microRNA-146a对牙龈卟啉单胞菌脂多糖刺激下淋巴细胞分泌细胞因子的调节作用

doi:10.7518/hxkq.2021.01.004

华西口腔医学杂志 ›› 2021, Vol. 39 ›› Issue (1): 26-31.doi: 10.7518/hxkq.2021.01.004

microRNA-146a对牙龈卟啉单胞菌脂多糖刺激下淋巴细胞分泌细胞因子的调节作用

司雨婷¹^,²(), 宋金花¹^,², 方珍², 韩晓哲³, 蒋少云¹^,²()

^1.天津医科大学口腔医院牙周科，天津 300070
^2.北京大学深圳医院口腔医学中心牙周科，深圳 518036
^3.美国哈佛大学牙学院Forsyth研究所，美国剑桥 02146

收稿日期:2020-02-17 修回日期:2020-10-23 出版日期:2021-02-01 发布日期:2021-03-02
通讯作者: 蒋少云 E-mail:siyutingsyt401@163.com;jiangshaoyun11@126.com
作者简介:司雨婷，硕士，E-mail：siyutingsyt401@163.com
基金资助:
国家自然科学基金(81670990);北京大学深圳医院院内基础研究基金(JCYJ2019004RC);美国国立口腔与颅面研究基金(R01DE025255)

MicroRNA-146a regulates the production of cytokines in lymphocytes stimulated by Porphyromonas gingivalis lipopolysaccharide

Si Yuting¹^,²(), Song Jinhua¹^,², Fang Zhen², Han Xiaozhe³, Jiang Shaoyun¹^,²()

^1.Dept. of Periodontology, Hospital of Stomatology, Tianjin Medical University, Tianjin 300070, China
^2.Dept. of Periodontology, Stomatological Center, Peking University Shenzhen Hospital, Shenzhen 518036, China
^3.The Forsyth Institute, Harvard School of Dental Medicine, Cambridge 02146, USA

Received:2020-02-17 Revised:2020-10-23 Online:2021-02-01 Published:2021-03-02
Contact: Jiang Shaoyun E-mail:siyutingsyt401@163.com;jiangshaoyun11@126.com
Supported by:
The National Natural Science Foundation of China(81670990);Basic Research Grand of Peking University Shenzhen Hospital(JCYJ2019004RC);National Institute of Dental and Craniofacial Research, The United States of America(R01DE025255)

摘要/Abstract

摘要： 目的

探讨microRNA-146a（miR-146a）对牙龈卟啉单胞菌（P.gingivalis）脂多糖（LPS）刺激下淋巴细胞分泌细胞因子的作用。

方法

从小鼠脾脏收集淋巴细胞。实时定量聚合酶链反应和酶联免疫反应用于检测淋巴细胞在P.gingivalis LPS、miR-146a模拟物或抑制剂处理后细胞因子的表达。

结果

与P.gingivalis LPS未刺激组相比，P.gingivalis LPS能促进白细胞介素（interleukin，IL）-1β、IL-6、细胞核因子κB受体活化因子配体（RANKL）和IL-10表达（P<0.05），虽抑制骨保护素（OPG）mRNA水平（P<0.05），但分泌水平差异无统计学意义（P>0.05）。与阴性对照组相比，P.gingivalis LPS处理的淋巴细胞中miR-146a模拟物抑制IL-1β、IL-6和RANKL表达（P<0.05），促进IL-10和OPG表达（P<0.05），miR-146a抑制剂对这些细胞因子产生相反的效果（P<0.05）。

结论

miR-146a可通过抑制淋巴细胞IL-1β、IL-6和RANKL表达，促进IL-10和OPG表达，从而抑制P.gingivalis LPS的炎性作用，为骨改建提供良好的成骨环境。

关键词: 牙周炎, microRNA-146a, 细胞因子, 淋巴细胞, 脂多糖

Abstract: Objective

This study aimed to investigate the effects of microRNA-146a (miR-146a) on the production of cytokines in lymphocytes stimulated by Porphyromonas gingivalis (P.gingivalis) lipopolysaccharide (LPS).

Methods

Lymphocytes were harvested from mouse spleen and cultured in vitro. The cells were treated with P. gingivalis LPS, miR-146a mimic, or miR-146a inhibitor. Scramble RNA served as the negative control of mimic and inhibitor. The production of inflammatory cytokines was detected by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay.

Results

Compared with non-LPS-stimulated group, P. gingivalis LPS could increase the levels of interleukin (IL)-1β, IL-6, receptor activator NF-κB ligand (RANKL), and IL-10 (P<0.05) and decrease the mRNA level of osteoprotectin (OPG) (P<0.05). However, it did not significantly change the secretion of OPG. Compared with the negative control group, miR-146a mimic upregulated the levels of IL-10 and OPG (P<0.05), downregulated IL-1β, IL-6, and RANKL (P<0.05). Meanwhile, miR-146a inhibitor had a reverse effect on these cytokines (P<0.05) in P.gingivalis LPS-treated-lymphocytes.

Conclusion

MiR-146a can provide a suitable microenvironment for bone formation by preventing the inflammatory effects of P.gingivalis LPS through the inhibition of IL-1β, IL-6, and RNAKL, thereby enhancing IL-10 and OPG.

Key words: periodontitis, microRNA-146a, cytokine, lymphocytes, lipopolysaccharide

中图分类号:

Q 257

司雨婷, 宋金花, 方珍, 韩晓哲, 蒋少云. microRNA-146a对牙龈卟啉单胞菌脂多糖刺激下淋巴细胞分泌细胞因子的调节作用[J]. 华西口腔医学杂志, 2021, 39(1): 26-31.

Si Yuting, Song Jinhua, Fang Zhen, Han Xiaozhe, Jiang Shaoyun. MicroRNA-146a regulates the production of cytokines in lymphocytes stimulated by Porphyromonas gingivalis lipopolysaccharide[J]. West China Journal of Stomatology, 2021, 39(1): 26-31.

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参考文献 29

1	Cekici A, Kantarci A, Hasturk H, et al. Inflammatory and immune pathways in the pathogenesis of periodontal disease[J]. Periodontol 2000, 2014, 64(1): 57-80.
2	Gonzales JR. T- and B-cell subsets in periodontitis[J]. Periodontol 2000, 2015, 69(1): 181-200.
3	Oliver-Bell J, Butcher JP, Malcolm J, et al. Periodontitis in the absence of B cells and specific anti-bacterial antibody[J]. Mol Oral Microbiol, 2014, 30(2): 160-169.
4	Zouali M. The emerging roles of B cells as partners and targets in periodontitis[J]. Autoimmunity, 2017, 50(1): 61-70.
5	Marques-Rocha JL, Samblas M, Milagro FI, et al. Noncoding RNAs, cytokines, and inflammation-related diseases[J]. FASEB J, 2015, 29(9): 3595-3611.
6	Tomofuji T, Yoneda T, Machida T, et al. MicroRNAs as serum biomarkers for periodontitis[J]. J Clin Periodontol, 2016, 43(5): 418-425.
7	Bourbour S, Beheshti M, Kazemian H, et al. Effects of microRNAs and their targets in periodontal diseases[J]. Infect Disord Drug Targets, 2018, 18(3): 183-191.
8	Xie YF, Shu R, Jiang SY, et al. Comparison of micro-RNA profiles of human periodontal diseased and healthy gingival tissues[J]. Int J Oral Sci, 2011, 3(3): 125-134.
9	Luan X, Zhou X, Naqvi A, et al. MicroRNAs and immunity in periodontal health and disease[J]. Int J Oral Sci, 2018, 10(3): 24.
10	Na HS, Park MH, Song YR, et al. Elevated microRNA-128 in periodontitis mitigates tumor necrosis factor-α responsevia p38 signaling pathway in macrophages[J]. J Periodontol, 2016, 87(9): e173-e182.
11	Fordham JB, Naqvi AR, Nares S. miR-24 regulates macrophage polarization and plasticity[J]. J Clin Cell Immunol, 2015, 6(5): 362.
12	Pratama A, Srivastava M, Williams NJ, et al. MicroRNA-146a regulates ICOS-ICOSL signaling to limit accumulation of T follicular helper cells and germinal centres[J]. Nat Commun, 2015, 6: 6436.
13	Zhang J, Jia G, Liu Q, et al. Silencing miR-146a influences B cells and ameliorates experimental autoimmune myasthenia gravis[J]. Immunology, 2015, 144(1): 56-67.
14	Han X, Lin X, Yu X, et al. Porphyromonas gingivalis infection-associated periodontal bone resorption is dependent on receptor activator of NF-κB ligand[J]. Infect Immun, 2013, 81(5): 1502-1509.
15	Jiang SY, Xue D, Xie YF, et al. The negative feedback regulation of microRNA-146a in human periodontal ligament cells after Porphyromonas gingivalis lipopolysaccharide stimulation[J]. Inflamm Res, 2015, 64(6): 441-451.
16	Xie YF, Shu R, Jiang SY, et al. MicroRNA-146 inhibits pro-inflammatory cytokine secretion through IL-1 receptor-associated kinase 1 inhuman gingival fibroblasts[J]. J Inflamm, 2013, 10(1): 20.
17	Jiang SY, Hu Y, Deng S, et al. miR-146a regulates inflammatory cytokine production in Porphyromonas gingivalis lipopolysaccharide-stimulated B cells by targeting IRAK1 but not TRAF6[J]. Biochim Biophys Acta Mol Basis Dis, 2018, 1864(3): 925-933.
18	Yu X, Hu Y, Freire M, et al. Role of toll-like receptor 2 in inflammation and alveolar bone loss in experimental peri-implantitis versus periodontitis[J]. J Periodont Res, 2018, 53(1): 98-106.
19	Lin J, Bi L, Yu X, et al. Porphyromonas gingivalis exacerbates ligature-induced, RANKL-dependent alveolar bone resorption via differential regulation of Toll-like receptor 2 (TLR2) and TLR4[J]. Infect Immun, 2014, 82(10): 4127-4134.
20	Liukkonen J, Gürsoy UK, Pussinen PJ, et al. Salivary concentrations of interleukin (IL)-1β, IL-17A, and IL-23 vary in relation to periodontal status[J]. J Periodontol, 2016, 87(12): 1484-1491.
21	Stadler AF, Angst PD, Arce RM, et al. Gingival crevicular fluid levels of cytokines/chemokines in chronic periodontitis: a meta-analysis[J]. J Clin Periodontol, 2016, 43(9): 727-745.
22	Mao CY, Wang YG, Zhang X, et al. Double-edged-sword effect of IL-1β on the osteogenesis of periodontal ligament stem cells via crosstalk between the NF-κB, MAPK and BMP/Smad signaling pathways[J]. Cell Death Dis, 2016, 7(7): e2296.
23	Yu T, Zhao L, Huang X, et al. Enhanced activity of the macrophage M1/M2 phenotypes and phenotypic switch to M1 in periodontal infection[J]. J Periodontol, 2016, 87(9): 1092-1102.
24	Zhang Q, Chen B, Zhu D, et al. Biomarker levels in gingival crevicular fluid of subjects with different periodontal conditions: a cross-sectional study[J]. Arch Oral Biol, 2016, 72: 92-98.
25	Zhang Q, Chen B, Yan F, et al. Interleukin-10 inhibits bone resorption: a potential therapeutic strategy in periodontitis and other bone loss diseases[J]. Biomed Res Int, 2014, 2014: 284836.
26	Yu P, Hu Y, Liu Z, et al. Local induction of B cell interleukin-10 competency alleviates inflammation and bone loss in ligature-induced experimental periodontitis in mice[J]. Infect Immun, 2016, 85(1): e00645-16.
27	Kobayashi R, Kono T, Bolerjack BA, et al. Induction of IL-10-producing CD4+ T-cells in chronic periodontitis[J]. J Dent Res, 2011, 90(5): 653-658.
28	Weitzmann MN. Bone and the immune system[J]. Toxicol Pathol, 2017, 45(7): 911-924.
29	朱东望, 薛栋, 赖文, 等. 微RNA-146a反转牙龈卟啉单胞菌脂多糖对人牙周膜细胞成骨的抑制作用[J]. 中华口腔医学杂志, 2018, 53(11): 753-759.
	Zhu DW, Xue D, Lai W, et al. microRNA-146a reverses the inhibitory effects of Porphyromonas gingivalis lipopolysaccharide onosteogenesis of human periodontal ligament cells[J]. Chin J Stomatol, 2018, 53(11): 753-759.

基因	引物序列
IL-1β	F：5’-ATGCCTTCCCCAGGGCATGT-3’ R：5’-CTGAGCGACCTGTCTTGGCCG-3’
IL-6	F：5’-TCCAGTTGCCTTCTTGGGAC-3’ R：5’-GTACTCCAGAAGACCAGAGG-3’
IL-10	F：5’-GACCAGCTGGACAACATACTGCTAA-3’ R：5’-GATAAGGCTTGGCAACCCAAGTAA-3’
OPG	F：5’-AGCAGGAGTGCAACCGCACC-3’ R：5’-TTCCAGCTTGCACCACGCCG-3’
RANKL	F：5’-CATGTGCCACTGAGAACCTTGAA-3’ R：5’-CAGGTCCCAGCGCAATGTAAC-3’
GAPDH	F：5’-CCCCAGCAAGGACACTGAGCAA-3’ R：5’-GTGGGTGCAGCGAACTTTATTGATG-3’
miR-146a	F：5’-GGGTGAGAACTGAATTCCA-3’ R：5’-CAGTGCGTGTCGTGGAGT-3’
U6 small nuclear RNA	F：5’-GCTTCGGCAGCACATATACTAAAAT-3’ R：5’-CGCTTCACGAATTTGCGTGTCAT-3’

细胞因子	P.gingivalis LPS 未刺激组	P.gingivalis LPS 刺激组	t值	P值
IL-1β	1.03±0.26	1.52±0.02	3.199	0.032 9
IL-6	1.03±0.28	2.60±0.77	3.325	0.029 2
IL-10	1.01±0.20	2.37±0.10	10.59	0.000 4
RANKL	1.00±0.03	3.85±1.07	4.604	0.010 0
OPG	1.01±0.20	0.59±0.03	3.631	0.022 1

细胞因子	P.gingivalis LPS未刺激组/pg·mL^-1	P.gingivalis LPS刺激组/pg·mL^-1	t值	P值
IL-1β	3.14±0.31	21.66±3.00	5.319	0.006 0
IL-6	349.09±26.10	498.26±20.52	7.781	0.001 5
IL-10	305.56±27.78	531.96±34.47	8.858	0.000 1
RANKL	16.36±0.91	48.20±0.66	24.50	<0.000 1
OPG	12.70±1.07	12.16±0.62	0.756	0.491 8

细胞因子	P.gingivalis LPS/ 模拟物阴性 RNA对照组	P.gingivalis LPS/miR-146a模拟物组	t值	P值
IL-1β	1.84±0.25	0.64±0.04	8.130	0.001 2
IL-6	2.32±0.28	1.60±0.28	3.111	0.035 8
IL-10	2.51±0.18	3.26±0.29	3.799	0.019 1
RANKL	2.94±0.46	0.90±0.27	6.620	0.002 7
OPG	0.63±0.05	3.25±0.29	15.71	<0.000 1

细胞因子	P.gingivalis LPS/ 模拟物阴性RNA对照组/pg·mL^-1	P.gingivalis LPS/miR-146a模拟物组/pg·mL^-1	t值	P值
IL-1β	18.20±2.60	6.06±1.50	3.50	0.024 9
IL-6	506.52±25.84	417.85±17.40	4.026	0.015 8
IL-10	566.21±53.20	684.93±24.70	3.506	0.024 8
RANKL	47.78±0.84	14.66±1.85	14.10	0.000 1
OPG	11.70±0.40	17.60±0.23	11.00	0.000 4

microRNA-146a对牙龈卟啉单胞菌脂多糖刺激下淋巴细胞分泌细胞因子的调节作用

MicroRNA-146a regulates the production of cytokines in lymphocytes stimulated by Porphyromonas gingivalis lipopolysaccharide

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细胞因子	P.gingivalis LPS/ 抑制剂阴性 RNA对照组	P.gingivalis LPS/miR-146a抑制物组	t值	P值
IL-1β	1.64±0.33	3.07±0.52	4.019	0.015 9
IL-6	2.09±0.37	3.58±0.54	3.982	0.016 4
IL-10	3.22±0.20	1.95±0.50	4.101	0.014 8
RANKL	3.69±0.66	9.09±1.79	4.901	0.008 0
OPG	0.81±0.05	0.38±0.06	9.144	0.000 8

细胞因子	P.gingivalis LPS/抑制剂阴性RNA对照组/pg·mL^-1	P.gingivalis LPS/ miR-146a/抑制物组/pg·mL^-1	t值	P值
IL-1β	16.46±1.50	62.40±3.47	10.53	0.000 5
IL-6	483.28±2.98	725.65±64.78	6.474	0.002 9
IL-10	577.63±10.46	351.85±89.29	4.350	0.012 2
RANKL	49.26±2.42	86.40±4.28	6.541	0.002 8
OPG	10.95±1.23	8.40±0.47	3.359	0.028 3

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