ObjectiveMacrophages could be fully polarized and acquire specific phenotype like M1, which considered to be essential for the alveolar bone destruction during the development of periodontitis. However, the molecular mechanisms underlying the effects of M1 macrophages on the alveolar bone destruction are still not clear yet.MethodsMouse periodontitis model was established to determine the involvement of M1 macrophages in the pathogenic process. Condition medium of the M1 macrophages (M1‐CM) was incubated with pre‐osteoblasts to evaluate its effects on the osteoblastogenesis. Cells after treatment with CM were used for RNA‐sequencing, quantitative PCR, Western blotting, and immunofluorescence staining to figure out pathways involved in the inhibition of osteoblastogenesis.ResultsIncreased infiltration of M1 macrophages was associated with alveolar bone destruction in periodontitis. M1‐CM markedly suppressed the generation of osteoblasts as evidenced by decreased expressions of Runx2 and Ocn, as well as reduced activity of ALP. Interestingly, RNA‐sequencing indicated the activation of TLR4/AP1 signaling pathway in pre‐osteoblasts treated with CM. Inhibition of TLR4 reduced the translocation of AP1 and rescued the osteoblastogenesis reduced by M1‐CM.ConclusionM1 macrophages induce TLR4/AP1 signaling of pre‐osteoblasts to inhibit the osteoblastogenesis via paracrine, at least partially contributing to alveolar bone destruction in periodontitis.

Objective Experiments were performed to evaluate CYLD expression in human gingival tissue samples and to examine the effects of CYLD on inflammatory responses in lipopolysaccharide (LPS)‐ or TNF‐α‐stimulated human gingival fibroblasts (HGFs). Methods Immunohistochemistry for CYLD and p65 expression was performed with healthy and inflamed gingival tissue samples. siRNA was used to knock down the expression of CYLD in HGFs. Upon LPS or TNF‐α stimulation, NF‐κB activation was detected in control and CYLD‐knockdown HGFs. RT‐PCR was applied to determine gene expression. Western blot analyses were employed to assess protein expression. Immunofluorescence staining was carried out to evaluate the nuclear translocation of p65. Results Immunohistochemical staining showed the expression of CYLD in human gingival tissues. In addition, CYLD protein expression was reduced in inflamed gingival tissue samples compared with healthy tissue samples. CYLD knockdown greatly enhanced the mRNA expression of proinflammatory cytokines in LPS‐ or TNF‐α‐stimulated HGFs. Furthermore, knocking down CYLD expression increased LPS‐stimulated NF‐κB activation in HGFs. Unexpectedly, CYLD knockdown did not affect TNF‐α‐induced NF‐κB activation. Conclusions Our results suggest that CYLD participates in periodontal inflammatory responses by negatively regulating LPS‐induced NF‐κB signalling.

Metformin serves an important role in improving the functions of endothelial progenitor cells (EPcs). MicroRNAs (miRNAs), small non-coding RNAs, have been investigated as significant regulators of EPC vascular functions. The present study investigated the molecular crosstalk between metformin and miRNA-130a (miR-130a) in the functions of EPcs exposed to palmitic acid (PA). Isolated EPcs were treated with metformin, PA, and metformin + PA, respectively. cell counting Kit-8, Transwell and Matrigel assays were performed to detect the proliferation, migration and tube formation ability of EPcs following different treatments. The expression of miR-130a, phosphatase and tensin homolog (PTEN) and phosphorylated-AKT was analyzed by reverse transcription-quantitative polymerase chain reaction and western blotting. The specific mechanism underlying the function of metformin in EPcs was further elucidated by transfecting miR-130a mimics and inhibitor to overexpress and inhibit the expression of miR-130a in EPcs, respectively. EPcs exhibited impaired functions of proliferation (P<0.01 compared with the control), migration (P<0.01 compared with the control) and tube formation (P<0.01 compared with the control) following treatment with PA, and the expression levels of miR-130a and PTEN were decreased and increased, respectively. However, the presence of metformin, or the overexpression of miR-130a using miR-130a mimic alleviated the impairment of angiogenesis and proliferation, decreased the expression of PTEN and activated the phosphoinositide-3 kinase/AKT pathway in EPcs exposed to PA. By contrast, downregulating the expression of miR-130a with a miR-130a inhibitor reversed the metformin-mediated protection. These results demonstrate the beneficial effect of miR-130a/PTEN on EPc functions, which can be regulated by metformin. The effects of metformin on improving PA-induced EPc dysfunction are mediated by miR-130a and PTEN, which may assist in the prevention and/or treatment of diabetic vascular disease.