敲除巨噬细胞Zfp260通过抑制M1型极化减轻小鼠牙周炎症

doi:10.12439/kqhm.1005-4979.2024.04.002

摘要/Abstract

摘要：

目的：研究锌指蛋白260（zinc finger protein 260，Zfp260/ZNF260）对巨噬细胞极化和牙周炎牙槽骨吸收的影响。方法：实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）检测人/野生型小鼠炎症牙周组织中ZNF260/Zfp260的表达。RT-qPCR检测RAW264.7细胞在炎症和非炎症环境中Zfp260的表达。繁育巨噬细胞内特异性敲除Zfp260小鼠，分别构建flox/flox对照鼠（Zfp260^f/f；Lyz2-cre^-）和条件性敲除（conditional knockout，cKO）鼠（Zfp260^f/f；Lyz2-cre⁺）牙周炎模型，micro-CT、苏木精-伊红（hematoxylin and eosin，HE）染色观察牙槽骨形态变化。RT-qPCR、蛋白质印迹法观察应用特异性小干扰RNA（small interfering RNA, siRNA）沉默Zfp260表达对RAW264.7极化的影响。RT-qPCR、免疫荧光染色观察牙周组织中M1、M2型极化标志分子的表达。结果：人/野生型小鼠炎症牙周组织中ZNF260/Zfp260的表达明显高于健康组。炎症环境下RAW264.7细胞中Zfp260的表达明显上调。cKO小鼠的牙周炎侧骨吸收小于flox/flox小鼠。沉默Zfp260表达可抑制RAW264.7在炎症环境下的M1型极化，且cKO小鼠的炎症牙周组织中M1型极化标志分子的表达明显少于flox/flox小鼠。结论：干扰巨噬细胞Zfp260的表达可抑制M1型极化并减轻牙周炎导致的牙槽骨吸收。

关键词: 锌指蛋白, 牙周炎, 巨噬细胞, 极化

Abstract:

Objective: To explore the effect of zinc finger protein 260 (Zfp260/ZNF260) on macrophage polarization and alveolar bone resorption due to periodontitis. Methods: Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of ZNF260/Zfp260 in inflammatory periodontal tissues of human/wild-type mice. The expression of Zfp260 in RAW264.7 cells in inflammatory or non-inflammatory environments was detected by RT-qPCR. Mice with specific knockout of Zfp260 were bred, and control mice (Zfp260^f/f; Lyz2-cre^-) and conditional knockout (cKO) mice (Zfp260^f/f; Lyz2-cre⁺) periodontitis models were established respectively. Morphological changes of alveolar bone were detected by micro-CT and hematoxylin-eosin (HE) staining. The polarization of RAW264.7 cells when Zfp260 was knocked down via specific small interfering RNA (siRNA) in an inflammatory environment was detected by RT-qPCR and western blotting. RT-qPCR and immunofluorescence staining were used to observe the expression of M1 and M2 macrophage-associated markers in periodontal tissues. Results: The expression of ZNF260/Zfp260 in inflammatory periodontal tissues of human/wild-type mice were significantly higher than that of healthy periodontal tissues. The expression of Zfp260 in RAW264.7 cells was significantly increased in an inflammatory environment. Alveolar bone resorption in the ligatured side of cKO mice was significantly less than that of flox/flox mice. The knockdown of Zfp260 in RAW264.7 cells could inhibit its M1 polarization in an inflammatory environment. The expression of M1-related markers in inflammatory periodontal tissues was significantly lower than that of flox/flox mice. Conclusion: Inhibition of Zfp260 in macrophage decreased M1 polarization and rescued the bone loss due to periodontitis.

Key words: zinc finger protein, periodontitis, macrophage, polarization

中图分类号:

R782

熊瑾, 王佐林. 敲除巨噬细胞Zfp260通过抑制M1型极化减轻小鼠牙周炎症[J]. 《口腔颌面外科杂志》, 2024, 34(4): 259-267.

XIONG Jin, WANG Zuolin. Knockout of macrophage Zfp260 attenuates periodontitis in mice via inhibiting M1-type polarization[J]. 《Journal of Oral and Maxillofacial Surgery》, 2024, 34(4): 259-267.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2024/V34/I4/259

图/表 7

表1 特异性小干扰RNA序列

Table 1 Specific siRNA sequence

siRNA名称		序列
si-Zfp260	靶向序列	GAATGTAAGGAGACCTTCA
	正义链	5'-GAAUGUAAGGAGACCUUCATT-3'
	反义链	5'-UGAAGGUCUCCUUACAUUCTT-3'
si-NC	靶向序列	无
	正义链	5'-UUCUGGGAACACGAGUGCUdTdT-3'
	反义链	5'-AGCACUCGUGUUCCCAGAAdTdT-3'

表2 RT-qPCR引物序列

Table 2 RT-qPCR primer sequence

基因名称	正向引物5'-3'	反向引物5'-3'
hsa-ZNF260	ATGTGGTAAAGTGTGCTCTCG	GGTGTCTGATGATGGTTGGCA
hsa-GAPDH	ACAACTTTGGTATCGTGGAAGG	GCCATCACGCCACAGTTTC
mmu-Zfp260	ATCTTCTGCATGACGAACCTG	GGCTTGTGAGGACTCTTCGC
mmu-GAPDH	AGGTCGGTGTGAACGGATTTG	TGTAGACCATGTAGTTGAGGTCA
mmu-IL-1β	GCAACTGTTCCTGAACTCAACT	ATCTTTTGGGGTCCGTCAACT
mmu-TNF-α	GGCAGGTTCTGTCCCTTTCA	CTGTGCTCATGGTGTCTTTTCTG
mmu-IL-6	TAGTCCTTCCTACCCCAATTTCC	TTGGTCCTTAGCCACTCCTTC
mmu-iNOS	GTTCTCAGCCCAACAATACAAGA	GTGGACGGGTCGATGTCAC
mmu-Arg1	GTCTGGCAGTTGGAAGCATCT	GCATCCACCCAAATGACACA
mmu-IL-10	GCTCTTACTGACTGGCATGAG	CGCAGCTCTAGGAGCATGTG
mmu-CD206	GGTGGAAGAAGAAGTAGCCT	GAAGGGTCAGTCTGTGTTTG
mmu-Ym1	CAGGTCTGGCAATTCTTCTGAA	GTCTTGCTCATGTGTGTAAGTGA

图1 ZNF260/Zfp260在牙周组织中的相对表达情况 A.健康组和牙周炎组人牙周组织中ZNF260（与小鼠Zfp260基因对应的人同源基因）的相对表达水平；B.健康组和牙周炎组小鼠牙周组织中Zfp260的相对表达水平。**表示P<0.01，***表示P<0.001，与健康组比较。

Figure 1 The relative expression of ZNF260/Zfp260 in periodontal tissues

图2 炎症和非炎症环境中Zfp260在RAW264.7细胞中的相对表达情况 A. M1极化标志分子IL-1β、TNF-α、IL-6、iNOS经Pg-LPS处理后在RAW264.7细胞中的相对表达量；B. Zfp260经Pg-LPS处理后在RAW264.7细胞中的相对表达量。***表示P<0.001，与空白对照组比较。

Figure 2 The relative expression of Zfp260 in RAW264.7 cells in inflammatory or non-inflammatory environments

图3 牙槽骨吸收情况 A.各组小鼠下颌骨三维重建结果，从左至右依次为颊侧面、冠状面、舌侧面及水平面的截图，CEJ-ABC的距离用红色细线表示；B. micro-CT结果示牙槽骨吸收统计结果；C.各组小鼠下颌第一磨牙根分叉区剩余牙槽骨面积统计结果；D. HE染色结果的牙槽骨吸收统计结果；E.各组小鼠下颌牙周组织的HE染色结果，黑色箭头表示CEJ-ABC的距离(×40，×100)。***表示P<0.001，与牙周炎组比较；#表示P<0.05，与flox/flox小鼠牙周炎组比较。

Figure 3 Alveolar bone resorption

图4 沉默Zfp260表达后RAW264.7细胞M1、M2极化标志分子的表达情况 A. Zfp260在各组细胞中的相对表达量；B.炎症环境中IL-1β、TNF-α、IL-6、IL-10和CD206在各组细胞中的相对表达量；C.炎症环境中各组细胞CD86蛋白表达条带；D.炎症环境中各组细胞CD86蛋白表达量的统计学分析；E.炎症环境中各组细胞Arg1蛋白表达条带；F.炎症环境中各组细胞Arg1蛋白表达量的统计学分析。**表示P<0.01，***表示P<0.001，与si-NC转染组比较。

Figure 4 The expression of M1, M2-related markers in RAW264.7 cells after knockdown of Zfp260

图5 牙周组织中M1、M2型标志分子表达情况 A.各组小鼠下颌第一磨牙牙周组织中的M1、M2型标志分子的相对表达量；B、C.各组小鼠下颌牙周组织的免疫荧光染色；蓝色为细胞核，红色为CD86阳性信号（图5B），绿色为CD206阳性信号（图5C），阳性信号均用白色箭头指示（×400）。D. 2组小鼠CD86⁺细胞统计学分析；E. 2组小鼠CD206⁺细胞统计学分析。*表示P<0.05，***表示P<0.001，与flox/flox牙周炎组比较。

Figure 5 The expression of M1, M2-related markers in periodontal tissues

参考文献 23

[1]	Tonetti MS, Greenwell H, Kornman KS. Staging and grading of periodontitis: Framework and proposal of a new classification and case definition[J]. J Periodontol, 2018, 89(Suppl 1): S159-S172.
[2]	Renn TY, Huang YK, Feng SW, et al. Prophylactic supplement with melatonin successfully suppresses the pathogenesis of periodontitis through normalizing RANKL/OPG ratio and depressing the TLR4/MyD88 signaling pathway[J]. J Pineal Res, 2018, 64(3): e12464.
[3]	Janakiram C, Dye BA. A public health approach for prevention of periodontal disease[J]. Periodontol 2000, 2020, 84(1): 202-214. doi: 10.1111/prd.12337 pmid: 32844412
[4]	Chukkapalli SS, Ambadapadi S, Varkoly K, et al. Impaired innate immune signaling due to combined Toll-like receptor 2 and 4 deficiency affects both periodontitis and atherosclerosis in response to polybacterial infection[J]. Pathog Dis, 2018, 76(8): fty076.
[5]	Sun XY, Mao YX, Dai PP, et al. Mitochondrial dysfunction is involved in the aggravation of periodontitis by diabetes[J]. J Clin Periodontol, 2017, 44(5): 463-471. doi: 10.1111/jcpe.12711 pmid: 28207937
[6]	Locati M, Curtale G, Mantovani A. Diversity, mechanisms, and significance of macrophage plasticity[J]. Annu Rev Pathol, 2020, 15: 123-147. doi: 10.1146/annurev-pathmechdis-012418-012718 pmid: 31530089
[7]	Murray PJ, Allen JE, Biswas SK, et al. Macrophage activation and polarization: Nomenclature and experimental guidelines[J]. Immunity, 2014, 41(1): 14-20. doi: 10.1016/j.immuni.2014.06.008 pmid: 25035950
[8]	Cheng YH, Tian YY, Xia JL, et al. The role of PTEN in regulation of hepatic macrophages activation and function in progression and reversal of liver fibrosis[J]. Toxicol Appl Pharmacol, 2017, 317: 51-62.
[9]	Yao YL, Xu XH, Jin LP. Macrophage polarization in physiological and pathological pregnancy[J]. Front Immunol, 2019, 10: 792. doi: 10.3389/fimmu.2019.00792 pmid: 31037072
[10]	Debrus S, Rahbani L, Marttila M, et al. The zinc finger-only protein Zfp260 is a novel cardiac regulator and a nuclear effector of alpha1-adrenergic signaling[J]. Mol Cell Biol, 2005, 25(19): 8669-8682. pmid: 16166646
[11]	Grisanti LA, Woster AP, Dahlman J, et al. α1-adrenergic receptors positively regulate Toll-like receptor cytokine production from human monocytes and macrophages[J]. J Pharmacol Exp Ther, 2011, 338(2): 648-657. doi: 10.1124/jpet.110.178012 pmid: 21571945
[12]	Sima C, Glogauer M. Macrophage subsets and osteoimmunology: Tuning of the immunological recognition and effector systems that maintain alveolar bone[J]. Periodontol 2000, 2013, 63(1): 80-101. doi: 10.1111/prd.12032 pmid: 23931056
[13]	Gamonal J, Acevedo A, Bascones A, et al. Levels of interleukin-1 beta, -8, and-10 and RANTES in gingival crevicular fluid and cell populations in adult periodontitis patients and the effect of periodontal treatment[J]. J Periodontol, 2000, 71(10): 1535-1545. doi: 10.1902/jop.2000.71.10.1535 pmid: 11063385
[14]	孙妩弋, 孙家昌, 厉歆然, 等. Arrb2基因敲除小鼠的繁育及基因型鉴定[J]. 中国药理学通报, 2018, 34(6): 878-881.
[15]	Jurado Acosta A, Rysä J, Szabo Z, et al. Transcription factor PEX1 modulates extracellular matrix turnover through regulation of MMP-9 expression[J]. Cell Tissue Res, 2017, 367(2): 369-385. doi: 10.1007/s00441-016-2527-2 pmid: 27826738
[16]	Komati H, Maharsy W, Beauregard J, et al. Zfp260 is an inducer of cardiac hypertrophy and a nuclear mediator of endothelin-1 signaling[J]. J Biol Chem, 2011, 286(2): 1508-1516. doi: 10.1074/jbc.M110.162966 pmid: 21051538
[17]	Khader YS, Dauod AS, El-Qaderi SS, et al. Periodontal status of diabetics compared with nondiabetics: A meta-analysis[J]. J Diabetes Complications, 2006, 20(1): 59-68.
[18]	Orlandi M, Graziani F, D'Aiuto F. Periodontal therapy and cardiovascular risk[J]. Periodontol 2000, 2020, 83(1): 107-124. doi: 10.1111/prd.12299 pmid: 32385887
[19]	Bally M, Dendukuri N, Rich B, et al. Risk of acute myocardial infarction with NSAIDs in real world use: Bayesian meta-analysis of individual patient data[J]. BMJ, 2017, 357: j1909.
[20]	Chun YH, Chun KR, Olguin D, et al. Biological foundation for periodontitis as a potential risk factor for atherosclerosis[J]. J Periodontal Res, 2005, 40(1): 87-95.
[21]	Viniegra A, Goldberg H, Çil Ç, et al. Resolving macrophages counter osteolysis by anabolic actions on bone cells[J]. J Dent Res, 2018, 97(10): 1160-1169. doi: 10.1177/0022034518777973 pmid: 29993312
[22]	Zhuang Z, Yoshizawa-Smith S, Glowacki A, et al. Induction of M2 macrophages prevents bone loss in murine periodontitis models[J]. J Dent Res, 2019, 98(2): 200-208. doi: 10.1177/0022034518805984 pmid: 30392438
[23]	Wu X, Chen H, Wang Y, et al. Akt2 affects periodontal inflammation via altering the M1/M2 ratio[J]. J Dent Res, 2020, 99(5): 577-587. doi: 10.1177/0022034520910127 pmid: 32228353

选择文件类型/文献管理软件名称

选择包含的内容