Low-intensity Pulsed Ultrasound regulates alveolar bone homeostasis in experimental Periodontitis by diminishing Oxidative Stress

Abstract

Periodontitis is a widespread oral disease that results in the loss of alveolar bone. Low-intensity pulsed ultrasound (LIPUS), which is a new therapeutic option, promotes alveolar bone regeneration in periodontal bone injury models. This study investigated the protective effect of LIPUS on oxidative stress in periodontitis and the mechanism underlying this process. Methods: An experimental periodontitis model was induced by administering a ligature. Immunohistochemistry was performed to detect the expression levels of oxidative stress, osteogenic, and osteoclastogenic markers in vivo. Cell viability and osteogenic differentiation were analyzed using the Cell Counting Kit-8, alkaline phosphatase, and Alizarin Red staining assays. A reactive oxygen species assay kit, lipid peroxidation MDA assay kit, and western blotting were used to determine oxidative stress status in vitro. To verify the role of nuclear factor erythroid 2-related factor 2 (Nrf2), an oxidative regulator, during LIPUS treatment, the siRNA technique and Nrf2^-/- mice were used. The PI3K/Akt inhibitor LY294002 was utilized to identify the effects of the PI3K-Akt/Nrf2 signaling pathway. Results: Alveolar bone resorption, which was experimentally induced by periodontitis in vivo, was alleviated by LIPUS via activation of Nrf2. Oxidative stress, induced via H₂O₂ treatment in vitro, inhibited cell viability and suppressed osteogenic differentiation. These effects were also alleviated by LIPUS treatment via Nrf2 activation. Nrf2 silencing blocked the antioxidant effect of LIPUS by diminishing heme oxygenase-1 expression. Nrf2^-/- mice were susceptible to ligature-induced periodontitis, and the protective effect of LIPUS on alveolar bone dysfunction was weaker in these mice. Activation of Nrf2 by LIPUS was accompanied by activation of the PI3K/Akt pathway. The oxidative defense function of LIPUS was inhibited by exposure to LY294002 in vitro. Conclusions: These results demonstrated that LIPUS regulates alveolar bone homeostasis in periodontitis by attenuating oxidative stress via the regulation of PI3K-Akt/Nrf2 signaling. Thus, Nrf2 plays a pivotal role in the protective effect exerted by LIPUS against ligature-induced experimental periodontitis.

Keywords: alveolar bone; oxidative stress; periodontitis.

Figure 1

LIPUS alleviates ligature-induced alveolar bone destruction in periodontitis. (A) SD rats were subjected to subcutaneous ligature insertion for 14 d, with or without LIPUS treatment (30 min/day, with 200 ms pulses and 1.5 MHz) for 14 d. (B) Micro-CT image of the alveolar bone (Scale bar = 1 mm), (C) CEJ-ABC distance, (D) BV/TV, and (E) Tb.Th. Control (no treatment), Ligature (ligature-induced experimental periodontitis), LIPUS (only LIPUS treatment), Ligature + LIPUS (experimental periodontitis before LIPUS treatment). Data are presented as the mean ± SEM (n = 3). *, p < 0.05; **, p < 0.005; ***, p < 0.0005; ****, p < 0.00005.

Figure 2

LIPUS alleviates ligature-induced oxidative stress via Nrf2 Pathway. (A) H&E staining at 200× and 400× magnification. TRAP staining, immunohistochemical staining of OCN, 3-NT, 8-OHdG, Nrf2, HO-1 at 200× and 400× (red box) magnification (Scale bar = 100 µm), (B) MPO flow analysis. Red arrows indicate positive staining.

Figure 3

H2O2 inhibits cell viability and osteogenic differentiation. (A) Cell viability detected by the CCK-8 assay at different concentration of H2O2. Osteogenic differentiation at different exposure periods for H2O2 detected by ALP staining (Scale bar = 1 mm) (B) and ALP activity assay (C). Data are presented as the mean ± SEM (n = 3). *, p < 0.05; **, p < 0.005; ***, p < 0.0005; ****, p < 0.00005.

Figure 4

LIPUS mitigates H2O2-induced inhibited cell viability and suppressed osteogenesis. (A) Cell viability detected by the CCK-8 assay at different treatment periods for LIPUS. (B) Cell viability detected by the CCK-8 assay under H₂O₂ exposure and LIPUS treatment. Osteogenic differentiation detected by (C) ALP staining (Scale bar = 1 mm), (D) ALP activity, (E) Alizarin Red staining (Scale bar = 1 mm), (F) quantification of ARS staining, and gene expression of (G) ALP, (H) OCN, (I) RANKL, (J) IL-6, and (K) TNF-α. Data are presented as the mean ± SEM (n = 3). *, p < 0.05; **, p < 0.005; ***, p < 0.0005; ****, p < 0.00005.

Figure 5

LIPUS mitigates H2O2-induced oxidative stress. Intracellular ROS measured by (A) DCFH-DA staining (Scale bar = 400 µm) and (B) DCFH-DA fluorescence intensity. (C) The MDA content of PDLCs was measured by MDA assay kit. Expression of 3-NT and 4-HNE were evaluated by (D) western blot. The representative expression of (E) 3-NT and (F) 4-HNE were measured by semi-quantitative analysis. Data are presented as the mean ± SEM (n = 3). *, p < 0.05; **, p < 0.005; ***, p < 0.0005; ****, p < 0.00005.

Figure 6

LIPUS mitigates H₂O₂-induced oxidative stress via Nrf2 signaling pathway. After H₂O₂-induced oxidative stress and LIPUS treatment, mRNA expression of (A) Nrf2 and (B) HO-1 was examined by qPCR. (C) Activation of phospho-Nrf2 and HO-1 were examined by western blot. The representative activation of (D) phospho-Nrf2 and (E) HO-1 were measured by semi-quantitative analysis. (F) Expression of Nrf2 in the nucleus was examined by western blot. (G) The quantitative expression of Nrf2 in the nucleus was measured by semi-quantitative analysis. Data are presented as the mean ± SEM (n = 3). *, p < 0.05; **, p < 0.005; ***, p < 0.0005; ****, p < 0.00005.

Figure 7

Knockdown of Nrf2 gene abrogates the protection of LIPUS against H₂O₂-induced oxidative stress. Transfection effects of small interfering RNA (siRNA) or negative control siRNA (NC) were analyzed by (A) qPCR and (B) western bloting. (C) The quantitative expression of Nrf2 in the nucleus was measured by semi-quantitative analysis. (D) After transfection with siRNA, activation of phospho-Nrf2 and HO-1 were examined by western blotting. The respective activation of (E) phospho-Nrf2 and (F) HO-1 were measured by semi-quantitative analysis. (G) Expression of Nrf2 in the nucleus was examined by western blot. (H) The quantitative expression of Nrf2 in the nucleus was measured by semi-quantitative analysis. Data are presented as the mean ± SEM (n = 3). *, p < 0.05; **, p < 0.005; ***, p < 0.0005; ****, p < 0.00005.

Figure 8

Knockout of Nrf2 weakens the protection effect exerted by LIPUS on ligature-induced alveolar bone destruction in periodontitis. Nrf2^+/+ mice and Nrf2^-/- mice were subjected to ligature insertion, with or without LIPUS treatment (30 min/day, with 200 ms pulses and 1.5 MHz) for 8 days simultaneously as described in Fig. 8B. Expression of Nrf2 and HO-1 was examined by western bloting. Activation of Nrf2 and HO-1 (A) quantitative levels (C). Three-dimensional image of the alveolar bone (Scale bar = 1 mm) (D). (E) CEJ-ABC distance, (F) BV/TV, and (G) Tb.Th. Nrf2 (I) and HO-1 (J) quantitative levels (H). Nrf2^+/+ (WT with no treatment), Nrf2^-/- Control (Nrf2^-/- with no treatment), Nrf2^-/- Ligature (Nrf2^-/- with ligature-induced experimental periodontitis), Nrf2^-/- LIPUS (Nrf2^-/- with LIPUS treatment only), Nrf2^-/- Ligature+LIPUS (Nrf2^-/- with experimental periodontitis and LIPUS treatment). Data are presented as the mean ± SEM (n = 3). *, p < 0.05; **, p < 0.005; ***, p < 0.0005; ****, p < 0.00005.

Figure 9

Knockout of Nrf2 abolishes the protection of LIPUS against ligature-induced oxidative stress. (A) TRAP staining, immunohistochemical staining of OCN, 3-NT, 8-OHdG, Nrf2, and HO-1 at 400× magnification in periodontal tissue (Scale bar = 100 µm). (B) MPO flow analysis. Red arrows indicate positive staining.

Figure 10

Inhibition of PI3K/Akt abrogated the protection of LIPUS against H₂O₂-induced oxidative stress. (A) Activation of phospho-Akt was examined by western blotting. (D) The quantitative activation of Nrf2 was measured by semi-quantitative analysis. (B) Pretreatment of LY294002 (10 µM), activation of phospho-Akt, phospho-Nrf2 and HO-1 were examined by western blotting. The respective activations of phospho-Akt (E), phospho-Nrf2 (F) and HO-1 (G) were measured by semi-quantitative analysis. (C) Expression of Nrf2 in the nucleus was examined by western blot. (H) The quantitative expression of Nrf2 in the nucleus was measured by semi-quantitative analysis. (I) Nrf2 translocation was determined by immunofluorescence at 200× magnification (Scale bar = 200 µm). Red: Nrf2-staining, blue: nucleus (DAPI), and pink: merger of blue and red indicating nuclear localization of Nrf2. Data are presented as the mean ± SEM (n = 3). *, p < 0.05; **, p < 0.005; ***, p < 0.0005; ****, p < 0.00005.