基于人消化道微生态体外模拟系统观察纳米二氧化钛对肠道菌群的影响

doi:10.19723/j.issn.1671-167X.2022.03.011

摘要/Abstract

摘要：

目的: 通过消化道微生态体外模拟系统探索纳米二氧化钛(titanium dioxide nanoparticles，TiO₂ NPs)对人源肠道菌群组成和结构的影响。方法: 对TiO₂ NPs进行粒径、形状、晶型和团聚程度的表征。通过模拟胃、小肠、结肠的液体环境和物理条件建立体外人消化道微生态模拟系统，并对模拟系统的稳定性进行评价。从人粪便中提取菌群，采用该模拟系统稳定培养，分别暴露于0、20、100、500 mg/L TiO₂ NPs中，收集染毒24 h后的菌液，通过16S rRNA测序技术分析TiO₂ NPs对人源肠道菌群组成和结构的影响，利用线性判别效应量分析(linear discriminant analysis effect size，LEfSe)筛选差异细菌，根据京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes，KEGG)数据库进行功能预测。结果: TiO₂ NPs为球形粒径，直径(25.12±5.64) nm，晶体结构为锐钛矿，在超纯水中水合粒径为(609.43±60.35) nm，Zeta电位为(-8.33±0.22) mV。体外消化道模拟系统培养人源肠道菌群24 h后达到相对稳定状态，肠球菌(Enterococci)、大肠杆菌(Enterobacterium)和乳酸杆菌(Lactobacillus)计数可分别达到(1.60±0.85)×10⁷个、(5.60±0.82)×10⁷个和(2.70±1.32)×10⁷个。16S rRNA测序结果显示，与对照组相比，TiO₂ NPs染毒组(20、100和500 mg/L)在门、纲、目、科、属水平上，肠道菌群的物种数量和均匀度未受到明显影响，但部分菌种的相对丰度发生显著变化。TiO₂ NPs染毒组(20、100和500 mg/L)与对照组之间共筛选出42种不同的差异细菌(线性判别分析分数，linear discriminant analysis score，LDA score>3)，以肠杆菌属(Enterobacter)、拟杆菌科(Bacteroidaceae)、乳酸杆菌科(Lactobacillaceae)、双歧杆菌科(Bifidobacteriaceae)和梭菌属(Clostridium)等为代表。进一步的肠道菌群功能预测分析显示，TiO₂ NPs可能影响肠道菌群的氧化磷酸化、能量代谢、磷酸盐和磷酸盐代谢、甲烷代谢等代谢和功能(P < 0.05)。结论: 体外人消化道微生态模拟系统下，TiO₂ NPs可以显著改变人源肠道菌群的组成和结构，以肠杆菌属和益生菌为代表，并可能进而影响机体多种物质的代谢和功能。

关键词: 纳米粒子, 二氧化钛, 胃肠道微生物组, 模型, 生物学, 16S rRNA测序技术

Abstract:

Objective: To explore the effects of oral exposure to titanium dioxide nanoparticles (TiO₂ NPs) on the composition and structure of human gut microbiota. Methods: The particle size, shape, crystal shape and degree of agglomeration in ultrapure water of TiO₂ NPs were characterized. The in vitro human digestive tract microecological simulation system was established by simulating the fluid environment and physical conditions of stomach, small intestine and colon, and the stability of the simulation system was evaluated. The bacterial communities were extracted from human feces and cultured stably in the simulated system. They were exposed to 0, 20, 100 and 500 mg/L TiO₂ NPs, respectively, and the bacterial fluids were collected after 24 h of exposure. The effect of TiO₂ NPs on the composition and structure of human gut microbiota was analyzed by 16S rRNA sequencing technology. Linear discriminant analysis effect size (LEfSe) was used to screen differential bacteria, and the Kyoto encyclopedia of genes and genomes (KEGG) database for functional prediction. Results: The spherical and anatase TiO₂ NPs were (25.12±5.64) nm in particle size, while in ultra-pure water hydrated particle size was (609.43±60.35) nm and Zeta potential was (-8.33±0.22) mV. The in vitro digestive tract microecology simulation system reached a relatively stable state after 24 hours, and the counts of Enterococci, Enterobacte-rium, and Lactobacillus reached (1.6±0.85)×10⁷, (5.6±0.82)×10⁷ and (2.7±1.32)×10⁷, respectively. 16S rRNA sequencing results showed that compared with the control group, the number and evenness of gut microbiota were not significantly affected at phylum, class, order, family and genus levels in TiO₂ NPs groups (20, 100 and 500 mg/L). The relative abundance of some species was significantly changed, and a total of 42 different bacteria were screened between the TiO₂ NPs groups (20, 100 and 500 mg/L) and the control group [linear discriminant analysis(LDA) score>3], represented by Enterobacter, Bacteroidaceae, Lactobacillaceae, Bifidobacteriaceae and Clostridium. Further predictive analysis of gut microbiota function showed that TiO₂ NPs might affect oxidative phosphorylation, energy meta-bolism, phosphonate and phosphonate metabolism, and methane metabolism (P < 0.05). Conclusion: In human digestive tract microecological simulation system, TiO₂ NPs could significantly change the composition and structure of human gut microbiota, represented by Enterobacter and probiotics, and may further affect a variety of metabolism and function of the body.

Key words: Nanoparticles, Titanium dioxide, Gastrointestinal microbiome, Models, biological, 16S rRNA sequencing technology

中图分类号:

R155.5

张家赫,史佳琪,陈章健,贾光. 基于人消化道微生态体外模拟系统观察纳米二氧化钛对肠道菌群的影响[J]. 北京大学学报（医学版）, 2022, 54(3): 468-476.

Jia-he ZHANG,Jia-qi SHI,Zhang-jian CHEN,Guang JIA. Effects of nano titanium dioxide on gut microbiota based on human digestive tract microecology simulation system in vitro[J]. Journal of Peking University (Health Sciences), 2022, 54(3): 468-476.

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参考文献 27

1	Aguilar F, Crebelli R, Di Domenico A, et al. Re-evaluation of titanium dioxide (E 171) as a food additive[J/OL]. EFSA Journal, 2016, 14(9): 4545. https://doi.org/10.2903/j.efsa.2016.4545.
2	Yang Y , Doudrick K , Bi X , et al. Characterization of food-grade titanium dioxide: The presence of nanosized particles[J]. Environ Sci Technol, 2014, 48 (11): 6391- 6400. doi: 10.1021/es500436x
3	Chen ZJ , Han S , Zhou SP , et al. Review of health safety aspects of titanium dioxide nanoparticles in food application[J]. Nanoimpact, 2020, 18, 100224. doi: 10.1016/j.impact.2020.100224
4	Shakeel M , Jabeen F , Shabbir S , et al. Toxicity of nano-titanium dioxide (TiO₂-NP) through various routes of exposure: A review[J]. Biol Trace Elem Res, 2016, 172 (1): 1- 36. doi: 10.1007/s12011-015-0550-x
5	Alavi M , Karimi N . Characterization, antibacterial, total antioxidant, scavenging, reducing power and ion chelating activities of green synthesized silver, copper and titanium dioxide nanoparticles using Artemisia haussknechtii leaf extract[J]. Artif Cells Nanomed Biotechnol, 2018, 46 (8): 2066- 2081.
6	Hajipour MJ , Fromm KM , Ashkarran AA , et al. Antibacterial properties of nanoparticles[J]. Trends Biotechnol, 2012, 30 (10): 499- 511. doi: 10.1016/j.tibtech.2012.06.004
7	Daou I , Moukrad N , Zegaoui O , et al. Antimicrobial activity of ZnO-TiO₂ nanomaterials synthesized from three different precursors of ZnO: Influence of ZnO/TiO₂ weight ratio[J]. Water Sci Technol, 2018, 77 (5/6): 1238- 1249.
8	Chen L , Guo Y , Hu C , et al. Dysbiosis of gut microbiota by chronic coexposure to titanium dioxide nanoparticles and bisphenol A: Implications for host health in zebrafish[J]. Environ Pollut, 2018, 234, 307- 317. doi: 10.1016/j.envpol.2017.11.074
9	Chen Z , Han S , Zhou D , et al. Effects of oral exposure to tita-nium dioxide nanoparticles on gut microbiota and gut-associated metabolism in vivo[J]. Nanoscale, 2019, 11 (46): 22398- 22412. doi: 10.1039/C9NR07580A
10	Chen Z , Zhou D , Han S , et al. Hepatotoxicity and the role of the gut-liver axis in rats after oral administration of titanium dioxide nanoparticles[J]. Part Fibre Toxicol, 2019, 16 (1): 48. doi: 10.1186/s12989-019-0332-2
11	Mu W , Wang Y , Huang C , et al. Effect of long-term intake of dietary titanium dioxide nanoparticles on intestine inflammation in mice[J]. J Agric Food Chem, 2019, 67 (33): 9382- 9389. doi: 10.1021/acs.jafc.9b02391
12	Li J , Yang S , Lei R , et al. Oral administration of rutile and anatase TiO₂ nanoparticles shifts mouse gut microbiota structure[J]. Nanoscale, 2018, 10 (16): 7736- 7745. doi: 10.1039/C8NR00386F
13	刘倩, 姜建辉, 吴瑛. 纳米二氧化钛对果蝇肠道共生菌的影响[J]. 黑龙江农业科学, 2017, (9): 94- 97.
14	Brodkorb A , Egger L , Alminger M , et al. INFOGEST static in vitro simulation of gastrointestinal food digestion[J]. Nat Protoc, 2019, 14 (4): 991- 1014. doi: 10.1038/s41596-018-0119-1
15	Molly K , Woestyne MV , Verstraete W . Development of a 5-step multichamber reactor as a simulation of the human intestinal microbial ecosystem[J]. Appl Microbiol Biot, 1993, 39 (2): 254- 258. doi: 10.1007/BF00228615
16	杨立娜, 黄靖航, 赵亚凡, 等. 胃肠道体外模拟系统在调控肠道菌群研究中的应用进展[J]. 渤海大学学报(自然科学版), 2018, 39 (4): 320- 329. doi: 10.3969/j.issn.1673-0569.2018.04.007
17	Laird BD , van de Wiele TR , Corriveau MC , et al. Gastrointestinal microbes increase arsenic bioaccessibility of ingested mine tai-lings using the simulator of the human intestinal microbial ecosystem[J]. Environ Sci Technol, 2007, 41 (15): 5542- 5547. doi: 10.1021/es062410e
18	叶峰, 王晓艳. 粪菌保存液及其保存粪菌的方法, CN105385599A[P/OL]. (2016-03-09)[2022-02-16]. https://pss-system.cponline.cnipa.gov.cn/documents/detail?prevPageTit=chagngui.
19	Schiller C , Frohlich CP , Giessmann T , et al. Intestinal fluid vo-lumes and transit of dosage forms as assessed by magnetic resonance imaging[J]. Aliment Pharm Ther, 2005, 22 (10): 971- 979. doi: 10.1111/j.1365-2036.2005.02683.x
20	Khan ST , Saleem S , Ahamed M , et al. Survival of probiotic bacteria in the presence of food grade nanoparticles from chocolates: An in vitro and in vivo study[J]. Appl Microbiol Biot, 2019, 103 (16): 6689- 6700. doi: 10.1007/s00253-019-09918-5
21	Baranowska-Wójcik E, Szwajgier D, Gustaw K. Effect of TiO₂ on selected pathogenic and opportunistic intestinal bacteria[J/OL]. Biol Trace Elem Res, (2021-07-23)[2021-12-08]. doi: 10.1007/s12011-021-02843-7.
22	Lucas-González R , Viuda-Martos M , Pérez-Alvarez JA , et al. In vitro digestion models suitable for foods: Opportunities for new fields of application and challenges[J]. Food Research International, 2018, 107, 423- 436. doi: 10.1016/j.foodres.2018.02.055
23	Dudefoi W , Moniz K , Allen-Vercoe E , et al. Impact of food grade and nano-TiO₂ particles on a human intestinal community[J]. Food Chem Toxicol, 2017, 106 (Pt A): 242- 249.
24	Gomaa EZ . Human gut microbiota/microbiome in health and di-seases: A review[J]. Antonie van Leeuwenhoek, 2020, 113 (12): 2019- 2040. doi: 10.1007/s10482-020-01474-7
25	Nogueira CM , de Azevedo WM , Dagli ML , et al. Titanium dio-xide induced inflammation in the small intestine[J]. World J Gastroenterol, 2012, 18 (34): 4729- 4735. doi: 10.3748/wjg.v18.i34.4729
26	刘静. 纳米TiO₂对肠上皮紧密连接蛋白Occludin和ZO-1的表达及相关细胞信号通道的影响[D]. 南京: 东南大学, 2019.
27	陈章健, 王云, 贾光. 纳米二氧化钛食品安全性研究进展[J]. 卫生研究, 2015, 44 (6): 1036- 1041.

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