Abstract:

Objective To discuss the effects of sirtuin 3 （SIRT3） deficiency on the pathological damage of periodontal tissues， osteoclast activity， and macrophage polarization in the mice with periodontitis， and to clarify the molecular mechanism. Methods Twelve C57BL/6 mice were randomly divided into SIRT3 wild-type （WT） group（SIRT3^+/+ group） and SIRT3 knockout group（SIRT3^-/- group）， with 6 mice in each group. A mouse periodontitis model was established， and the samples were collected on the 7th and 14th days of modeling. HE staining， Safranin O-Fast Green staining， and tartrate-resistant acid phosphatase （TRAP） staining were used to observe the patholmorphology and osteoclast distribution in periodontal tissues of the mice in two groups； immunohistochemistry was used to detect the expression levels of inducible nitric oxide synthase （iNOS） and cluster of differentiation 163 （CD163） proteins in periodontal tissues of the mice in two groups； Western blotting method was used to detect the expression levels of matrix metalloproteinase-9 （MMP-9）， phosphorylated-P65（p-P65）， and cathepsin K （CTSK） proteins in aperiodontal tissues of the mice in two groups； real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the expression levels of acid phosphatase 5 （ACP5）， MMP-9， nuclear factor of activated T cells cytoplasmic 1 （NFATC1）， and tumor necrosis factor α （TNF-α） mRNA in periodontal tissues of the mice in two groups； Micro-CT scanning was used to analyze the changes of alveolar bone microstructure and the surface area of alveolar bone damage in periodontal tissues of the mice in two groups. Results The HE staining results showed that in SIRT3^+/+ group， the periodontal ligament fibers in periodontal tissues were arranged neatly， with less inflammatory infiltration and bone resorption lacunae； in SIRT3^-/- group， the periodontal ligament fibers in periodontal tissues were arranged disorderly， with pervodontal ligament edema and widening， a large number of neutrophils and lymphocytes diffusely infiltrated， and the alveolar bone margin showed moth-eaten defects with multinucleated osteoclasts. Compared with SIRT3^+/+ group on the 7th day of modeling， the percentage of inflammatory area in periodontal ligament region of the mice in SIRT3^-/- group was significantly increased （P<0.05）； at 14th day of modeling， compared with SIRT3^+/+ group， the percentage of inflammatory area in periodontal ligament region of the mice in SIRT3^-/- group was significantly increased （P<0.01）. The Safranin O-Fast Green staining results showed that in SIRT3^+/+ group， the bone matrix of alveolar bone was uniformly green， the trabecular surface was smooth and continuous， a few scattered red granules were observed in the bone lacunae， the boundary between bone matrix and bone marrow cavity was clear without significant staining loss， and the collagen fibers were densely and neatly arranged without signs of fracture or dissolution； in SIRT3^-/- group， the green staining of alveolar bone matrix was significantly lighter and showed patchy loss， the collagen fibers were fractured and dissolved， the trabecular surface was rough and irregular， the red safranin positive area was significantly reduced or even disappeared， and multiple unstained blank areas appeared in the bone matrix， especially around bone resorption lacunae. Compared with SIRT3^+/+ group on the 7th and 14th days of modeling， the percentages of bone matrix degradation area of the mice in SIRT3^-/- group were significantly increased （P<0.01）. The TRAP staining results showed that on the 7th day of modeling， compared with SIRT3^+/+ group， the steoclast activity in periodontal tissue of the mice in SIRT3^-/- group was significantly increased （P<0.05）； on the 14th day of modeling， compared with SIRT3^+/+ group， the steoclast activity in periodontal tissue of the mice in SIRT3^-/- group was significantly increased （P<0.01）. The immunohistochemistry results showed that a large number of iNOS strongly positive cells were observed in periodontal tissue of the mice in SIRT3^-/- group， distributed in patches； while in SIRT3^+/+ group， there were few positive cells with light staining； CD163 positive cells in periodontal tissue of the mice in two groups were scattered and weakly stained. Compared with SIRT3^+/+ group， the expression levels of iNOS protein in periodontal tissue of the mice in SIRT3^-/- group was significantly increased （P<0.01）. The Western blotting results showed that compared with SIRT3^+/+ group， the expression levels of MMP-9 and p-P65 proteins in periodontal tissue of the mice in SIRT3^-/- group were significantly increased （P<0.05）， and the expression level of CTSK protein was significantly decreased （P<0.05）. The RT-qPCR method results showed that at 3rd day of modeling， compared with SIRT3^+/+ group， the expression levels of TNF-α， ACP5， MMP-9， and NFATC1 mRNA in periodontal tissues of the mice in SIRT3^-/- group were significantly increased （P<0.01）； on the 5th and 7th days of modeling， compared with SIRT3^+/+ group， the expression levels of ACP5 and MMP-9 mRNA in periodontal tissues of the mice in SIRT3^-/- group were significantly increased （P<0.01）. The Micro-CT scanning results showed that on the 14th day of periodontitis model， obvious alveolar bone resorption and relaxation and erosion of gingiva tissue were observed in the alveolar bone tissue of the mice. Compared with SIRT3^+/+ group， the surface area of alveolar bone damage of the mice in SIRT3^-/- group was significantly increased （P<0.05）. Conclusion SIRT3 deficiency aggravates alveolar bone resorption and periodontal tissue damage in a mouse periodontitis model， and the mechanism may be related to upregulation of MMP-9 and nuclear factor-kappa B（NF-κB） signaling pathways， promotion of macrophage polarization toward the pro-inflammatory M1 phenotype， and subsequent enhancement of osteoclast activity and bone matrix degradation.

Key words: Periodontitis, Sirtuin 3, Osteoclasts, Macrophages, Inducible nitric oxide synthase