Metformin ameliorates HMGB1-mediated oxidative stress through mTOR pathway in experimental periodontitis

Abstract

Periodontitis is an oral chronic inflammatory disease. Inhibiting tissue destruction and promoting tissue regeneration are important means for the treatment of periodontitis. Metformin not only has hypoglycemic effect but also has anti-inflammatory effect. Metformin has been shown to inhibit oxidative stress and activate autophagy through AMPK/mTOR pathway. High mobility group box 1 (HMGB1) has been implicated in the pathogenesis of many inflammatory diseases including periodontitis, it can participate in the induction of oxidative stress. HMGB1 is an autophagy regulator under oxidative stress, which can activate mTOR pathway. However, it is not clear whether metformin is related to HMGB1 and its mechanism in the process of periodontitis. Cell viability and expression of inflammatory cytokines were clarified by Cell Counting Kit-8, real-time PCR and enzyme-linked immunosorbent assay. Western blot and immunofluorescence were conducted to determine HMGB1 intracellular localization and expression of autophagy-associated proteins in vitro. Experimental periodontitis mice model was induced by administering a ligature. Immunohistochemistry was performed to detect the expression and localization of HMGB1 in vivo. The results of CCK-8, real-time PCR, enzyme-linked immunosorbent assay, Western blot and immunofluorescence showed lipopolysaccharide (LPS) treatment inhibited cell viability, and increased HMGB1 expression at a dose-independent manner. Metformin can reduce the effect of LPS. It also improves autophagy pathway inhibited by LPS and down-regulates mTOR expression. In addition, metformin attenuated alveolar bone resorption induced by ligation. This study provides new evidence for that metformin is a potential drug for the treatment of periodontitis and HMGB1 may be a potential target for periodontal intervention.

Keywords: Autophagy; Metformin; Oxidative stress; Periodontal ligament cells; Periodontitis.

Figure 1

LPS inhibits hPDLCs viability and induces inflammation. (A) Cell viability detected by the CCK-8 assay at different concentration of LPS. Inflammatory cytokines mRNA expression of IL-6 (B) and IL-8 (C) were examined by real-time PCR. Inflammatory cytokines production of IL-6 (D) and IL-8 (E) were examined by ELISA in culture medium. Data are presented as the mean ± SD (n = 3). ∗, P < 0.05; ∗∗, P < 0.005; ∗∗∗, P < 0.0005; ∗∗∗∗, P < 0.00005.

Figure 2

HMGB1 is highly expressed in LPS-induced hPDLCs. (A) HMGB1 mRNA expression were examined by qPCR. (B) HMGB1 production detected by ELISA in culture medium. Total (D) and nuclear (C) HMGB1 protein expression were examined by Western blot. Data are presented as the mean ± SD (n = 3). ∗, P < 0.05; ∗∗, P < 0.005; ∗∗∗, P < 0.0005; ∗∗∗∗, P < 0.00005.

Figure 3

Metformin alleviates LPS-induced hPDLCs. (A) Cell viability detected by the CCK-8 assay at different concentration of metformin. Inflammatory cytokines mRNA expression of HMGB1 (B), IL-6 and IL-8 (C) were examined by real-time PCR. Inflammatory cytokines production of HMGB1 (D), IL-6 (E) and IL-8 (F) were examined by ELISA in culture medium. Data are presented as the mean ± SD (n = 3). ∗, P < 0.05; ∗∗, P < 0.005; ∗∗∗, P < 0.0005; ∗∗∗∗, P < 0.00005.

Figure 4

Metformin alleviates LPS-induced hPDLCs via inhabiting HMGB1 translocation and releasing. (A) Total and nuclear HMGB1 protein expressions were examined by Western blot. (B) HMGB1 translocation and releasing was determined by immunofluorescence at 400× magnification. Red: HMGB1-staining, Blue: nucleus (DAPI) and Pink: merge of blue and red indicating nuclear localization of HMGB1. Scale bar = 100 μm. Data are presented as the mean ± SD (n = 3). ∗, P < 0.05; ∗∗, P < 0.005; ∗∗∗, P < 0.0005; ∗∗∗∗, P < 0.00005.

Figure 5

Metformin alleviates ligature induced-periodontitis. (A) Mice were subjected to ligature insertion for 14 d, with or without metformin treatment (every day, with 200 mg/kg). (B) CEJ-ABC distance. Control (no treatment), Ligature (ligature induced experimental periodontitis), Ligature + Met (experimental periodontitis with metformin treatment). (C) Micro CT image of the alveolar bone. (D) Immunohistochemical staining of HMGB1 at a 400x (scale bar = 100 μm) magnification. (E) Histological analysis of Hype staining showed that the bone resorption in the ligation group was higher than that in the control group. Data are presented as the mean ± SD (n = 6). ∗, P < 0.05; ∗∗, P < 0.005; ∗∗∗, P < 0.0005; ∗∗∗∗, P < 0.00005.

Figure 6

mTOR pathway is involved in the antioxidant stress of metformin. (A) hPDLCs was pretreated with autophagy inhibitors 3-MA and Bafilomycin A1, and then treated with metformin for 12 h, then the expression of autophagy related proteins was observed. (B) hPDLCs was pretreated with autophagy inhibitors 3-MA and Bafilomycin A1, and then treated with metformin for 24 h, then the expression of autophagy related proteins was observed. Pretreatment of periodontal ligament stem cells with different autophagy inhibitors bafilomycin a1 (C) and 3-MA (D). Then rapamycin was added to make positive control. Finally, periodontal ligament stem cells were treated with met for 24 h. Autophagy-related proteins were examined by Western blot. Data are presented as the mean ± SD (n = 3). ∗, P < 0.05; ∗∗, P < 0.005; ∗∗∗, P < 0.0005; ∗∗∗∗, P < 0.00005.

Supplementary Figure 1

Quantifications of western blots in Fig. 6. (A), (B), (C) and (D) HMGB1, mTOR and LC3-II/LC3-I ratio were examined by western blot. Western blot's data are quantified. Data are presented as the mean ± SD (n = 3). ∗, P < 0.05; ∗∗, P < 0.005; ∗∗∗, P < 0.0005; ∗∗∗∗, P < 0.00005.