Mecanismos envolvidos na perda óssea alveolar induzida pela doença periodontal em ratos espontaneamente hipertensos: papel do sistema renina-angiotensina e microRNAs
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Data
2023-06-23
Autores
Brito, Victor Gustavo Balera [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A doença periodontal (DP) é uma desordem inflamatória prevalente que afeta os tecidos de suporte e proteção dos dentes. Devido sua alta prevalência, é frequentemente associada a comorbidades, como hipertensão, que pode contribuir para sua progressão. O rato espontaneamente hipertenso (SHR) é um modelo que apresenta características semelhantes à hipertensão essencial humana, bem como comprometimento ósseo intrínseco e maior perda óssea alveolar associada à DP. Portanto, o objetivo deste estudo foi investigar dois mecanismos, o sistema renina-angiotensina (SRA) e a expressão de microRNAs (miRNAs), para melhor compreender o aumento da perda óssea alveolar induzida pela DP nesse modelo. Para isso, utilizamos ratos machos Wistar e SHR com 10 semanas de idade e a DP foi induzida por meio de ligadura bilateral inserida nos primeiros molares inferiores e mantida por 15 dias. Para avaliar a participação do SRA, os animais foram tratados com telmisartan (10 mg/Kg/dia) e foram realizadas análises da arquitetura óssea (microCT), expressão dos componentes do SRA, marcadores de formação, remodelamento e reabsorção óssea, bem como a produção de mediadores inflamatórios na mandíbula (RT-qPCR, ELISA e IHC-P). Para investigar a expressão diferencial de miRNAs associado a DP, foi utilizado ensaio de microarranjo e posteriormente análises bioinformáticas para identificar potenciais vias reguladas. Os resultados mostraram que o rato SHR apresentou maior perda óssea alveolar, em comparação ao Wistar (normotenso), o que foi parcialmente prevenido pelo tratamento com telmisartan. Observamos uma redução nos marcadores de osteoclastos (remodelamento e reabsorção), o que foi associada à menor produção de mediadores inflamatórios, inibição do receptor de angiotensina II tipo 1 (Agtr1) e maior expressão do receptor tipo 2 (Agt2r). Entretanto, observamos que a modulação dos componentes do SRA foi similar entre ratos Wistar e SHR, não explicando as diferenças observadas entre os modelos. De forma interessante, a análise de microarranjo revelou um perfil distinto de miRNAs relacionados à resposta imune e metabolismo ósseo nos SHR, bem como miRNAs diferencialmente modulados entre os modelos, previamente não associados à homeostase ou desordens periodontais. Resumidamente, mostramos que os componentes do SRA contribuem significativamente para a perda óssea alveolar, mas não explicam completamente a progressão aumentada observada nos SHR. No entanto, a modulação diferencial de miRNAs nos animais hipertensos sugerem seu envolvimento nessas diferenças, além de destacar potenciais alvos terapêuticos para DP, e outras desordens ósseas, que podem ser de interesse para orientar futuras pesquisas. Nesse sentido, a partir dos miRNAs diferencialmente expressos na DP, elegemos um miRNA de interesse, o miR-127-3p, que apresenta sequência conservada entre múltiplas espécies (incluindo ratos, camundongos e humanos), cuja função na osteogênese são havia sido completamente elucidada. Demonstramos então um efeito inibitório do miR-127-3p na diferenciação osteogênica de células osteoprogenitoras humanas (células mesenquimais estromais da medula óssea), por meio da superexpressão in vitro por meio de um mímico de miRNA. Estudos adicionais são necessários para confirmar um efeito in vivo, mas nossos resultados contribuem para a compreensão da função do miR-127-3p na biologia óssea. Financiamentos: CAPES (código 001), FAPESP (2015/03965-2, 2018/23676-3 e 2021/09597-6).
Periodontal disease (PD) is a prevalent inflammatory disorder affecting the teeth' supporting and protective tissues. Due to its high prevalence, it is often associated with other comorbidities such as hypertension, which can contribute to its progression. The spontaneously hypertensive rat (SHR) is a model that exhibits similarities to essential hypertension in humans, as well as intrinsic bone impairment and increased alveolar bone loss associated with PD. Therefore, the aim of this study was to investigate two mechanisms, the renin-angiotensin system (RAS) and microRNA (miRNA) expression, to better understand the increased alveolar bone loss induced by PD in this model. Male Wistar and SHR rats at 10 weeks of age were used, and PD was induced by bilateral ligature placement on the lower first molars and maintained for 15 days. To evaluate the involvement of the RAS, the animals were treated with telmisartan (10 mg/kg/day), and we analyzed the alveolar bone architecture (micro-CT), RAS component expression (RT-qPCR and IHC-P), bone formation, remodeling, and resorption markers expression (RT-qPCR), and the production of inflammatory mediators in the mandible (ELISA). A microarray assay was used to investigate the differential expression of miRNAs associated with PD, followed by bioinformatics analysis to identify potential regulated pathways. The results showed that the SHR rat exhibited greater alveolar bone loss than the normotensive Wistar rat, partially prevented by telmisartan treatment. We observed a reduction in osteoclast markers, associated with decreased production of inflammatory mediators, inhibition of the angiotensin II type 1 receptor (Agtr1), and increased expression of the type 2 receptor (Agt2r) in the telmisartan-treated animals. However, we found that the modulation of RAS components was similar between Wistar and SHR rats, which did not explain the observed differences between the models. Interestingly, microarray analysis revealed a distinct profile of miRNAs related to immune response and bone metabolism in SHR rats, as well as differentially modulated miRNAs between the models that were not previously associated with periodontal homeostasis or disorders. In summary, we demonstrated that RAS components significantly contribute to alveolar bone loss but do not fully explain the increased progression observed in SHR rats. However, the differential modulation of miRNAs in hypertensive animals suggests their involvement in these differences, highlighting potential therapeutic targets for PD and other bone disorders as well, which may be of interest for guiding future research. In this context, from the differentially expressed miRNAs in PD, we selected a miRNA of interest, miR-127-3p, which presents a conserved sequences among multiple species (including rats, mice, and humans) and which role in osteogenesis has not been fully elucidated. We demonstrated an inhibitory effect of miR-127-3p on the osteogenic differentiation of human osteoprogenitor cells (bone marrow-derived mesenchymal stromal cells) through in vitro overexpression using a miRNA mimic. Further studies are needed to confirm an in vivo effect, but our results contribute to understanding the function of miR-127-3p in bone biology. Funding: CAPES (code 001), FAPESP (2015/03965-2, 2018/23676-3, and 2021/09597-6).
Periodontal disease (PD) is a prevalent inflammatory disorder affecting the teeth' supporting and protective tissues. Due to its high prevalence, it is often associated with other comorbidities such as hypertension, which can contribute to its progression. The spontaneously hypertensive rat (SHR) is a model that exhibits similarities to essential hypertension in humans, as well as intrinsic bone impairment and increased alveolar bone loss associated with PD. Therefore, the aim of this study was to investigate two mechanisms, the renin-angiotensin system (RAS) and microRNA (miRNA) expression, to better understand the increased alveolar bone loss induced by PD in this model. Male Wistar and SHR rats at 10 weeks of age were used, and PD was induced by bilateral ligature placement on the lower first molars and maintained for 15 days. To evaluate the involvement of the RAS, the animals were treated with telmisartan (10 mg/kg/day), and we analyzed the alveolar bone architecture (micro-CT), RAS component expression (RT-qPCR and IHC-P), bone formation, remodeling, and resorption markers expression (RT-qPCR), and the production of inflammatory mediators in the mandible (ELISA). A microarray assay was used to investigate the differential expression of miRNAs associated with PD, followed by bioinformatics analysis to identify potential regulated pathways. The results showed that the SHR rat exhibited greater alveolar bone loss than the normotensive Wistar rat, partially prevented by telmisartan treatment. We observed a reduction in osteoclast markers, associated with decreased production of inflammatory mediators, inhibition of the angiotensin II type 1 receptor (Agtr1), and increased expression of the type 2 receptor (Agt2r) in the telmisartan-treated animals. However, we found that the modulation of RAS components was similar between Wistar and SHR rats, which did not explain the observed differences between the models. Interestingly, microarray analysis revealed a distinct profile of miRNAs related to immune response and bone metabolism in SHR rats, as well as differentially modulated miRNAs between the models that were not previously associated with periodontal homeostasis or disorders. In summary, we demonstrated that RAS components significantly contribute to alveolar bone loss but do not fully explain the increased progression observed in SHR rats. However, the differential modulation of miRNAs in hypertensive animals suggests their involvement in these differences, highlighting potential therapeutic targets for PD and other bone disorders as well, which may be of interest for guiding future research. In this context, from the differentially expressed miRNAs in PD, we selected a miRNA of interest, miR-127-3p, which presents a conserved sequences among multiple species (including rats, mice, and humans) and which role in osteogenesis has not been fully elucidated. We demonstrated an inhibitory effect of miR-127-3p on the osteogenic differentiation of human osteoprogenitor cells (bone marrow-derived mesenchymal stromal cells) through in vitro overexpression using a miRNA mimic. Further studies are needed to confirm an in vivo effect, but our results contribute to understanding the function of miR-127-3p in bone biology. Funding: CAPES (code 001), FAPESP (2015/03965-2, 2018/23676-3, and 2021/09597-6).
Descrição
Palavras-chave
Doenças periodontais, Hipertensão, Sistema renina-angiotensina, Inflamação, MicroRNAs, Periodontal diseases