牙髓卟啉单胞菌内毒素对成骨细胞分化的抑制作用

doi:10.19439/j.sjos.2021.04.003

上海口腔医学 ›› 2021, Vol. 30 ›› Issue (4): 350-354.doi: 10.19439/j.sjos.2021.04.003

牙髓卟啉单胞菌内毒素对成骨细胞分化的抑制作用

王瑞, 杨谛, 于雅琼, 郭佳杰, 仇丽鸿

中国医科大学口腔医学院·附属口腔医院牙体牙髓病科,辽宁省口腔疾病重点实验室, 中国医科大学口腔医学院中心实验室, 辽宁沈阳 110002

收稿日期:2020-01-09 修回日期:2020-03-26 出版日期:2021-08-25 发布日期:2021-09-23
通讯作者: 杨谛,E-mail:missyangdi@hotmail.com
作者简介:王瑞（1993-）,女,医师,硕士,E-mail:737419245@qq.com
基金资助:
辽宁省自然科学基金（2019-MS-375）

Effect of inhibition of Porphyromonas endodontalis on osteoblast differentiation

WANG Rui, YANG Di, YU Ya-qiong, GUO Jia-jie, QIU Li-hong

Department of Endodontics, School and Hospital of Stomatology, China Medical University, Liaoning Provincial Key Laboratory of Oral Disease, Cental Laboratory, School of Stomatology, China Medical University. Shenyang 110002,Liaoning Province, China

Received:2020-01-09 Revised:2020-03-26 Online:2021-08-25 Published:2021-09-23

摘要/Abstract

摘要： 目的: 研究牙髓卟啉单胞菌脂多糖（P.e-LPS）对成骨细胞分化的影响,探讨P.e-LPS在根尖周骨吸收疾病中的致病机制。方法: 厌氧条件下培养P.e,应用热酚水法提取P.e-LPS,采用凝胶鲎试剂法对所提取的LPS进行定性分析。应用成骨细胞分化培养基（50 μg/mL抗坏血酸、6 mmol/L β甘油磷酸钠）诱导前成骨细胞系MC3T3-E1向成骨细胞分化。实时定量PCR（RT-PCR）检测成骨分化基因Distal-less homeobox 5（DLX5）、Runt-related transcription factor 2(Runx2)、Osterix、骨涎蛋白(bone sialoprotein,BSP)、骨钙素（osteocalcin,OCN）以及胶原（Collagen）的表达。碱性磷酸酶（ALP）活性测定、茜素红染色以及Von Kossa染色检测成骨细胞的矿化水平。应用siRNA 转染,沉默P.e-LPS的受体Toll-like receptor-4（TLR-4）在成骨细胞中的表达。采用SPSS 11.0软件包对数据进行统计学分析。结果: P.e-LPS（10 μg/mL）作用于MC3T3-E1细胞3 d,与对照组相比,成骨分化基因DLX5、Runx2、Osterix、OCN、BSP、胶原的mRNA表达水平显著下降（P<0.05）。P.e-LPS（10 μg/mL）分别作用于MC3T3-E1细胞7 d和14 d,ALP活性及茜素红染色强度下降。P.e-LPS分别作用于siRNA-TLR-4转染组及对照组7、14、21 d,与对照组相比,si-TLR-4组细胞的成骨分化基因表达水平、ALP活性、茜素红染色强度以及Von Kossa 染色强度均显著高于对照组（P<0.05）。结论: P.e-LPS通过受体TLR-4抑制成骨细胞的分化,从而参与根尖周病变的骨吸收过程。

关键词: 牙髓卟啉单胞菌, 脂多糖, Toll样受体4, 成骨分化

Abstract: PURPOSE: Porphyromonas endodontalis (P.e) is the dominant bacterium in the infected canal of pulpal and periapical disease.Lipopolysaccharides (LPS) in the outer membrane of the cell wall is an important toxicity factor of P.e. In this study, the effect of P.e-LPS on osteoblast differentiation was studied, and the pathogenic mechanism of P.e-LPS in periapical bone resorption disease was explored. METHODS: Porphyromonas endodontalis was cultured under anaerobic conditions. P.e-LPS was extracted by thermophenol water method, and then the extracted LPS was qualitatively analyzed by gel limulireagent method. Preosteoblast cell line MC3T3-E1 were induced to differentiate into osteoblasts by osteoblast differentiation medium (50 μg/mL ascorbic acid,6 mmol/L beta-glycerphosphate). Expressions of osteogenic differentiation genes including distal-less homeobox 5（DLX5）, runt-related transcription factor 2(Runx2), Osterix, bone sialoprotein (BSP), OCN(osteocalcin) and Collagen were detected by RT-PCR. The activity of alkaline phosphatase(ALP), alizarin red staining and Von Kossa staining were used to determine the mineralization level of osteoblasts.The expression of TOLL-like receptor-4 (TLR-4), the receptor of P.e-LPS, was silenced by siRNA transfection. SPSS 11.0 software package was used for statistical analysis of the data. RESULTS: The mRNA expressions of osteogenic differentiation genes including DLX5, Runx2, Osterix, OCN, BSP, and Collagen were significantly decreased after treated with P.e-LPS (10 μg/mL) for 3 d, compared with the control group（P< 0.05）.After treated with P.e-LPS (10 μg/mL) for 7 d or 14 d, respectively, ALP and alizarin red staining intensity was decreased. P.e-LPS was applied to the si-TLR-4 transfection group and the control group for 7,14 and 21 d, respectively. Compared with the control group, the expression level of osteogenic differentiation genes, ALP, alizarin red staining and Von Kossa staining intensity of si-TLR-4 group were significantly higher than those of the control group (P< 0.05). CONCLUSIONS: P.e-LPS inhibits the differentiation of osteoblasts through TLR-4 receptor, thus participating in bone resorption process of periapical lesions.

Key words: Porphyromonas endodontalis, Lipopolysaccharides, Toll-like receptor 4, Osteoblast differentiation

中图分类号:

R781.3

王瑞, 杨谛, 于雅琼, 郭佳杰, 仇丽鸿. 牙髓卟啉单胞菌内毒素对成骨细胞分化的抑制作用[J]. 上海口腔医学, 2021, 30(4): 350-354.

WANG Rui, YANG Di, YU Ya-qiong, GUO Jia-jie, QIU Li-hong. Effect of inhibition of Porphyromonas endodontalis on osteoblast differentiation[J]. Shanghai Journal of Stomatology, 2021, 30(4): 350-354.

参考文献

[1] 杨谛, 李任, 仇丽鸿, 等. 牙髓卟啉单胞菌内毒素对成骨细胞表达IL-1βmRNA和IL-6 mRNA的影响[J].上海口腔医学, 2009, 18(2): 194-197.
[2] Yu Y, Qiu L, Guo J.TRIB3 mediates the expression of Wnt5a and activation of nuclear factor-κB in Porphyromonas endodontalis lipopolysaccharide-treated osteoblasts[J]. Mol Oral Microbiol, 2015, 30(4): 295-306.
[3] George N, Flamiatos E, Kawasaki K, et al.Oral microbiota species in acute apical endodontic abscesses[J]. J Oral Microbiol, 2016, 8:30989.
[4] 刘琨, 侯本祥. 牙髓卟啉单胞菌的毒力因子及其致病性[J].北京口腔医学, 2007, 15(1): 50-52.
[5] 李任, 仇丽鸿. 牙髓卟啉单胞菌内毒素致病性研究进展[J]. 中国实用口腔科杂志, 2009, 2(12): 756-758.
[6] Koga T, Nishihara T, Fujiwara T.Biochemical and immunebiological properties of lipopolysaccharide (LPS) from Bacteroides gingivalis and comparison with LPS from Escherichia coli[J]. Infect Immun, 1985, 47(3): 638-647.
[7] Okamura H, Yoshida K, Yang D, et al.Double-stranded RNA-dependent protein kinase is required for bone calcification in MC3T3-E1 cells in vitro osterix transcription factor[J]. Exp Cell Res, 2005, 311(1): 117-125.
[8] Guo J, Yang D, Okamura H.Calcium hydroxide suppresses Porphyromonas endodontalis lipopolysaccharide-induced bone destruction[J]. J Dent Res, 2014, 93(5): 508-513.
[9] Yang D, Okamura H, Qiu L.Upregulated osterix expression elicited by Runx2 and Dlx5 is required for the accelerated osteoblast differentiation in PP2A Cα-knockdown cells[J]. Cell Biol Int, 2018, 42(4): 403-410.
[10] Okamura H, Yang D, Yoshida K.Protein phosphatase 2A Cα is involved in osteoclastogenesis by regulating RANKL and OPG expression in osteoblasts[J]. FEBS Lett, 2013, 587(1): 48-53.
[11] 于雅琼, 曲柳, 仇丽鸿, 等. TNF-α在慢性根尖周炎骨破坏中的机制探讨[J].上海口腔医学, 2016, 25(4): 414-419.
[12] 严杰, 方平楚. 幽门螺杆菌脂多糖生物学活性的研究[J]. 中华微生物学和免疫学杂志, 1994, 14(3): 196-198.
[13] 于雅琼, 李晓琳, 仇丽鸿, 等. 白藜芦醇对牙髓卟啉单胞菌脂多糖诱导成骨细胞产生MIP-2的影响[J]. 上海口腔医学, 2019, 28(2): 128-132.
[14] Yang D, Okamura H, Nakashima Y, et al.Histone demethylase Jmjd3 regulates osteoblast differentiation via transcription factors Runx2 and osterix[J]. J Biol Chem, 2013, 288(47): 33530-33541.
[15] Nakashima K, Zhou X, Kunkel G, et al.The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation[J]. Cell, 2002, 108(1): 17-29.
[16] Hill TP, Spater D, Taketo MM, et al.Canonical Wnt/β-catenin signaling prevents osteoblasts from differentiating into chondrocytes[J]. Dev Cell, 2005, 8(5): 727-738.
[17] Komori T.Regulation of osteoblast differentiation by transcription factors[J]. J Cell Bio chem, 2006, 99(5): 1233-1239.
[18] Komori T.Regulation of bone development and maintenance by Runx2[J]. Front Biosci, 2008, 13:898-903.
[19] Okamura H, Yoshida K, Yang D, et al.Protein phosphatase 2A Cα regulates osteoblast differentiation and the expressions of bone sialoprotein and osteocalcin via osterix transcript-tion factor[J]. J Cell Physiol, 2013, 228(5): 1031-1037.
[20] 付文举,张德志,仲兴,等.脂多糖对体外培养成骨细胞凋亡影响的初步实验研究[J].中国现代医学杂志,2014,24(13):8-12.
[21] Liu YH, Huang D, Li ZJ, et al.Toll-like receptor-4- dependence of the lipopolysaccharide-mediated inhibition of osteoblast differentiation[J]. Genet Mol Res, 2016, 15(2): 15027191
[22] 曹佳, 侯本祥, 张琛. CD14、TLR4在大鼠尖周炎症组织中的表达[J]. 牙体牙髓牙周病学杂志, 2008, 18(3): 130-134.
[23] 贾舸, 仇丽鸿. 口腔致病菌膜表面相关受体研究进展[J].中国实用口腔科杂志, 2010, 3(5): 313-315.
[24] Kikkert R, Laine ML, Aarden LA, et al.Activation of toll-like receptors 2 and 4 by gram-negative periodontal bacteria[J]. Oral Microbiol Immunol, 2007, 22(3): 145-151.
[25] 贾舸,薛明,李任,等.不同卟啉单胞菌脂多糖对小鼠成骨细胞CD14、TLRs表达的影响[J]. 上海口腔医学, 2011, 20(6): 598-602.
[26] Mo IF, Yip KH, Chan WK, et al.Prolonged exposure to bacterial toxins downregulated expression of toll-like receptors in mesenchymal stromal cell-derived osteoprogenitors[J]. BMC Cell Biol, 2008, 9:52.
[27] 张婧婧,张楠,吴伟,等. mTORC1信号促进前成骨细胞MC3T3-E1的分化成熟[J].中国免疫学杂志,2016, 32(11): 1573-1577.
[28] Murakami Y, Hanazawa S, Tanaka S, et al.A possible mechanism of maxillofacial abscess formation: involvement of Porphyromonas endodontalis lipopolysaccharide via the expression of inflammatory cytokines[J]. Oral Microbiol Immunol, 2001, 16(6): 321-325.

牙髓卟啉单胞菌内毒素对成骨细胞分化的抑制作用

Effect of inhibition of Porphyromonas endodontalis on osteoblast differentiation

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