Effects Of All-trans Retinoic Acid On M1 Macrophage-mediated Immune Status In Mice With Periodontitis

ObjectiveThe effect of all-trans retinoic acid（ATRA）on the immune status of M1macrophages in periodontitis mice was studied by using Porphyromonas gingival（P.g）with high bacterial content to infect the oral cavity of mice.MethodsFifteen 7-week-old female C57BL/6 mice were randomly divided into 3groups（n=5）:Sham group,P.g+sodium carboxymethyl cellulose（P.g+CMC）group and all-trans retinoic acid（P.g+ATRA）group.1×10¹⁰P.gingivalis W83was used to infect the oral cavity of mice for 7 consecutive days to establish the animal model of chronic periodontitis.P.g+CMC group was intragastric gavage of 1%Sodium carboxymethyl cellulose（CMC）solution without ATRA in the same volume on other days,and P.g+ATRA group was intragastric gavage on other days（25μg/g body weight）for experimental study.The body weight of three groups of mice was dynamically monitored,and samples were collected from D42.The periodontal condition of mice was observed by stereomicroscope,and the gingival index and the degree of tooth looseness were detected.The distance from the cemento-enameljunction（CEJ）to the crest of alveolar bone（ABC）was measured to evaluate the absorption of alveolar bone.Hematoxylin and eosin（HE）staining were used to observe the loss of periodontal connective tissue attachment and alveolar bone resorption.F4/80⁺iNOS⁺M1 macrophages in gingival tissue,spleen and abdominal cavity of mice were detected by flow cytometry.Real-time Quantitative polymerase chain reaction method was used to detect iNOS（in-ducible Nitric Oxide Synthase）and Interleukin-10（interleukin-10）in the gingival tissue,spleen and abdominal cavity of mice.And Transforming Growth factor-β1（TGF-β1）mRNA expression.Results1.The measurement and analysis results of alveolar bone showed that compared with Sham group,the distance between CEJ and ABC in P.g+CMC group was significantly increased（P<0.05）,and the absorption rate of alveolar bone was increased（P<0.05）.Compared with P.g+CMC group,the distance from enamel cementum to alveolar crest of mice in P.g+ATRA group was significantly decreased（P<0.01）,and the absorption rate of alveolar bone was decreased（P<0.05）,and there was no statistical significance between P.g+ATRA group and Sham group（P>0.05）.2.The results of HE staining showed that,compared with the Sham group,P.g+CMC group had a large number of inflammatory cells infiltrating in the gingival tissue,the binding epithelial attachment was destroyed,the height of alveolar bone was reduced,and bone resorption lacunae appeared.The infiltration of inflammatory cells in the gingival tissue of mice treated with ATRA was less than that of P.g+CMC group,and the damage of binding epithelial attachment and alveolar bone resorption were not obvious.3.Flow cytometry showed that compared with Sham group,the proportion and number of F4/80⁺iNOS⁺M1 macrophages in gingival tissue,spleen and abdominal cavity of P.g+CMC group were significantly increased（P<0.05）.Compared with P.g+CMC group,the proportion and number of macrophages of F4/80⁺iNOS⁺M1 in P.g+ATRA group were significantly decreased（P<0.01）.4.The results of q PCR showed that,compared with Sham group,the expression level of iNOS mRNA in gingival tissue,spleen and abdominal cavity of P.g+CMC group was significantly increased（P<0.05）,and the expression levels of anti-inflammatory cytokines IL-10 and TGF-β1 mRNA were significantly increased（P<0.05）.Compared with P.g+CMC group,the expression level of iNOS mRNA in M1 macrophages in P.g+ATRA group was significantly decreased（P<0.01）,and the expression levels of anti-inflammatory cytokines IL-10 and TGF-β1 mRNA were significantly increased（P<0.01）.Conclusions1.In periodontitis,the immune response mediated by M1 macrophages is enhanced,which is manifested as the number and proportion of cells of M1 macrophages are significantly increased,and the mRNA expression level of related cytokine iNOS is increased.2.In periodontitis,ATRA can down-regulate the immune response mediated by M1 macrophages:ATRA down-regulates the cell number and cell proportion of M1 macrophages as well as the related pro-inflammatory factor iNOS,and up-regulates the expression of anti-inflammatory factors IL-10 and TGF-β1,thus playing a protective role in periodontitis.