Please wait a minute...
浙江大学学报（医学版）
浙江大学学报（医学版）  2021, Vol. 50 Issue (2): 171-178    DOI: 10.3724/zdxbyxb-2021-0109
专题报道     
牙龈卟啉单胞菌重组牙龈蛋白酶刺激牙龈成纤维细胞内钙离子浓度变化及其机制
张迪亚1(),卢可心2,李盛来3,吴燕岷2,*()
1.浙江大学医学院附属邵逸夫医院牙科，浙江 杭州 310016
2.浙江大学医学院附属第二医院牙周病专科，浙江 杭州 310009
3.浙江大学医学院附属口腔医院口腔外科，浙江 杭州 310006
Ca2+ mobilization and signaling pathways induced by Porphyromonas gingivalis rRgpB in human gingival fibroblast
ZHANG Diya1(),LU Kexin2,LI Shenglai3,WU Yanmin2,*()
1. Dental Department, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310016, China;
2. Department of Periodontology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China;
3. Oral Surgery, Stomatology Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China
 全文: PDF(5266 KB)   HTML( 12 )
摘要：

目的：探讨牙龈卟啉单胞菌重组牙龈蛋白酶（rRgpB）刺激蛋白酶激活受体（PAR）介导的人牙龈成纤维细胞（HGF）内钙离子浓度（[Ca2+]i）的动态变化及细胞内信号转导通路。方法：流式细胞术检测 HGF 表达的 PAR 类型，CCK-8 法检测细胞增殖，以牙龈卟啉单胞菌rRgpB作用于 HGF，激光共聚焦显微镜观察 HGF 内[Ca2+]i的动态变化及 PAR-1 拮抗剂的阻断作用；蛋白质印迹法检测 HGF 内 c-Jun 氨基末端激酶（JNK）、胞外信号调节激酶 1/2（ERK1/2）、p38 丝裂原激活的蛋白激酶（p38 MAPK）、p65 蛋白水平的变化。结果：HGF 表达 PAR-1 和 PAR-3，且 rRgpB 可促进 HGF 生长。细胞受到 rRgpB 作用后能激发瞬时增强的[Ca2+]i荧光信号，并且这种作用能够被 PAR-1 拮抗剂完全阻断；与空白对照组比较，rRgpB 诱导细胞6、12?h后，JNK、ERK1/2、p65 磷酸化蛋白表达均显著上调（均P<0.05），但 p38 MAPK 磷酸化蛋白水平未见明显变化（P>0.05）；PAR-1 拮抗剂成功抑制了 rRgpB 诱导的 JNK、ERK1/2、p65 磷酸化蛋白表达的上调。结论：rRgpB 通过 PAR-1 诱导 HGF 内[Ca2+]i增加，并激活细胞内 JNK、ERK1/2、核因子κB 信号通路。
关键词： 牙龈蛋白酶;  人牙龈成纤维细胞;  蛋白酶激活受体;  钙离子;  信号通路    
Abstract:

Objective: To assess the Porphyromonas gingivalis (Pg) recombinant gingivalis gingipain R2 (rRgpB)-induced Ca2+ mobilization in human gingival fibroblast (HGF) mediated by protease-activated receptor (PAR) and its downstream signal transduction pathways. Methods: Flow cytometry was used to detect the expression of PAR in HGF. The proliferation of HGF was measured by CCK-8. The dynamic changes of intracellular Ca2+ concentration ([Ca2+]i) in HGF induced by rRgpB and the blocking effect of PAR-1 antagonist were observed by laser confocal microscopy. Western blot was performed to determine the phosphorylation levels of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen-activated protein kinase (p38 MAPK) and p65 in HGF. Results: PAR-1 and PAR-3 were expressed in HGF, and the rRgpB could promote the proliferation of HGF. rRgpB caused a transient increase in [Ca2+]i, which could be completely suppressed by vorapaxar, a PAR-1 antagonist. The phosphorylation levels of JNK, ERK1/2 and p65 were significantly up-regulated after the induction of rRgpB for 6?h and 12?h (all P<0.05), which was completely inhibited by vorapaxar. However, the phosphorylation level of p38 MAPK had no significant change after rRgpB stimulation.Conclusions: rRgpB causes an increase in [Ca2+]i in HGF mediated by PAR-1. JNK, ERK1/2 and nuclear factor-κB may be involved in intracellular signal transduction after PAR-1 activation.
Key words: Gingipains    Human gingival fibroblasts    Protease-activated receptor    Calciumion    Signaling pathways
收稿日期: 2021-01-14 出版日期: 2021-06-18
CLC:  R781.4  
基金资助: 浙江省自然科学基金（LY17H140002）； 浙江省新一轮卫生高层次人才培养工程
通讯作者: 吴燕岷     E-mail: zhangdiya@zju.edu.cn;wuyanmin@zju.edu.cn
作者简介: 张迪亚，副主任医师，主要从事牙周炎和种植体周围炎发病机制和治疗研究；E-mail：zhangdiya@zju.edu.cn；https：//orcid.org/0000-0003-4471-9669. 卢可心，硕士研究生，主要从事牙周炎和种植体周围炎发病机制和治疗研究；E-mail：3130102947@zju.edu.cn；https：//orcid.org/0000-0002-0924-9713
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
张迪亚
卢可心
李盛来
吴燕岷

引用本文:

张迪亚,卢可心,李盛来,吴燕岷. 牙龈卟啉单胞菌重组牙龈蛋白酶刺激牙龈成纤维细胞内钙离子浓度变化及其机制[J]. 浙江大学学报（医学版）, 2021, 50(2): 171-178.

ZHANG Diya,LU Kexin,LI Shenglai,WU Yanmin. Ca2+ mobilization and signaling pathways induced by Porphyromonas gingivalis rRgpB in human gingival fibroblast. J Zhejiang Univ (Med Sci), 2021, 50(2): 171-178.

链接本文:

http://www.zjujournals.com/med/CN/10.3724/zdxbyxb-2021-0109    或    http://www.zjujournals.com/med/CN/Y2021/V50/I2/171

图 1  蛋白酶激活受体（PAR）在人牙龈成纤维细胞表面的表达结果
图 2  重组牙龈蛋白酶 R2（rRgpB）对人牙龈成纤维细胞增殖的影响与空白对照组比较，< 0.05，< 0.01.
图 3  各组人牙龈成纤维细胞内钙离子荧光图空白对照组细胞内钙离子荧光（绿色荧光）信号较弱，凝血酶组和 rRgpB 组细胞内钙离子荧光信号较强，rRgpB +沃拉帕沙组钙离子荧光信号强度与空白对照组相近. 标尺=20 μm.rRgpB：重组牙龈蛋白酶R2.
图 4  各组人牙龈成纤维细胞内钙离子荧光强度变化曲线rRgpB：重组牙龈蛋白酶R2.
图 5  重组牙龈蛋白酶 R2（rRgpB）刺激人牙龈成纤维细胞内信号通路相关蛋白表达的变化与空白对照组比较，< 0.05. JNK：c-Jun 氨基末端激酶；ERK：胞外信号调节激酶；GAPDH：甘油醛-3-磷酸脱氢酶.
图 6  沃拉帕沙对重组牙龈蛋白酶 R2（rRgpB）作用下人牙龈成纤维细胞内信号通路相关蛋白表达的影响与空白对照组比较，<0.05；与 rRgpB+沃拉帕沙组比较，< 0.05. JNK：c-Jun 氨基末端激酶；ERK：胞外信号调节激酶；GAPDH：甘油醛-3-磷酸脱氢酶.
1 HAFFAJEEA D, SOCRANSKYS S. Microbial etiological agents of destructive periodontal diseases[J]Periodontol 2000, 1994, 5( 1): 78-111.
doi: 10.1111/j.1600-0757.1994.tb00020.x
2 ZHANGD, CHENL, LIS, et al.Lipopolysaccharide (LPS) of Porphyromonas gingivalis induces IL-1β, TNF-α and IL-6 production by THP-1 cells in a way different from that of Escherichia coli LPS[J]Innate Immun, 2008, 14( 2): 99-107.
doi: 10.1177/1753425907088244
3 O-BRIEN-SIMPSONN M, VEITHP D, DASHPERS G, et al.Porphyromonas gingivalis gingipains: the molecular teeth of a microbial vampire[J]Curr Protein Pept Sci, 2003, 4( 6): 409-426.
doi: 10.2174/1389203033487009
4 GIACAMANR A, ASRANIA C, ROSSK F, et al.Cleavage of protease-activated receptors on an immortalized oral epithelial cell line by Porphyromonas gingivalis gingipains[J]Microbiology, 2009, 155( 10): 3238-3246.
doi: 10.1099/mic.0.029132-0
5 OSSOVSKAYAV S, BUNNETTN W. Protease-activated receptors: contribution to physiology and disease[J]Physiol Rev, 2004, 84( 2): 579-621.
doi: 10.1152/physrev.00028.2003
6 TANAKAN, MORITAT, NEZUA, et al.Thrombin-induced Ca2+ mobilization in human gingival fibroblasts is mediated by protease-activated receptor-1 (PAR-1)[J]Life Sci, 2003, 73( 3): 301-310.
doi: 10.1016/s0024-3205(03)00269-8
7 CHANGM C, CHANC P, WUH L, et al.Thrombin-stimulated growth, clustering, and collagen lattice contraction of human gingival fibroblasts is associated with its protease activity[J]J Periodontol, 2001, 72( 3): 303-313.
doi: 10.1902/jop.2001.72.3.303
8 HOUL, RAVENALLS, MACEYM G, et al.Protease-activated receptors and their role in IL-6 and NF-IL-6 expression in human gingival fibroblasts[J]J Periodontal Res, 1998, 33( 4): 205-211.
doi: 10.1111/j.1600-0765.1998.tb02192.x
9 张迪亚, 胡玲静, 李盛来, 等. 牙龈成纤维细胞表达蛋白酶激活受体的类型及作用研究[J]. 中华口腔医学杂志, 2014, 49(1): 21-26
ZHANG Diya, HU Lingjing, LI Shenglai, et al. Expressions of protease-activated receptors in human gingival fibroblasts and its functions in periodontitis[J]. Chinese Journal of Stomatology, 2014, 49(1): 21-26. (in Chinese)
10 SAMBRANOG R, WEISSE J, ZHENGY W, et al.Role of thrombin signalling in platelets in haemostasis and thrombosis[J]Nature, 2001, 413( 6851): 74-78.
doi: 10.1038/35092573
11 HUANGP, HEX Y, XUM. Protease-activated receptor 1 inhibitor improves brain edema in rats with intracerebral hemorrhage[J]Bratisl Lek Listy, 2020, 121( 8): 600-604.
doi: 10.4149/BLL_2020_099
12 SAEEDM A, NGG Z, D?BRITZJ, et al.Protease-activated receptor 1 plays a proinflammatory role in colitis by promoting Th17-related immunity[J]Inflamm Bowel Dis, 2017, 23( 4): 593-602.
doi: 10.1097/MIB.0000000000001045
13 OLIANASM C, DEDONIS, ONALIP. Proteinase-activated receptors 1 and 2 in rat olfactory system: layer-specific regulation of multiple signaling pathways in the main olfactory bulb and induction of neurite retraction in olfactory sensory neurons[J]Neuroscience, 2007, 146( 3): 1289-1301.
doi: 10.1016/j.neuroscience.2007.02.059
14 IMAMURAT. The role of gingipains in the pathogenesis of periodontal disease[J]J Periodontol, 2003, 74( 1): 111-118.
doi: 10.1902/jop.2003.74.1.111
15 HOESCHR E, YIENGERK, WEINREICHD, et al.Coexistence of functional IP3 and ryanodine receptors in vagal sensory neurons and their activation by ATP[J]J NeuroPhysiol, 2002, 88( 3): 1212-1219.
doi: 10.1152/jn.2002.88.3.1212
16 ROVAI E S, HOLZHAUSEN M. The role of proteinase-activated receptors 1 and 2 in the regulation of periodontal tissue metabolism and disease[J]. J Immunol Res, 2017, 2017: 5193572
17 KLARSTR?ME K, KHALAFH, K?LVEGRENH, et al.The role of Porphyromonas gingivalis gingipains in platelet activation and innate immune modulation[J]Mol Oral Microbiol, 2015, 30( 1): 62-73.
doi: 10.1111/omi.12067
18 PETZOLDT, THIENELM, DANNENBERGL, et al.Rivaroxaban reduces arterial thrombosis by inhibition of FXa-driven platelet activation via protease activated receptor-1[J]Circ Res, 2020, 126( 4): 486-500.
doi: 10.1161/CIRCRESAHA.119.315099
19 LOURBAKOSA, POTEMPAJ, TRAVISJ, et al.Arginine-specific protease from Porphyromonas gingivalis activates protease-activated receptors on human oral epithelial cells and induces interleukin-6 secretion[J]Infect Immun, 2001, 69( 8): 5121-5130.
doi: 10.1128/IAI.69.8.5121-5130.2001
20 JAYAPRAKASHK, DEMIRELI, KHALAFH, et al.The role of phagocytosis, oxidative burst and neutrophil extracellular traps in the interaction between neutrophils and the periodontal pathogen Porphyromonas gingivalis[J]Mol oral Microbiol, 2015, 30( 5): 361-375.
doi: 10.1111/omi.12099
21 KANNOY, ISHISAKIA, KAWASHITAE, et al.uPA attenuated LPS-induced inflammatory osteoclastogenesis through the plasmin/PAR-1/Ca2+/CaMKK/AMPK Axis[J]Int J Biol Sci, 2016, 12( 1): 63-71.
doi: 10.7150/ijbs.12690
22 LOURBAKOSA, YUANY P, JENKINSA L, et al.Activation of protease-activated receptors by gingipains from Porphyromonas gingivalis leads to platelet aggregation: a new trait in microbial pathogenicity[J]Blood, 2001, 97( 12): 3790-3797.
doi: 10.1182/blood.V97.12.3790
23 YANGW H, DENGY T, HSIEHY P, et al.Thrombin activates latent TGFβ1 via integrin αvβ1 in gingival fibroblasts[J]J Dent Res, 2016, 95( 8): 939-945.
doi: 10.1177/0022034516634288
24 TADAH, NISHIOKAT, TAKASEA, et al.Porphyromonas gingivalis induces the production of interleukin-31 by human mast cells, resulting in dysfunction of the gingival epithelial barrier[J/OL]Cell MicroBiol, 2019, 21( 3): e12972.
doi: 10.1111/cmi.12972
25 JAYAPRAKASHK, DEMIRELI, GUNALTAYS, et al.PKC, ERK/p38 MAP kinases and NF-κB targeted signalling play a role in the expression and release of IL-1β and CXCL8 in Porphyromonas gingivalis-infected THP1 cells[J]APMIS, 2017, 125( 7): 623-633.
doi: 10.1111/apm.12701
[1] 毛红美,孙毅. 原纤毛及其在肿瘤发生发展中的作用[J]. 浙江大学学报（医学版）, 2021, 50(2): 245-260.
[2] 黄耀凭,杨凤,周天华,谢珊珊. Hippo信号通路及其在消化系统肿瘤中的作用研究进展[J]. 浙江大学学报（医学版）, 2020, 49(1): 35-43.
[3] 曾玲晖，丁美萍. mTOR信号通路在癫痫发生中的作用[J]. 浙江大学学报（医学版）, 2013, 42(6): 597-601.
[4] 张潇芸, 姜英, 杨军. p53非依赖性信号通路在DNA损伤致细胞凋亡中的研究进展[J]. 浙江大学学报（医学版）, 2013, 42(2): 217-223.
[5] . Wnt/β-catenin信号通路对间充质干细胞衰老的影响及其作用机制[J]. 浙江大学学报（医学版）, 2011, 40(6): 630-640.
[6] 蔡蓓蕾;黄雪琴;董晓武;方三华;卢韵碧;张纬萍;胡永洲;魏尔清 . null[J]. 浙江大学学报（医学版）, 2009, 38(6): 598-604.
[7] 应淑琴，徐耕，单江. 缺血再灌注对大鼠细胞黏附分子和单核细胞趋化蛋白-1表达的影响及硫酸镁的作用[J]. 浙江大学学报（医学版）, 2003, 32(3): 223-226.