Quercetin through miR-147-5p/Clip3 axis reducing Th17 cell differentiation to alleviate periodontitis.

ABSTRACT

Background: Quercetin (QU) plays an important role in treating periodontitis; however, the mechanism through which microRNAs regulate Th17 cell differentiation has not been determined. Methods: QU was administered intragastrically to periodontitis rats once a day for one month. The morphology of alveolar bone was observed by micro-CT, gingival tissue structure was observed by HE staining, IL-6, TNF-α, IL-17A, RORγt, FOXP3 and IL-10 were detected by immunohistochemical staining, and Th17 and Treg cells in the peripheral blood were detected by flow cytometry. CD4+T cells were induced to differentiate into Th17 cells in vitro. Cell viability was determined by CCK8, and IL-17A and RORγt were detected by qPCR. Th17 cells were detected by flow cytometry, microRNA sequencing and bioinformatics analysis were used to screen key microRNAs, the phenotypic changes of Th17 cells were observed after overexpressed microRNAs via mimics. TargetScan database, in situ hybridization, and dual-luciferase reporter experiment were used to predict and prove target genes of microRNAs. The phenotype of Th17 cells was observed after overexpression of microRNA and target gene. Results: Compared with periodontitis group, the distance from cementoenamel junction(CEJ) to alveolar bone(AB) was decreased, the structure of gingival papilla was improved, IL-6, TNF-α, IL-17, and RORγt were downregulated, FOXP3 and IL-10 were upregulated, the proportion of Th17 decreased and Treg increased in peripheral blood after QU treatment. Compared with Th17 cell group, mRNA levels of IL-17A and RORγt were decreased, and proportion of Th17 cells was significantly lower in the coculture group. MiR-147-5p was low in control group, upregulated in Th17 cell group, and downregulated after QU intervention, it's eight bases were inversely related to 3'UTR of Clip3, miR-147-5p with Clip3 were co-located in cells of periodontal tissue. Compared with those in Th17-mimicsNC + QU cells, the mRNA levels of RORγt and IL-17A upregulated, and proportion of Th17 cells increased in Th17-miR-147-5p + QU cells. The miR-147-5p mimics inhibited the luciferase activity of the WT Clip3 3'UTR but had no effect on the Mut Clip3 3'UTR. Clip3 was significantly downregulated after the overexpression of miR-147-5p. Mimics transfected with miR-147-5p reversed the decrease in the proportion of Th17 cells induced by QU, while the overexpression of Clip3 antagonized the effect of miR-147-5p and further reduced the proportion of Th17 cells. Moreover, the overexpression of miR-147-5p reversed the decreases in the mRNA levels of IL-17 and RORγt induced by QU treatment, while pcDNA3.1 Clip3 treatment further decreased the mRNA levels of IL-17 and RORγt. Conclusion: QU reducing inflammatory response and promoting alveolar bone injury and repair, which closely relative to inhibit the differentiation of CD4+T cells into Th17 cells by downregulating miR-147-5p to promote the activation of Clip3.