Plasmid encoding microRNA-200c ameliorates periodontitis and systemic inflammation in obese mice

Abstract

The present study was conducted to characterize microRNA-200c (miR-200c) and its regulators in adipogenic differentiation, obesity, and periodontitis in obese subjects (PiOSs), and to determine the therapeutic efficacy of plasmid DNA encoding miR-200c as a treatment for PiOSs. We report that highly expressed miR-200c in gingival tissues was downregulated in diet-induced obese (DIO) mice and during adipogenic differentiation of human bone marrow mesenchymal stromal cells (hBMSCs). Local injection of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) in the maxilla interdental gingiva of DIO mice reduced miR-200c in gingival and adipose tissues and induced periodontal inflammation associated with systemic elevation of interleukin-6 (IL-6) and impaired glucose tolerance. The inhibitory functions of Pg-LPS and IL-6 on miR-200c and their effectiveness on Zeb1 were confirmed in vitro. Injection of naked plasmid DNA encoding miR-200c into the gingiva effectively rescued miR-200c downregulation, prevented periodontal and systemic inflammation, and alleviated the impaired glucose metabolism in obese mice with LPS-induced periodontitis. Increased circulating exosomal miR-200c and its function on suppressing proinflammatory cytokines and adipogenesis explained the mechanism(s) of gingival application of miR-200c in attenuating systemic inflammation in PiOSs. These results demonstrated that miR-200c reduced by Pg-LPS and IL-6 in periodontitis and obesity might lead to the pathogenesis of PiOSs, and upregulation of miR-200c in the gingiva presents a therapeutic approach for PiOSs.

Keywords: IL-6; Leptin; Pg-LPS; Zeb1; gene therapy; glucose intolerance; microRNA-200c; mouse; obesity; periodontitis.

Graphical abstract

Figure 1

Relative fold changes of miR-200c expression in different tissues and organs of 22-week-old C57BL/6J mice ∗p < 0.05, n = 3.

Figure 2

Characteristics of DIO mice fed with an HFD after 6 and 16 weeks (A) Body weights of mice fed with an HFD for 6 and 16 weeks. (B) Relative fold changes of miR-200c in gingiva and WAT in mice fed with an HFD for 6 weeks. (C and D) Relative transcript level changes of noggin, Zeb1, IL-6, and IL-8 in gingival tissues and WAT in mice fed with an HFD for 6 weeks. (E) Relative fold changes of miR-200c in gingiva and WAT in mice fed with an HFD for 16 weeks. (F and G) Relative transcript change of noggin, Zeb1, IL-6, and IL-8 in gingival tissues and WAT in mice fed with an HFD for 16 weeks. (H) Concentrations of IL-6 in blood serum in mice fed with an HFD for 16 weeks. (I) Blood glucose concentrations of a GTT test in mice fed with an HFD for 16 weeks. ∗p < 0.05 versus wild-type (WT), n = 3–9.

Figure 3

Characteristics of adipogenesis of hBMSCs on miR-200c expression (A) Microphotographs of hBMSCs with Oil Red O staining 3 weeks after adipogenic differentiation and control. (B) Relative transcript changes of noggin, Zeb1, LPL, PPAR-γ, and leptin of hBMSCs 1 week after adipogenic differentiation compared to controls. (C) Relative fold change of miR-200c hBMSCs 3 and 7 days after differentiation and controls. (D) Normalized fold changes of miR-200c expression in hBMSCs 3 days after treatment with PMIS-miR-200c. (E) Relative transcript changes of PPAR-γ, LPL, and noggin in adipogenic differentiated hBMSCs pretreated with PMIS-miR-200c. ∗p < 0.05, performed in triplicate.

Figure 4

Pathophysiological characteristics of Pg-LPS injection into the gingival sulcus between maxillary M1/M2 of obese mice after 16 weeks of being fed an HFD (A) μCT scan of maxillary bones on the palatal side of obese mice 2 weeks after receiving Pg-LPS at different concentrations. (B–D) Quantitative measurement of AB loss, BMD, and BV/TV at maxillary M1/M2 of mice. (E) Relative fold change of miR-200c in gingival tissues of mice with different treatments. (F and G) Relative transcript changes of IL-6, IL-8, and Zeb1 in gingiva and WAT of mice after different treatments. (H) Serum glucose concentration measured by a GTT test in mice with different treatment. ∗p < 0.05 versus PBS injection; n = 3–6.

Figure 5

Representative μCT scan and microphotographs of histological cross-sections of obese mice 4 weeks after receiving Pg-LPS with miR-200c treatment and controls (A) μCT images on the palatal side and cross-section of maxillary bones. (B) H&E-stained histological cross-section of mice after different treatments. AB, alveolar bone; arrow, granulation tissues; scale bar, 100 μm. (C) TRAP-stained histological cross-section of mice after different treatments. Arrow, activated osteoclasts; scale bar, 100 μm.

Figure 6

Quantitative measurements of the effectiveness of miR-200c on attenuating AB loss and systemic inflammation in obese mice with periodontitis (A–C) Quantitative μCT measurement of AB loss (A), BMD (B), and BV/TV (C) at maxillary M2/M3 of obese mice with Pg-LPS injection 4 weeks after treatment with miR-200c at different concentrations and controls. (D) Serum concentration of IL-6 in mice 4 weeks after different treatments. (E and F) Relative transcript changes of miR-200c, IL-6, and IL-8 in gingiva and WAT of mice with different treatments. (G) Serum glucose concentrations from a GTT test in mice 4 weeks after receiving different treatments. ∗p < 0.05 versus LPS-EV, n = 4–6.

Figure 7

Exosomal miR-200c on inflammation and adipogenesis (A) Relative fold change of miR-200c in serum exosomes isolated from mice receiving plasmid miR-200c injection in gingival tissues. p = 0.075, n = 3. (B) TEM image of exosomes isolated from HEPM cells with an overexpression of miR-200c and western blot using anti-CD63 and CD-9. (C) Relative fold changes of miR-200c in human ADSCs after treatment with exosomes isolated from HEPM cells with miR-200c overexpression and controls. (D) Relative transcript changes of IL-6, IL-8, and LPL in human ADSCs after treatment with exosomes isolated from HEPM cells with miR-200c overexpression and controls. ∗p < 0.05, performed in triplicate.

Figure 8

Effectiveness of IL-6 and LPS on miR-200c and Zeb1 (A and B) Relative fold changes of miR-200c expression in HEPM cells treated with IL-6 and LPS at different concentrations. (C and D) Relative transcript changes of Zeb1 in HEPM cells treated with IL-6 and Pg-LPS at different concentrations. ∗p < 0.05, performed in triplicate.

Figure 9

Potential roles of miR-200c in pathogenesis and treatment of PiOSs