国际口腔医学杂志

国际口腔医学杂志 ›› 2024, Vol. 51 ›› Issue (5): 538-549.doi: 10.7518/gjkq.2024067

• 口腔黏膜病专栏 • 上一篇    下一篇

槟榔提取物对口腔上皮细胞生物功能影响的研究

李明1,2(),唐瞻贵1,2(),原振英3   

  1. 1.中南大学湘雅口腔医（学）院 长沙 410000
    2.湖南省口腔健康研究重点实验室 长沙 410000
    3.长沙市口腔医院五一路院区牙体牙髓科 长沙 410000
  • 收稿日期:2023-10-16 修回日期:2024-04-28 出版日期:2024-09-01 发布日期:2024-09-14
  • 通讯作者: 唐瞻贵
  • 作者简介:李明，主治医师，博士，Email：limingq248@163.com
  • 基金资助:
    国家自然科学基金(82170972)

Effect of areca nut extract on the biological function of oral epithelial cells

Ming Li1,2(),Zhangui Tang1,2(),Zhenying Yuan3   

  1. 1.Xiangya School/Hospital of Stomatology, Central South University, Changsha 410000, China
    2.Hunan Key Labora-tory of Oral Health Research, Changsha 410000, China
    3.Dept. of Cariology and Endodontics, Wuyi Road District, Changsha Stomatological Hospital, Changsha 410000, China
  • Received:2023-10-16 Revised:2024-04-28 Online:2024-09-01 Published:2024-09-14
  • Contact: Zhangui Tang
  • Supported by:
    National Natural Science Foundation of China(82170972)

RichHTML

20

PDF (PC)

369

摘要：

目的 探讨槟榔提取物（ANE）对人口腔上皮细胞的生物学功能影响。 方法 本研究首先分析了梯度浓度ANE下对人口腔角质细胞（HOK）增殖的影响；采用第二代RNA测序分析对照组与ANE干预组HOK细胞的差异表达基因。随后，流式细胞术用于检测HOK细胞周期和凋亡；β-半乳糖苷酶（β-Gal）染色试剂盒用于检测细胞衰老；免疫荧光实验检测HOK细胞中Ki67和γH2AX蛋白的表达；实时聚合酶链反应（RT-PCR）用于检测衰老相关表型基因白细胞介素（IL）-6、基质金属蛋白酶-2（MMP2）和TGFB1表达；免疫印迹用于检测自噬相关蛋白（LC3-Ⅰ/Ⅱ 和p62）和上皮间充质转化（EMT）相关蛋白（SMAD2、p-SMAD2、E-Cadherin和Vimentin）的表达；酶联免疫吸附实验用于检测HOK细胞外分泌蛋白TGFB1表达。 结果 测序结果显示ANE可引起HOK细胞细胞周期、p53、自噬、FoxO、细胞衰老等信号通路相关基因异常表达。流式细胞术显示ANE可诱导HOK细胞凋亡和G2/M期阻滞。与对照组相比，ANE处理后的HOK细胞衰老细胞数量增加，衰老相关表型基因IL-6、MMP2和TGFB1表达上调。此外，相较于对照组，ANE组中HOK细胞的γH2AX、LC3-Ⅱ、TGFB1、p-SMAD2和Vimentin蛋白表达增加，Ki67蛋白、LC3-Ⅰ和E-Cadherin蛋白表达下降。 结论 ANE可诱导HOK细胞凋亡、G2/M期阻滞、衰老、DNA损伤、自噬和EMT，形成促癌的细胞微环境。

关键词: 槟榔, 槟榔提取物, 口腔角质细胞, 细胞衰老, 周期阻滞, 自噬, 上皮间充质转化

Abstract:

Objective　This study aimed to investigate the effects of areca nut extract (ANE) on the biological function of human oral epithelial cells. Methods　First, we analyzed the effects of ANE with gradient concentrations on the proli-feration of human oral keratinocytes (HOKs). Next-generation RNA sequencing was used to examine the differential genes between control HOKs and those treated with ANE. Flow cytometry was employed to evaluate the apoptosis and cycle arrest of HOKs. β-galactosidase staining kit was used to detect cell senescence. Immunofluorescence was applied to detect the expression of Ki67 and γH2AX protein in HOKs. Real-time PCR (RT-PCR) was utilized to detect the expression of senescence-related phenotypic genes interleukin (IL)-6, matrix metalloproteinase-2 (MMP2), and TGFB1. Wes-tern blot was adopted to detect the expression of autophagy-related proteins (LC3-Ⅰ/Ⅱ and p62) and epithelial-mesenchymal transition (EMT)-related proteins (SMAD2, p-SMAD2, E-cadherin, and Vimentin). Enzyme-linked immunosorbent assay was employed to detect the expression of exocrine TGFB1 protein in HOKs. Results　Sequencing results showed that ANE caused abnormality in cell cycle, p53, autophagy, FoxO, cellular senescence, and other signal pathways. Flow cytometry showed that ANE induced HOK apoptosis and G2/M phase arrest. The number of senescent cells and the expression of senescent phenotypic secretion genes (IL-6, MMP2, and TGFB1) increased in the ANE group. Compared with those in the control group, the expression levels of γH2AX, LC3-Ⅱ, TGFB1, p-SMAD2, and Vimentin protein increased and those of Ki67, LC3-Ⅰ, and E-cadherin protein decreased in the ANE group. Conclusion　ANE could induce HOK apoptosis, G2/M phase arrest, senescence, DNA double-strand damage, autophagy, and EMT, possibly forming a cell microenvironment that promotes tumor occurrence.

Key words: areca nut, areca nut extract, oral keratinocyte, cell senescence, cycle arrest, autophagy, epithelial-mesenchymal transformation

中图分类号: 

  • Q257

图 1

高效液相色谱检测槟榔提取物中4种槟榔生物碱的含量左：高效液相色谱图；右：4种槟榔生物碱的含量。"

图 2

CCK-8实验检测ANE对HOK细胞的增殖能力的影响*：P<0.05；**：P<0.01。"

图 3

对照HOK与10 mg/mL ANE干预的HOK细胞差异基因的火山图"

图 4

KEGG和GO分别分析对照组HOK与10 mg/mL ANE组HOK细胞差异基因涉及的信号通路和基因注释A：KEGG分析结果；B：GO分析结果。"

图 5

流式细胞术结果显示ANE可促进HOK细胞凋亡和G2/M期细胞比例增加A：对照组HOK细胞凋亡图；B：10 mg/mL ANE组HOK细胞凋亡图；C：对照组与10 mg/mL ANE组HOK细胞凋亡结果统计；D：对照组HOK细胞周期图；E：10 mg/mL ANE组HOK细胞周期图；F：对照组与10 mg/mL ANE组HOK细胞周期结果统计。*：P<0.05；***：P<0.001。"

图 6

10 mg/mL ANE组较对照组HOK细胞Ki67蛋白染色细胞数减少 免疫荧光 ×100"

图 7

10 mg/mL ANE可促进HOK细胞衰老，Ki67蛋白表达降低，衰老相关表型基因表达增加A：10 mg/mL ANE组较对照组HOK细胞染色数量增加 β-Gal染色 ×100；B：RT-PCR显示10 mg/mL ANE组HOK细胞中IL-6、MMP2、TGFB1 mRNA的表达较对照组上调。***：P<0.001。"

图 8

ANE可诱导HOK细胞DNA双链损伤和自噬A：10 mg/mL ANE增加了HOK细胞中γH2AX蛋白表达 免疫荧光 ×100；B：免疫印迹结果显示，相较于对照组，ANE诱导LC3-Ⅰ/LC3-Ⅱ蛋白表达比例下调，p62蛋白表达下调。***：P<0.001。"

图 9

ANE可诱导HOK细胞TGFB1蛋白分泌，促进EMTA：ELISA实验显示，2 mg/mL ANE长期处理HOK细胞后可上调TGFB1外分泌蛋白表达；B：ANE长期处理的HOK细胞其体积和间隙增大，细胞聚集性下降，细胞形态呈梭形或不规则条形，并可见较多圆形或卵圆形的细胞凋亡小体 显微镜 ×100；C：相较于对照组，ANE长期处理HOK细胞可诱导E-Cadherin蛋白表达下调，p-SMAD2和Vimentin蛋白表达增加。**：P<0.01；***：P<0.001。"

1 Warnakulasuriya S, Chen THH. Areca nut and oral cancer: evidence from studies conducted in humans[J]. J Dent Res, 2022, 101(10): 1139-1146.
2 Mehrtash H, Duncan K, Parascandola M, et al. Defining a global research and policy agenda for betel quid and areca nut[J]. Lancet Oncol, 2017, 18(12): e767-e775.
3 Burton-Bradley BG. Arecaidinism: betel chewing in transcultural perspective[J]. Can J Psychiatry, 1979, 24(5): 481-488.
4 Wang YY, Tail YH, Wang WC, et al. Malignant transformation in 5 071 southern Taiwanese patients with potentially malignant oral mucosal disorders [J]. BMC Oral Health, 2014, 14: 99.
5 Zain RB, Ikeda N, Razak IA, et al. A national epidemiological survey of oral mucosal lesions in Malaysia[J]. Community Dent Oral Epidemiol, 1997, 25(5): 377-383.
6 Cai XJ, Yao ZG, Liu G, et al. Oral submucous fibrosis: a clinicopathological study of 674 cases in China[J]. J Oral Pathol Med, 2019, 48(4): 321-325.
7 杜永秀, 孙东业, 翦新春, 等. 咀嚼槟榔种类与口腔黏膜疾病的流行病学调查分析[J]. 华西口腔医学杂志, 2016, 34(4): 391-394.
Du YX, Sun DY, Jian XC, et al. Epidemiological investigation of chewing fresh or dried betel nut and oral mucosal disease[J]. West China J Stomatol, 2016, 34(4): 391-394.
8 Gupta AK, Tulsyan S, Thakur N, et al. Chemistry, metabolism and pharmacology of carcinogenic alkaloids present in areca nut and factors affecting their concentration[J]. Regul Toxicol Pharmacol, 2020, 110: 104548.
9 Chang MC, Wu HL, Lee JJ, et al. The induction of prostaglandin E2 production, interleukin-6 production, cell cycle arrest, and cytotoxicity in primary oral keratinocytes and KB cancer cells by areca nut ingredients is differentially regulated by MEK/ERK activation[J]. J Biol Chem, 2004, 279(49): 50676-50683.
10 Gu LQ, Xie CQ, Peng Q, et al. Arecoline suppresses epithelial cell viability through the Akt/mTOR signaling pathway via upregulation of PHLPP2[J]. To-xicology, 2019, 419: 32-39.
11 Chang MC, Chan CP, Chen YJ, et al. Areca nut components stimulate ADAM17, IL-1α, PGE2 and 8-isoprostane production in oral keratinocyte: role of reactive oxygen species, EGF and JAK signaling[J]. Oncotarget, 2016, 7(13): 16879-16894.
12 Li YC, Chang JT, Chiu C, et al. Areca nut contri-butes to oral malignancy through facilitating the conversion of cancer stem cells[J]. Mol Carcinog, 2016, 55(5): 1012-1023.
13 Jeng JH, Wang YJ, Chiang BL, et al. Roles of keratinocyte inflammation in oral cancer: regulating the prostaglandin E2, interleukin-6 and TNF-alpha production of oral epithelial cells by areca nut extract and arecoline[J]. Carcinogenesis, 2003, 24(8): 1301-1315.
14 Al-Tayar BA, Ahmad A, Yusoff ME, et al. Cytoto-xic effects of betel quid and areca nut aqueous extracts on mouse fibroblast, human mouth-ordinary-epithelium 1 and human oral squamous cell carcinoma cell lines[J]. Asian Pac J Cancer Prev, 2020, 21(4): 1005-1009.
15 屈文佳, 贾哲, 辛洁萍, 等. 高效液相色谱法同时测定槟榔中4种生物碱的含量[J]. 中南药学, 2020, 18(3): 485-488.
Qu WJ, Jia Z, Xin JP, et al. Simultaneously determination of 4 alkaloids in Arecae Semen by HPLC[J]. Cent South Pharm, 2020, 18(3): 485-488.
16 Huang DW, Sherman BT, Tan QN, et al. DAVID bioinformatics resources: expanded annotation database and novel algorithms to better extract biology from large gene lists[J]. Nucleic Acids Res, 2007, 35(Web Server issue): W169-W175.
17 Herranz N, Gil J. Mechanisms and functions of cellular senescence[J]. J Clin Invest, 2018, 128(4): 1238-1246.
18 Sharma A, Almasan A. Autophagy and PTEN in DNA damage-induced senescence[J]. Adv Cancer Res, 2021, 150: 249-284.
19 Yoshii SR, Mizushima N. Monitoring and measu-ring autophagy[J]. Int J Mol Sci, 2017, 18(9): 1865.
20 Pankiv S, Clausen TH, Lamark T, et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy[J]. J Biol Chem, 2007, 282(33): 24131-24145.
21 Chen YJ, Chang JT, Liao CT, et al. Head and neck cancer in the betel quid chewing area: recent advances in molecular carcinogenesis[J]. Cancer Sci, 2008, 99(8): 1507-1514.
22 Chen PH, Mahmood Q, Mariottini GL, et al. Adverse health effects of betel quid and the risk of oral and pharyngeal cancers[J]. Biomed Res Int, 2017, 2017: 3904098.
23 Zhou ZS, Li M, Gao F, et al. Arecoline suppresses HaCaT cell proliferation through cell cycle regulatory molecules[J]. Oncol Rep, 2013, 29(6): 2438-2444.
24 Wang TY, Peng CY, Lee SS, et al. Acquisition cancer stemness, mesenchymal transdifferentiation, and chemoresistance properties by chronic exposure of oral epithelial cells to arecoline[J]. Oncotarget, 2016, 7(51): 84072-84081.
25 Chen XX, He YZ, Deng YR. Chemical composition, pharmacological, and toxicological effects of betel nut[J]. Evid Based Complement Alternat Med, 2021, 2021: 1808081.
26 Nagesh R, Kiran Kumar KM, Naveen Kumar M, et al. Stress activated p38 MAPK regulates cell cycle via AP-1 factors in areca extract exposed human lung epithelial cells[J]. Cytotechnology, 2019, 71(2): 507-520.
27 Yen CY, Lin MH, Liu SY, et al. Arecoline-mediated inhibition of AMP-activated protein kinase through reactive oxygen species is required for apoptosis induction[J]. Oral Oncol, 2011, 47(5): 345-351.
28 Farhan M, Wang HT, Gaur U, et al. FOXO signaling pathways as therapeutic targets in cancer[J]. Int J Biol Sci, 2017, 13(7): 815-827.
29 Hernández Borrero LJ, El-Deiry WS. Tumor suppressor p53: biology, signaling pathways, and therapeutic targeting[J]. Biochim Biophys Acta Rev Cancer, 2021, 1876(1): 188556.
30 Furukawa-Hibi Y, Kobayashi Y, Chen C, et al. FOXO transcription factors in cell-cycle regulation and the response to oxidative stress[J]. Antioxid Redox Signal, 2005, 7(5/6): 752-760.
31 Tseng SK, Chang MC, Su CY, et al. Arecoline induced cell cycle arrest, apoptosis, and cytotoxicity to human endothelial cells[J]. Clin Oral Investig, 2012, 16(4): 1267-1273.
32 Ullah M, Cox S, Kelly E, et al. Arecoline is cytoto-xic for human endothelial cells[J]. J Oral Pathol Med, 2014, 43(10): 761-769.
33 Rehman A, Ali S, Lone MA, et al. Areca nut alkaloids induce irreparable DNA damage and senescence in fibroblasts and may create a favourable environment for tumour progression[J]. J Oral Pathol Med, 2016, 45(5): 365-372.
34 Huang JL, Lu HH, Lu YN, et al. Enhancement of the genotoxicity of benzo[a]pyrene by arecoline through suppression of DNA repair in HEp-2 cells[J]. Toxicol In Vitro, 2016, 33: 80-87.
35 Basu AK. DNA damage, mutagenesis and cancer[J]. Int J Mol Sci, 2018, 19(4): 970.
36 Lai KC, Lee TC. Genetic damage in cultured human keratinocytes stressed by long-term exposure to areca nut extracts[J]. Mutat Res, 2006, 599(1/2): 66-75.
37 Liu SY, Lin MH, Yang SC, et al. Increased expression of matrix metalloproteinase-2 in oral cells after short-term stimulation and long-term usage of areca quid[J]. J Formos Med Assoc, 2005, 104(6): 390-397.
38 Dongre A, Weinberg RA. New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer[J]. Nat Rev Mol Cell Biol, 2019, 20(2): 69-84.
39 Tseng YH, Chang KW, Liu CJ, et al. Areca nut extract represses migration and differentiation while activating matrix metalloproteinase-9 of normal gingival epithelial cells[J]. J Periodontal Res, 2008, 43(5): 490-499.
40 Li XH, He SK, Ma BY. Autophagy and autophagy-related proteins in cancer[J]. Mol Cancer, 2020, 19(1): 12.
41 Xu Z, Huang CM, Shao Z, et al. Autophagy induced by areca nut extract contributes to decreasing cisplatin toxicity in oral squamous cell carcinoma cells: roles of reactive oxygen species/AMPK signaling[J]. Int J Mol Sci, 2017, 18(3): 524.
[1] 古丽其合热·阿布来提,秦旭,朱光勋. 线粒体自噬在牙周炎发生发展过程中的研究进展[J]. 国际口腔医学杂志, 2024, 51(1): 68-73.
[2] 叶玉琳,江莉婷,高益鸣. 舍格伦综合征唾液腺中自噬现象的研究进展[J]. 国际口腔医学杂志, 2022, 49(5): 556-560.
[3] 李归平,秦旭,朱光勋. 腺苷酸活化蛋白激酶在牙周病发生发展中的研究进展[J]. 国际口腔医学杂志, 2022, 49(3): 343-348.
[4] 方苓力,谭玺,叶雨丝,黄兰,何瑶. 颞下颌关节退行性变早期髁突软骨细胞行为改变的实验研究[J]. 国际口腔医学杂志, 2021, 48(4): 417-425.
[5] 周丰,陈野,陈晨,张奕宁,耿瑞蔓,刘戟. 沉默信息调节因子1调控牙周炎发生发展的机制[J]. 国际口腔医学杂志, 2021, 48(3): 341-346.
[6] 尹圆圆,马华钰,李昕怡,徐静晨,柳汀,陈嵩,何姝姝. 小鼠正畸牙移动中牙周组织自噬相关基因表达的初步研究[J]. 国际口腔医学杂志, 2020, 47(6): 627-634.
[7] 李辉莉,方厂云,苏征. 槟榔碱对颊黏膜成纤维细胞增殖及迁移的影响[J]. 国际口腔医学杂志, 2020, 47(1): 32-36.
[8] 朱俊瑾,周佳琦,伍颖颖. 哺乳动物雷帕霉素靶蛋白复合物1介导的自噬对骨代谢的调控[J]. 国际口腔医学杂志, 2020, 47(1): 84-89.
[9] 张鹏, 丁一, 王琪. 炎性衰老在糖尿病牙周炎中的作用机制及研究现状[J]. 国际口腔医学杂志, 2017, 44(6): 664-668.
[10] 陈冠辉 侯劲松. 低氧和自噬与肿瘤的发生发展[J]. 国际口腔医学杂志, 2016, 43(5): 584-588.
[11] 吴家顺，汤亚玲. 涎腺腺样囊性癌中上皮间充质转化分子调控机制的研究进展[J]. 国际口腔医学杂志, 2016, 43(4): 421-427.
[12] 任静宜1 刘歆婵1 丁烨1 于洪强1 周延民1 于维先2. 细胞自噬和炎症反应的相互调控与牙周炎[J]. 国际口腔医学杂志, 2016, 43(4): 462-467.
[13] 邵小钧 席庆. 食用槟榔及其与口腔癌间的关系[J]. 国际口腔医学杂志, 2015, 42(6): 668-672.
[14] 李明，彭解英，吴颖芳，张睿. 槟榔碱诱导上皮细胞基质金属蛋白酶9表达上调的分子机制[J]. 国际口腔医学杂志, 2015, 42(2): 166-169.
[15] 金淑芳 蒋灿华. 细胞自噬相关蛋白8及其连接系统与头颈部恶性肿瘤[J]. 国际口腔医学杂志, 2014, 41(2): 195-198.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
[1] 寇波1，李爱群1，王春玲2，郑效忠3，唐伟3. 三维激光扫描测量技术分析年轻成人快速上颌扩弓后腭部表面形态的变化[J]. 国际口腔医学杂志, 2009, 36(2): 144 -144～147 .
[2] 赖文莉,罗恩,丁寅,房兵. 正畸-正颌病例分析[J]. 国际口腔医学杂志, 2014, 41(2): 129 -132 .