NAT10对小鼠巨噬细胞极化影响的实验研究

doi:10.12439/kqhm.1005-4979.2025.04.002

摘要/Abstract

摘要：

目的：探讨N-乙酰转移酶10（N-acetyltransferase 10，NAT10）基因对小鼠巨噬细胞极化的影响。方法：体外培养小鼠单核细胞系RAW264.7细胞并对其进行巨噬细胞极化诱导，采用实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）检测NAT10的基因相对表达水平；选用Remodelin hydrobromide（NAT10抑制剂）抑制NAT10表达，并观察其对RAW264.7细胞极化的影响。采用丝线结扎法构建小鼠牙周炎模型并同期注射Remodelin hydrobromide，将其随机分为对照组、牙周炎组、对照给药组和牙周炎给药组，7 d后取下颌骨行RT-qPCR、micro-CT检查及组织学染色。结果：NAT10在小鼠M2型巨噬细胞中高表达；与对照组相比，牙周炎组牙槽骨组织中NAT10表达量下降，根分叉处NAT10阳性细胞占总细胞比例和NAT10、F4/80双阳细胞占比均降低。浓度梯度实验显示10 μmol/L的Remodelin hydrobromide抑制效率最好，加入Remodelin hydrobromide后，RAW264.7细胞中M1型极化相关基因表达增加，M2型极化相关基因表达下降。抑制NAT10表达后，小鼠牙周炎模型中牙槽骨破坏加剧；根分叉处CD86阳性细胞比例增加；CD163阳性细胞占比下降。结论：NAT10在RAW264.7细胞M2型极化后表达量显著增加，在小鼠牙周炎牙槽骨中表达量显著下降。抑制NAT10表达可促进巨噬细胞M1型极化，抑制M2型极化，且可以显著加剧牙周炎小鼠的牙槽骨破坏。

Abstract:

Objective: To investigate the effect of N-acetyltransferase 10 (NAT10) gene on the polarization of mouse macrophages in vitro and in vivo. Methods: Mouse monocyte line RAW264.7 cells were cultured in vitro and induced by polarization, and the expression level of NAT10 was observed using real-time quantitative polymerase chain reaction (RT-qPCR). The NAT10 inhibitor Remodelin hydrobromide was used to inhibit the expression of NAT10, and its effect on the polarization of RAW264.7 cells was observed. A model of periodontitis was established using silk thread ligation, while Remodelin hydrobromide was injected simultaneously. The mice were randomly divided into control group, periodontitis group, control medication group and periodontitis medication group. After 7 days, their mandibles were taken for RT-qPCR, micro-CT analysis and histological staining. Results: NAT10 was highly expressed in mouse M2 macrophages. Compared with the control group, the periodontitis group showed decreased NAT10 expression in alveolar bone tissue, along with reduced proportions of NAT10-positive cells in the furcation area and lower percentages of NAT10/F4/80 double-positive cells. The concentration gradient experiment showed that 10 μmol/L Remodelin hydrobromide had the best inhibition efficiency. After adding Remodelin hydrobromide, the expression of M1 macrophage-associated genes increased and M2 macrophage-associated genes decreased in RAW264.7 cells. Inhibition of NAT10 expression enhanced alveolar bone destruction in a mouse model of periodontitis; the proportion of CD86-positive cells at the root bifurcation increased; the proportion of CD163-positive cells decreased. Conclusion: The expression of NAT10 significantly increased in RAW264.7 cells after M2 polarization, and significantly decreased in the mouse periodontitis alveolar bone. Inhibiting the expression of NAT10 can promote M1 polarization of macrophages, inhibit M2 polarization, and notably exacerbate alveolar bone destruction in mice with periodontitis.

中图分类号:

张贤中, 王海丞, 陈永亮, 王佐林. NAT10对小鼠巨噬细胞极化影响的实验研究[J]. 《口腔颌面外科杂志》, 2025, 35(4): 251-259.

ZHANG Xianzhong, WANG Haicheng, CHEN Yongliang, WANG Zuolin. Study on the effect of NAT10 on the polarization of macrophages in mice[J]. 《Journal of Oral and Maxillofacial Surgery》, 2025, 35(4): 251-259.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2025/V35/I4/251

图/表 7

表1 RT-qPCR引物序列

Table 1 primer sequences of RT-qPCR

基因名称	正向引物(5'-3')	反向引物(5'-3')
β-actin	GGCTGTATTCCCCTCCATCG	CCAGTTGGTAACAATGCCATGT
NAT10	GCGGCAGAGGTCTCTTTTTGT	GTGACTGCTAAATCCCAGCTC
CD80	ACCCCCAACATAACTGAGTCT	TTCCAACCAAGAGAAGCGAGG
CD86	TGTTTCCGTGGAGACGCAAG	TTGAGCCTTTGTAAATGGGCA
INOS	GTTCTCAGCCCAACAATACAAGA	GTGGACGGGTCGATGTCAC
CD163	ATGGGTGGACACAGAATGGTT	CAGGAGCGTTAGTGACAGCAG
CD206	CTCTGTTCAGCTATTGGACGC	CGGAATTTCTGGGATTCAGCTTC
Arg1	CTCCAAGCCAAAGTCCTTAGAG	AGGAGCTGTCATTAGGGACATC

图1 NAT10在RAW264.7细胞中的表达 A.光学显微镜下RAW264.7细胞形态；B—G.诱导24 h后3组细胞表面标志物基因相对表达量；H.诱导24 h后NAT10在细胞中的基因相对表达量。*表示P<0.05，**表示P<0.01，***表示P<0.001，与M0组比较；###表示P<0.001，与M1组比较。

Figure 1 Expression of NAT10 in RAW264.7 cells

图2 NAT10在对照组和牙周炎组中的表达情况 A.造模7 d后小鼠下颌骨HE染色图（×40），黄色箭头表示邻间隙的牙槽骨；B.建模7 d后NAT10在下颌牙槽骨中的基因相对表达量；C. 2组小鼠下颌第一磨牙根分叉处免疫荧光染色图（×100），右侧图片（×250）为左侧矩形框内放大图，黄色箭头表示被NAT10抗体染色的细胞；D. 2组小鼠NAT10⁺细胞比例比较；E. 2组小鼠下颌第一磨牙根分叉处免疫荧光染色图（×100），右侧图片（×250）为左侧矩形框内放大图，黄色箭头表示同时被NAT10抗体及F4/80抗体染色的细胞；F. 2组小鼠双阳细胞占比比较。***表示P<0.001，与对照组比较。

Figure 2 Expression of NAT10 in the control group and periodontitis group

图3 不同浓度的Remodelin hydrobromide对NAT10表达的抑制效果 A.处理24 h后，不同浓度的Remodelin hydrobromide对NAT10表达的抑制效果，*表示P<0.01，***表示P<0.001，与0 μmol/L组比较；B. Remodelin hydrobromide的抑制效率。

Figure 3 Inhibitory effect of different concentrations of Remodelin hydrobromide on NAT10 expression

图4 抑制NAT10对RAW264.7细胞极化的影响 A、B. 10 μmol/L Remodelin hydrobromide抑制NAT10 24 h后，M1、M2型巨噬细胞相关基因的基因相对表达量。**表示P<0.01，***表示P<0.001，与0 μmol/L组比较。

Figure 4 Effect of Remodelin hydrobromide on polarization of RAW264.7 cells after inhibiting NAT10

图5 基于micro-CT测量的小鼠牙槽骨破坏情况 A.建模成功后4组小鼠micro-CT重建及测量方法示意图，绿色线段为小鼠下颌第一磨牙近远中根最膨隆处连线，红色线段为根分叉牙槽骨高度，蓝色线段为小鼠下颌第一磨牙釉牙骨质界连线，黄色线段为牙槽嵴顶骨吸收高度；B—D. 4组小鼠根分叉牙槽骨高度的比较；E—G. 4组小鼠牙槽嵴顶骨吸收高度的比较；H—J. 4组小鼠牙槽骨吸收面积的比较。*表示P<0.05，**表示P<0.01，***表示P<0.001，与对照组比较；#表示P<0.05，##表示P<0.01，与牙周炎组比较。

Figure 5 Alveolar bone destruction in mice assessed by micro-CT

图6 免疫荧光染色显示M1、M2型巨噬细胞在各组中的分布情况 A.建模成功后4组小鼠下颌第一磨牙根分叉处免疫荧光染色（×200），黄色箭头代表染色明显的细胞；B—D. M1型巨噬细胞占总细胞比例变化；E—G.M2型巨噬细胞占总细胞比例变化。*表示P<0.05，**表示P<0.01，***表示P<0.001，与对照组比较；#表示P<0.05，###表示P<0.001，与牙周炎组比较。

Figure 6 Quantitative analysis of M1 and M2 macrophage distribution in each group by immunofluorescence

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