do sinal insulínico (SI) no músculo gastrocnêmio (MG) e aumento na concentração plasmática de fator de necrose tumoral-α (TNF-α). O TNF-α pode ativar o fator de transcrição nuclear kappa B (NF-kB) que diminui a expressão gênica do transportador de glicose GLUT4. Nesse contexto, mais estudos são necessários para investigar se as alterações no SI observadas em ratos adultos, proles de ratas com AP também estão presentes na continuidade da cascata insulínica. Ademais, sabendo-se que o estresse oxidativo tem sido implicado como fator contribuinte tanto para o início quanto para a progressão do diabetes, torna-se fundamental verificar o grau de estresse oxidativo tecidual nesta prole adulta. Em vista disso, os objetivos deste estudo foram avaliar a RI, estresse oxidativo, etapa final do SI e via inflamatória no MG de ratos adultos, proles de ratas com AP. Para tanto, as 21 ratas Wistar (2 meses de idade) foram distribuídas em 3 grupos: 1) ratas controle; 2) ratas com 1 AP induzida em 1º molar superior direito; 3) ratas com 4 APs induzidas em 1os e 2os molares superiores e inferiores do lado direito. A AP foi induzida empregando-se broca em aço carbono dotada de esfera de 0,1 mm na extremidade. Após 30 dias da exposição pulpar, as ratas de todos os grupos foram colocadas para acasalamento. Quando os filhotes machos de todas as ratas completaram 75 dias de idade, foram realizadas as seguintes análises plasmáticas: 1) glicemia; 2) insulinemia; 3) RI. Ademais, foram realizados os seguintes experimentos no MG: 1) grau de fosforilação em serina/treonina da Akt, após o estímulo insulínico; 2) conteúdo proteico de GLUT4; 3) grau de metilação do DNA na região promotora do gene do GLUT4; 4) expressão gênica do GLUT4 e TNF-α; 5) conteúdo proteico de TNF-α e PGC-1α; 6) grau de fosforilação de NF-kB p50 e NF-kB p65; 7) estresse oxidativo (superóxido dismutase – SOD e espécies reativas ao tiobarbitúrico – TBARS). A análise estatística foi realizada pela análise de variância, seguida do teste de Tukey. O nível de significância adotado foi de 5%. Os resultados mostraram que AP materna (tanto 1 foco de infecção como 4 focos de infecções) promoveu hiperinsulinemia, RI e diminuição na atividade antioxidante da SOD e na concentração de TBARS no MG de sua prole adulta. Entretanto, somente a AP materna em quatro dentes promoveu aumento na massa corpórea, na ingestão alimentar e no grau de fosforilação das subunidades p50 e p65 do NF-kB no MG de proles adultas. Ademais, houve diminuição no grau de fosforilação em serina e treonina da Akt (após estímulo insulínico) e na expressão gênica e conteúdo proteico de GLUT4 no MG em proles adultas de ratas com 4APs. A AP materna não promoveu alteração na glicemia de jejum, conteúdo de PGC-1α, grau de metilação do DNA na região promotora do gene do GLUT4 e expressão gênica e conteúdo proteico de TNF-α no MG de proles adultas. Esses resultados revelam o impacto que a AP materna tem em longo prazo na predisposição às alterações metabólicas na fase adulta da prole. Isso reforça a importância que a manutenção da saúde bucal materna tem sobre a saúde geral da prole.
Fetal programming suggests that adverse stimuli when applied during early fetal development may alter metabolism of offspring, increasing the risk of disease in adulthood. DNA methylation is one of the epigenetic mechanisms involved in this programming and regulates gene expression. Previous studies have shown that maternal apical periodontitis (AP) in rats promotes in their adult offspring: insulin resistance (IR), alteration in the initial steps of the insulin signaling (IS) in the gastrocnemius muscle (GM) and increase in plasma concentration of tumor necrosis factor-α (TNF-α). TNF-α can activate nuclear transcription factor kappa B (NF-kB) thus decreasing gene expression of the GLUT4 glucose transporter. In this context, more studies are needed to investigate whether changes in IS observed in adult rats, offspring of rats with AP are also present in the continuity of the insulin cascade. In addition, since oxidative stress has been implicated as a contributing factor for both the onset and progression of diabetes, it is essential to verify the degree of tissue oxidative stress in this adult offspring. Therefore, the aims of this study were to evaluate IR, oxidative stress, final steps of IS and inflammatory pathway in the GM of adult rats, offspring of rats with AP. For this purpose, 21 female Wistar rats (2 months of age) were distributed into three groups: 1) control rats (CN); 2) group with one AP induced in the upper right first molar (1AP); 3) group with four APs induced in the first and second upper and lower right molars (4AP). AP was induced using a surgical round bur measuring 0.1 mm in diameter. After 30 days of pulp exposure, female rats of all groups were placed for mating. Pregnant rats were separated into individual cages. When the male offspring of all rats reached 75 days of age, the following plasma analyzes were performed: 1) glycemia; 2) insulinemia; 3) IR. Furthermore, the following analyzes were performed in the GM: 1) Akt serine/threonine phosphorylation status, after insulin stimulation; 2) GLUT4 content; 3) degree of DNA methylation in the proximal promoter region of the GLUT4 gene; 4) GLUT4 and TNF- 13 α gene expression; 5) TNF-α and PGC-1α content; 6) p50 and p65 subunits of NF-kB phosphorylation status; 7) oxidative stress (superoxide dismutase – SOD and thiobarbiturate reactive species – TBARS). Statistical analyzes were performed by analysis of variance, followed by the Tukey test. The level of significance adopted was 5%. Results showed that maternal AP (both 1 infection focus and 4 infection focus) promoted hyperinsulinemia, IR and decrease in SOD antioxidant activity and TBARS concentration in the GM of their adult offspring. However, only maternal AP in four teeth promoted an increase in body weight, food intake and p50 and p65 subunits of NF-kB phosphorylation status in the GM of adult offspring. Furthermore, there was a decrease in Akt serine and threonine phosphorylation status (after insulin stimulation) and in gene expression and protein content of GLUT4 in the GM in adult offspring of rats with 4APs. Maternal AP did not change fasting glycemia, PGC-1α content, degree of DNA methylation in the proximal promoter region of GLUT4, gene expression and protein content of TNF-α in the GM of adult offspring. These results demonstrate that maternal AP is associated with IR and promotes important alterations in IS and inflammation pathways in adult offspring. This reinforces the importance of maternal oral health on the overall health of offspring.
Fetal programming suggests that adverse stimuli when applied during early fetal development may alter metabolism of offspring, increasing the risk of disease in adulthood. DNA methylation is one of the epigenetic mechanisms involved in this programming and regulates gene expression. Previous studies have shown that maternal apical periodontitis (AP) in rats promotes in their adult offspring: insulin resistance (IR), alteration in the initial steps of the insulin signaling (IS) in the gastrocnemius muscle (GM) and increase in plasma concentration of tumor necrosis factor-α (TNF-α). TNF-α can activate nuclear transcription factor kappa B (NF-kB) thus decreasing gene expression of the GLUT4 glucose transporter. In this context, more studies are needed to investigate whether changes in IS observed in adult rats, offspring of rats with AP are also present in the continuity of the insulin cascade. In addition, since oxidative stress has been implicated as a contributing factor for both the onset and progression of diabetes, it is essential to verify the degree of tissue oxidative stress in this adult offspring. Therefore, the aims of this study were to evaluate IR, oxidative stress, final steps of IS and inflammatory pathway in the GM of adult rats, offspring of rats with AP. For this purpose, 21 female Wistar rats (2 months of age) were distributed into three groups: 1) control rats (CN); 2) group with one AP induced in the upper right first molar (1AP); 3) group with four APs induced in the first and second upper and lower right molars (4AP). AP was induced using a surgical round bur measuring 0.1 mm in diameter. After 30 days of pulp exposure, female rats of all groups were placed for mating. Pregnant rats were separated into individual cages. When the male offspring of all rats reached 75 days of age, the following plasma analyzes were performed: 1) glycemia; 2) insulinemia; 3) IR. Furthermore, the following analyzes were performed in the GM: 1) Akt serine/threonine phosphorylation status, after insulin stimulation; 2) GLUT4 content; 3) degree of DNA methylation in the proximal promoter region of the GLUT4 gene; 4) GLUT4 and TNF- 13 α gene expression; 5) TNF-α and PGC-1α content; 6) p50 and p65 subunits of NF-kB phosphorylation status; 7) oxidative stress (superoxide dismutase – SOD and thiobarbiturate reactive species – TBARS). Statistical analyzes were performed by analysis of variance, followed by the Tukey test. The level of significance adopted was 5%. Results showed that maternal AP (both 1 infection focus and 4 infection focus) promoted hyperinsulinemia, IR and decrease in SOD antioxidant activity and TBARS concentration in the GM of their adult offspring. However, only maternal AP in four teeth promoted an increase in body weight, food intake and p50 and p65 subunits of NF-kB phosphorylation status in the GM of adult offspring. Furthermore, there was a decrease in Akt serine and threonine phosphorylation status (after insulin stimulation) and in gene expression and protein content of GLUT4 in the GM in adult offspring of rats with 4APs. Maternal AP did not change fasting glycemia, PGC-1α content, degree of DNA methylation in the proximal promoter region of GLUT4, gene expression and protein content of TNF-α in the GM of adult offspring. These results demonstrate that maternal AP is associated with IR and promotes important alterations in IS and inflammation pathways in adult offspring. This reinforces the importance of maternal oral health on the overall health of offspring.
Descrição
Palavras-chave
Periodontite periapical, Desenvolvimento fetal, Resistência à insulina, Inflamação, Inflammation