Efeito da diacereína no tratamento da doença periodontal induzida em molares de ratos
Carregando...
Data
2020-03-27
Autores
Silva, Renata Cristina Lima
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
A periodontite (DP) é uma doença imunoinflamatória que promove o recrutamento de células específicas que liberam mediadores, dentre eles interleucina-1 (IL-1), fator de necrose tumoral-alfa (TNF-α) e metaloproteinases da matriz (MMPs). Há evidências de que a diacereína, uma medicação anti-IL-1, reduz a produção de MMPs e TNF-α. O objetivo do presente estudo foi avaliar se a diacereína acelera a regressão da inflamação e reduz a perda óssea na DP induzida. No total, foram utilizados 66 ratos Holtzman distribuídos, aleatoriamente, nos diferentes grupos. Em 42 ratos, a DP foi induzida com inserção de uma ligadura no 1º molar superior direito por 7 dias. Após a indução, 12 animais foram sacrificados: 6 animais do grupo GP (grupo periodontite) e 6 animais do grupo controle (GC), usado como parâmetro da DP induzida. No 7º dia de indução da DP, a ligadura colocada em 36 ratos foi removida e iniciou-se o tratamento com 100 mg/kg de massa corpórea de diacereína (GPD) ou solução fisiológica (GPS) por gavagem durante 7, 15 e 30 dias. Após 24 horas da administração da última dose, 6 animais de cada grupo (GPD, GPS e GC) foram sacrificados. Após eutanásia, fragmentos de maxila com molares do lado direito foram removidos e logo imersos em solução de formaldeído 4%. Em seguida, os espécimes foram descalcificados em EDTA 7% e processados para inclusão em parafina. De cada fragmento, 5 cortes sagitais semi-seriados foram corados com hematoxilina e eosina (HE) para análises morfológica e quantitativa dos seguintes parâmetros: densidade de volume de células inflamatórias (VvCI) e fibroblastos (VvFb) na mucosa gengival interdentária molares e mensuração das distâncias do epitélio juncional à junção cemento-esmalte (EJ-JCE) e da JCE à crista do processo alveolar (JCE-PA). A reação histoquímica para fosfatase ácida resistente ao tartarato (TRAP), usada como marcador de osteoclastos, foi realizada e o número de osteoclastos TRAP-positivos na superfície do processo alveolar computado. A densidade numérica de células IL-1β- e MMP-8-positivas, após reações de imuno-histoquímica, também foi avaliada. Os dados quantitativos foram submetidos à análise de variância de dois fatores (two-way ANOVA) e pós-teste de Tukey (p ≤ 0,05); quando GP e GC foram comparados, utilizou-se o teste t de Student não paramétrico. Os resultados revelaram um aumento significante nas distâncias de EJ-JCE e JCE-PA em GP, GPD e GPS comparados a GC, no qual o término do EJ coincidiu com a JCE em todos os períodos. Entretanto, não foram detectadas diferenças entre GPD e GPS de todos os períodos. Em todos os períodos, foi verificada uma redução significante na VvCI dos animais tratados com diacereína comparados ao GPS. Não foi detectada diferença no número de osteoclastos entre GPD e GC. Aos 7 e 15 dias, o número de osteoclastos foi significantemente menor no GPD do que no GPS, enquanto aos 30 dias, diferenças não foram detectadas. A imunoexpressão de IL-1β nos espécimes do GPD foi significantemente menor em comparação ao GPS, nos diferentes períodos. Após 15 e 30 dias, não foram detectadas diferenças entre os grupos GPD e GC. Uma acentuada imunoexpressão de MMP-8 foi detectada na mucosa com DP, exceto aos 30 dias. Neste período, não foi detectada diferença entre GPD e CG. O número de células MMP-8-imunopositivas foi significantemente menor no GPD em comparação ao GPS de todos os períodos. Nossos resultados apontam para uma ação inibitória da diacereína sobre a IL-1β na mucosa gengival com periodontite, promovendo redução no infiltrado inflamatório, no número de osteoclastos e na imunoexpressão de MMP-8, exercendo papel benéfico na regressão da doença periodontal.
Periodontitis (PD) is an immunoinflammatory disease that promotes the recruitment of specific cells. These cells release mediators, including interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-α) and matrix metalloproteinases (MMPs). There is evidence that diacerein, an anti-IL-1 drug, reduces the production of MMPs and TNF-α. The aim of the present study was to evaluate if diacerein accelerates the regression of inflammation and reduces bone loss in induced PD. Sixty-six Holtzman male rats were randomly distributed in different groups. Periodontal disease was induced for 7 days in the upper right first molar of 42 rats. After 7 days of PD induction, 12 animals were killed: 6 animals from the control group (CG) and 6 animals from the PG group (periodontitis group). Thirty-six rats with PD received 100 mg/kg of diacerein body mass (PDG) per gavage or saline solution (PSG) for 7, 15 and 30 days. In each period, 6 animals of PDG, PSG and 6 animals without any treatment (healthy periodontium) were killed (CG). After euthanasia, fragments of maxilla containing the molars were removed and immediately immersed in a 4% formaldehyde solution. After decalcification in 7% EDTA, the fragments were processed for paraffin-embedding. From each specimen, non-serial sagittal sections were stained with hematoxylin and eosin (HE) for morphological and quantitative analyses of the following parameters: volume density of inflammatory cells (VvIC) and fibroblasts (VvFb) in the lamina propria of the interdental gingiva, between the 1st and 2nd molars; the distances between the junctional epithelium to the cemento-enamel junction (JE-CEJ) and the CEJ to the alveolar process crest (CEJ-AP) were measured. The histochemical reaction for tartrateresistant acid phosphatase (TRAP), used as an osteoclast marker, was performed and the number of TRAP-positive osteoclasts on the alveolar process surface was computed. Moreover, the numerical density of IL-1β- and MMP-8-immunopositive cells was obtained. Quantitative data were subjected to two-way analysis of variance (two-way ANOVA) and Tukey's post-test (p ≤ 0.05); when PG and CG were compared, the non-parametric t Student test was used. The results revealed a significant increase in the distances of JE-CEJ and CEJ-AP in the animals of PG, PDG and PSG groups compared with the CG; in the CG, the most apical portion of JE was situated at the JCE, at all periods. Significant differences in the CEJ-PA and JE-CEJ measurements were not detected between the PDG and PSG groups, at all time points. Significant reduction in VvIC was observed in diacerein-treated animals compared to the GPS group, at all periods. At all periods, no significant difference was detected in the number of osteoclasts between the PDG and CG groups. At 7 and 15 days, the number of osteoclasts was significantly lower in the PDG than in PSG, while significant difference was not detected at 30 days. The immunoexpression of IL-1β in PDG specimens was significantly lower than in PSG, at all time points. After 15 and 30 days, no significant differences were detected between animals of PDG and CG groups. An enhanced immunoexpression of MMP-8 was seen in the gingival mucosa of animals with periodontal disease, except at 30 days. In this period, no significant difference was detected between animals of PDG and CG groups. The number of MMP-8-immunopositive cells was significantly reduced in PDG specimens in comparison with PSG, at all periods. Our results point to an inhibitory action of diacerein on IL-1β in the gingival mucosa of rat molars with periodontitis, promoting a reduction in the inflammatory infiltrate, in the number of osteoclasts and in the immunoexpression of MMP-8, thus playing a beneficial role in the regression of periodontal disease.
Periodontitis (PD) is an immunoinflammatory disease that promotes the recruitment of specific cells. These cells release mediators, including interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-α) and matrix metalloproteinases (MMPs). There is evidence that diacerein, an anti-IL-1 drug, reduces the production of MMPs and TNF-α. The aim of the present study was to evaluate if diacerein accelerates the regression of inflammation and reduces bone loss in induced PD. Sixty-six Holtzman male rats were randomly distributed in different groups. Periodontal disease was induced for 7 days in the upper right first molar of 42 rats. After 7 days of PD induction, 12 animals were killed: 6 animals from the control group (CG) and 6 animals from the PG group (periodontitis group). Thirty-six rats with PD received 100 mg/kg of diacerein body mass (PDG) per gavage or saline solution (PSG) for 7, 15 and 30 days. In each period, 6 animals of PDG, PSG and 6 animals without any treatment (healthy periodontium) were killed (CG). After euthanasia, fragments of maxilla containing the molars were removed and immediately immersed in a 4% formaldehyde solution. After decalcification in 7% EDTA, the fragments were processed for paraffin-embedding. From each specimen, non-serial sagittal sections were stained with hematoxylin and eosin (HE) for morphological and quantitative analyses of the following parameters: volume density of inflammatory cells (VvIC) and fibroblasts (VvFb) in the lamina propria of the interdental gingiva, between the 1st and 2nd molars; the distances between the junctional epithelium to the cemento-enamel junction (JE-CEJ) and the CEJ to the alveolar process crest (CEJ-AP) were measured. The histochemical reaction for tartrateresistant acid phosphatase (TRAP), used as an osteoclast marker, was performed and the number of TRAP-positive osteoclasts on the alveolar process surface was computed. Moreover, the numerical density of IL-1β- and MMP-8-immunopositive cells was obtained. Quantitative data were subjected to two-way analysis of variance (two-way ANOVA) and Tukey's post-test (p ≤ 0.05); when PG and CG were compared, the non-parametric t Student test was used. The results revealed a significant increase in the distances of JE-CEJ and CEJ-AP in the animals of PG, PDG and PSG groups compared with the CG; in the CG, the most apical portion of JE was situated at the JCE, at all periods. Significant differences in the CEJ-PA and JE-CEJ measurements were not detected between the PDG and PSG groups, at all time points. Significant reduction in VvIC was observed in diacerein-treated animals compared to the GPS group, at all periods. At all periods, no significant difference was detected in the number of osteoclasts between the PDG and CG groups. At 7 and 15 days, the number of osteoclasts was significantly lower in the PDG than in PSG, while significant difference was not detected at 30 days. The immunoexpression of IL-1β in PDG specimens was significantly lower than in PSG, at all time points. After 15 and 30 days, no significant differences were detected between animals of PDG and CG groups. An enhanced immunoexpression of MMP-8 was seen in the gingival mucosa of animals with periodontal disease, except at 30 days. In this period, no significant difference was detected between animals of PDG and CG groups. The number of MMP-8-immunopositive cells was significantly reduced in PDG specimens in comparison with PSG, at all periods. Our results point to an inhibitory action of diacerein on IL-1β in the gingival mucosa of rat molars with periodontitis, promoting a reduction in the inflammatory infiltrate, in the number of osteoclasts and in the immunoexpression of MMP-8, thus playing a beneficial role in the regression of periodontal disease.
Descrição
Palavras-chave
Doença periodontal, Citocinas, Metaloproteinases da matriz, Interleucina-1, Periodontite, Periodontal disease, Cytokines, Matrix metalloproteinases, Interleukin-1, Periodontitis