SIRT3对牙周炎模型小鼠破骨细胞分化和巨噬细胞活化的抑制作用及其机制

doi:10.13481/j.1671-587X.20260301

吉林大学学报(医学版) ›› 2026, Vol. 52 ›› Issue (3): 581-589.doi: 10.13481/j.1671-587X.20260301

• 基础研究 • 下一篇

SIRT3对牙周炎模型小鼠破骨细胞分化和巨噬细胞活化的抑制作用及其机制

沈佳琪¹,葛玘²,姚秀³,雷长海⁴()

^1.海军军医大学基础医学院组织学与胚胎学教研室，上海 200433
^2.海军军医大学第一附属医院口腔科，上海 200433
^3.上海口腔医院口腔医学院口腔种植科，上海 200001
^4.海军军医大学基础医学院生物物理系，上海 200433

收稿日期:2024-12-13 接受日期:2025-02-23 出版日期:2026-05-28 发布日期:2026-06-08
通讯作者: 雷长海 E-mail:lei@smmu.edu.cn
作者简介:沈佳琪（1996-），男，内蒙古自治区赤峰市人，在读硕士研究生，主要从事炎症反应与免疫机制方面的研究。
基金资助:
国家自然科学基金项目(32471123)

Inhibitory effect of SIRT3 on osteoclast differentiation and macrophage activation in periodontitis model of mice and its mechanism

Jiaqi SHEN¹,Qi GE²,Xiu YAO³,Changhai LEI⁴()

^1.Department of Histology and Embryology，School of Basic Medical Sciences，Naval Medical University，Shanghai 200433，China
^2.Department of Stomatology，First Affiliated Hospital，Naval Medical University，Shanghai 200433，China
^3.Department of Oral Implantology，School of Stomatology，Shanghai Stomatological Hospital，Shanghai 200001，China
^4.Department of Biophysics，School of Basic Medical Sciences，Naval Medical University，Shanghai 200433，China

Received:2024-12-13 Accepted:2025-02-23 Online:2026-05-28 Published:2026-06-08
Contact: Changhai LEI E-mail:lei@smmu.edu.cn

摘要/Abstract

摘要：

目的探讨沉默调节蛋白3（SIRT3）缺失对牙周炎小鼠牙周组织病理损伤、破骨细胞活性及巨噬细胞极化的影响，并初步阐明其分子机制。方法 12只C57BL/6小鼠随机分为SIRT3野生型（WT）小鼠组（SIRT3^+/+组）和SIRT3基因敲除小鼠组（SIRT3^―/―组），每组6只。建立小鼠牙周炎模型，分别于建模第7和14天取材。采用HE染色、番红O固绿染色和抗酒石酸酸性磷酸酶（TRAP）染色观察2组小鼠牙周组织病理形态表现及破骨细胞分布，免疫组织化学染色法检测2组小鼠牙周组织中诱导型一氧化氮合酶（iNOS）和分化集群163（CD163）蛋白表达水平，Western blotting法检测2组小鼠牙周组织中基质金属蛋白酶9（MMP-9）、磷酸化P65（p-P65）和组织蛋白酶K（CTSK）蛋白表达水平，实时荧光定量PCR（RT-qPCR）法检测2组小鼠牙周组织中耐酒石酸酸性磷酸酶（ACP5）、MMP-9、核因子活化7淋巴细胞胞质1（NFATC1）和肿瘤坏死因子α（TNF-α）mRNA表达水平，Micro-CT扫描分析2组小鼠牙周组织牙槽骨微观结构变化和牙槽骨损伤表面积。结果 HE染色，SIRT3^+/+组小鼠牙周组织中牙周膜排列整齐，炎性浸润及骨吸收陷窝较少；SIRT3^―/―组小鼠牙周组织中牙周膜纤维排列紊乱、牙周膜水肿增宽，大量中性粒细胞及淋巴细胞弥漫浸润，牙槽骨缘呈蚕食状缺损，可见多核破骨细胞；建模第7天，与SIRT3^+/+组比较，SIRT3^―/―组小鼠牙周膜区域炎症面积百分率明显升高（P<0.05）；建模第14天，与SIRT3^+/+组比较，SIRT3^―/―组小鼠牙周膜区域炎症面积百分率明显升高（P<0.01）。番红O固绿染色，SIRT3^+/+组小鼠牙槽骨骨基质呈均匀的绿色结构，骨小梁表面光滑连续，骨陷窝内可见少量散在的红色颗粒，骨基质与骨髓腔界限清晰，无显著染色缺失区，胶原纤维排列致密整齐，未见断裂或溶解迹象；SIRT3^―/―组小鼠牙槽骨骨基质绿色染色明显变淡、呈斑片状缺失，胶原纤维出现断裂、溶解，骨小梁表面粗糙不整，红色番红O阳性区域显著减少甚至消失，骨基质内出现多处无着色的空白区域，尤以骨吸收陷窝周围为著。建模第7和14天，与SIRT3^+/+组比较，SIRT3^―/―组小鼠骨基质降解面积百分率均明显升高（P<0.01）。TRAP染色，建模第7天，与SIRT3^+/+组比较，SIRT3^―/―组小鼠牙周组织中破骨细胞活性明显升高（P<0.05）；建模第14天，与SIRT3^+/+组比较，SIRT3^―/―组小鼠牙周组织中破骨细胞活性明显升高（P<0.01）。免疫组织化学染色法，SIRT3^―/―组小鼠牙周组织可见大量iNOS强阳性细胞，呈片状分布；SIRT3^+/+组小鼠牙周组织中阳性细胞少、染色浅；2组小鼠牙周组织中CD163阳性细胞均少量散在，染色强度弱。与SIRT3^+/+组比较，SIRT3^―/―组小鼠牙周组织中iNOS蛋白表达水平明显升高（P<0.01）。Western blotting法，与SIRT3^+/+组比较，SIRT3^―/―组小鼠牙周组织中MMP-9和p-P65蛋白表达水平均明显升高（P<0.05），CTSK蛋白表达水平明显降低（P<0.05）。RT-qPCR法，牙周炎模型构建第3天，与SIRT3^+/+组比较，SIRT3^―/―组小鼠牙周组织中TNF-α、ACP5、MMP-9和NFATC1 mRNA表达水平明显升高（P<0.01）；第5和7天时，与SIRT3^+/+组比较，SIRT3^―/―组小鼠牙周组织中ACP5和MMP-9 mRNA表达水平明显升高（P<0.01）。Micro-CT扫描，在建模第14天小鼠牙周组织中表现出明显的牙槽骨吸收和牙龈组织的松弛及侵蚀；与SIRT3^+/+组比较，SIRT3^―/―组小鼠牙槽骨损伤表面积明显增加（P<0.05）。结论 SIRT3缺失可加重大鼠牙周炎模型中的牙槽骨吸收及牙周组织损伤，其机制可能与上调MMP-9和核因子κB（NF-κB）信号通路，促进巨噬细胞向促炎的M1表型极化，进而增强破骨细胞活性及骨基质降解有关。

关键词: 牙周炎, 沉默调节蛋白3, 破骨细胞, 巨噬细胞, 诱导型一氧化氮合酶

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

中图分类号:

沈佳琪,葛玘,姚秀,雷长海. SIRT3对牙周炎模型小鼠破骨细胞分化和巨噬细胞活化的抑制作用及其机制[J]. 吉林大学学报(医学版), 2026, 52(3): 581-589.

Jiaqi SHEN,Qi GE,Xiu YAO,Changhai LEI. Inhibitory effect of SIRT3 on osteoclast differentiation and macrophage activation in periodontitis model of mice and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2026, 52(3): 581-589.

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参考文献 23

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Group	ACP5 mRNA	MMP-9 mRNA	NFATC1 mRNA	TNF-α mRNA
SIRT3^+/+
Day 1	0.003±0.009	0.011±0.002	0.000±0.000	0.000±0.000
Day 3	0.005±0.009	0.047±0.015	0.013±0.002	0.037±0.030
Day 5	2.274±0.013	0.684±0.004	0.007±0.001	0.018±0.028
Day 7	0.908±0.003	0.952±0.027	0.004±0.001	0.021±0.004
SIRT3^-/-
Day 1	0.009±0.004	0.004±0.002	0.009±0.000	0.002±0.002
Day 3	0.523±0.018^*	0.565±0.022^*	0.040±0.000^*	0.063±0.028^*
Day 5	2.720±0.011^*	0.810±0.002^*	0.007±0.000	0.015±0.003
Day 7	1.486±0.005^*	1.553±0.024^*	0.003±0.000	0.026±0.008

SIRT3对牙周炎模型小鼠破骨细胞分化和巨噬细胞活化的抑制作用及其机制

Inhibitory effect of SIRT3 on osteoclast differentiation and macrophage activation in periodontitis model of mice and its mechanism

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