人體皮膚致電微生物組通過調節鐵和自由基來減輕紫外線B引起的皮膚損傷。

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巴艾倫(Arun Balasubramaniam) 查詢紙本館藏

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生醫科學與工程學系

論文名稱

人體皮膚致電微生物組通過調節鐵和自由基來減輕紫外線B引起的皮膚損傷。
(The Electrogenic Human Skin Microbiome Attenuates Ultraviolet-B Induced Skin Damage via Regulation of Irons and Free Radicals)

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摘要(中)

人類皮膚中豐富的微生物組精通細胞外電子轉移 (EET)，並且天生就從事不同的分子任務。眾所周知，紫外線B (UV-B) 輻射會造成深層組織移位並減弱免疫反應。微生物影響之重要性對於紫外線引起之皮膚損傷是無庸置疑的。近年來，腸道微生物組藉由膜上電子傳遞蛋白產生電已經被證明，然而皮膚微生物組中的致電細菌表徵似乎完全沒有被表明。本研究藉由ferric-ferrozine方法概述了皮膚表皮葡萄球菌 (S. epidermidis) 是一種致電細菌菌株。甘油對於S. epidermidis的發酵之產電能力至關重要，在燃料電池中 (MFC) 通過測量電壓變化得知藉由5-甲基糠醛 (5-MF) 抑制顯著的減少了細菌產電量。本研究為了瞭解UV-B對S. epidermidis發電以及抗性的影響，開發了一個具有陽極和陰極的培養環境。儘管UV-B照射減少了細菌產電量，但長時間的甘油孵育會導致表S. epidermidis發酵，並增加產電量以抵消UV-B的影響。因此，人類皮膚原有之細菌群也許能作為生物標誌物即時的藉由產生之電量描繪出紫外線輻射。
　　皮膚益生元對皮膚益生菌的探究尚未明確，也許能通過對現有皮膚護理之化合物進行改造與激發來適應新的症狀。利用四種已被國際化妝品成分命名法 (INCI) 註冊的化合物，研究其誘導S. epidermidis (一種在人體皮膚中富集的細菌) 發酵的能力。最初用作於潤膚劑的Liquid coco-caprylate/caprate (LCC)有效地引發了S. epidermidis的發酵，產生短鏈脂肪酸 (SCFA) 並激發了強勁的電力。LCC加S. epidermidis在小鼠皮膚上的施用可顯著減少UV-B誘導的損傷，可通過形成之4-羥基壬烯醛(4-HNE) 、環丁烷嘧啶二聚體 (CPD) 和皮膚損傷來評估。研究發現，具有低表達丙酮酸脫氫酶 (pdh) 和磷酸乙酰基轉移酶 (pta) 基因之S. epidermidis S2的電原性很差。當將S. epidermidis S2以及LCC應用於小鼠皮膚時，S. epidermidis加LCC對UV-B誘導皮膚損傷的保護作用被大大抑制。探索LCC促進S. epidermidis抵抗UV-B的新適應症，提供了將INCI註冊化合物重新用作皮膚益生元的例子，使人類可以從皮膚微生物組普遍存在的細菌中受益。S. epidermidis加甘油在小鼠皮膚上的局部施用減輕了UV-B誘導的不穩定亞鐵離子(Fe2 +)、4-HNE和CPD的產生，表明細菌產生的電能減弱由UV-B引起的氧化損傷。在S. epidermidis中存在的親環蛋白A中，調節EET系統中II型NADH氫化酶 (ndh2) 的基因表達和脫甲基甲萘醌-8 (DMK-8)的產生至關重要。抑制親環蛋白A消除了電子生成，並大大降低了S. epidermidis發酵的抗UV活性。本研究中我們首次證明了致電皮膚細菌可促進親環蛋白A介導的途徑產電，從而抵禦紫外線輻射問題。

摘要(英)

A vibrant range of microbiome has been annexed in the skin that are proficient in extracellular electron transfer (EET) and beyond innately engaged in different molecular tasks. Ultraviolet-B (UV-B) radiation was indeed known to trigger deeper tissue shift as well as to attenuate the immune reaction. The importance of the microbiome was hardly debated in UV-induced skin insults. Bacteria have been using electron transport proteins throughout the membrane to generate electricity in the intestinal microbiome that has been identified recently. Although, the characterization of electrogenic bacteria in the skin microbiome seems to be almost completely unmapped. We have outlined the skin Staphylococcus epidermidis (S. epidermidis) as an electrogenic bacterial strain using a ferric-ferrozine investigation. The fermentation of glycerol is vital for the production of electricity in S. epidermidis while inhibition of 5-methyl furfural (5-MF) significantly reduced the bacterial electricity measured in a fuel cell by voltage changes (MFC). In order to investigate the impact of UV-B on electricity generation and UV-B bacterial resistance in S. epidermidis bacteria, a small-scale chamber with both anode and cathode was developed. Though UV-B decreased bacterial electricity, prolonged glycerol incubation led to fermentation of S. epidermidis and increased electricity to counteract UV-B effect. Electricity produced by human skin-based bacteria may be used as an adaptive biomarker to depict ultraviolet radiation in live time.
The exploration of skin prebiotics for skin probiotics have not been well defined and may be instigated by retrofitting existing skincare compounds for new indications. Four compounds that have been registered by the International Nomenclature of Cosmetic Ingredients (INCI) were included to study their abilities to induce the fermentation of Staphylococcus epidermidis (S. epidermidis), a bacterial species abundant in the human skin. Liquid coco-caprylate/caprate (LCC), originally used as an emollient, effectively initiated the fermentation of S. epidermidis, produced short-chain fatty acids (SCFAs), and provoked robust electricity. Application of LCC plus S. epidermidis on mouse skin significantly reduced ultraviolet B (UV-B)-induced injuries which were evaluated by the formation of 4-hydroxynonenal (4-HNE), cyclobutane pyrimidine dimers (CPD), and skin lesions. An S. epidermidis S2 isolate with low expressions of genes encoding pyruvate dehydrogenase (pdh), and phosphate acetyltransferase (pta) was found to be poorly electrogenic. The protective action of S. epidermidis plus LCC against UV-B-induced skin injuries was considerably suppressed when mouse skin was applied with LCC in combination with the S. epidermidis S2 isolate. Exploring new indication of LCC for promoting S. epidermidis against UV-B provided an example of repurposing INCI-registered compounds as skin prebiotics.
The host may benefit from the prevalent bacteria present in the skin microbiome. Topical application of S. epidermidis plus glycerol onto mouse skin mitigated the UV-B-induced production of labile ferrous ion (Fe2+), 4-HNE (4-hydroxy-2-nonenal), and cyclobutene pyrimidine dimers (CPD), demonstrating that bacterial electricity acts to attenuate oxidative damage caused by UV-B. The regulation of gene expression of Type II NADH hydrogenase gene (ndh2) and production of demethylmenaquinone-8 (DMK-8) in EET systems for the production of electricity was critical in Cyclophilin A present in S. epidermidis. Suppression of cyclophilin A axed electron generation and considerably reduced the anti-UV activity of S. epidermidis fermentation. Cumulatively, we demonstrate for the first time that electrogenic skin bacteria facilitate a cyclophilin A-mediated pathway to generate electricity as a defense against UV.

關鍵字(中)

★ 皮肤
★ 微生物组
★ 紫外线
★ 癌症
★ 益生菌
★ 预科

關鍵字(英)

★ Skin
★ Microbiome
★ Ultraviolet rays
★ Cancer
★ Probiotics
★ Prebiotics

論文目次

Abstract in Chinese ii
Abstract in English viii
Acknowledgement xi
List of tables xvii
List of figures xviii
Abbreviations xxvi
Chapter 1- Introduction 1
1.1. The Human skin microbiome 1
1.2. Host-microbiome Interaction 2
1.3. Skin probiotic bacteria 3
1.4. Skin prebiotics 4
1.5. Extracellular electron transfer 5
1.6. Electrons as antioxidant 5
1.7. Staphylococcus epidermidis 6
1.8. UV-B irradiation 7
Chapter 2 -Skin Bacteria Mediate Glycerol Fermentation to Produce Electricity and Resist UV-B 9
9
2.1. Introduction 9
2.2. Materials and methods 10
2.2.1. Ethics statement 10
2.2.2. Bacteria identification 11
2.2.3. 5-Methyl furfural mediated fermentation inhibition 11
2.2.4. Electrogenic bacteria screening 11
2.2.5. Minimum bactericidal concentration (MBC) assay 12
2.2.6. Microbial fuel cell construction 12
2.2.7. Electricity detection in vitro 13
2.2.8. Statistical analysis 13
2.3. Results 14
2.3.1. Electrogenic bacteria screening by TSB agar-based ferrozine overlay assay 14
2.3.2. The electricity generation from glycerol mediated S. epidermidis ATCC 12228 fermentation through microbial fuel cell (MFC) technology 16
2.4. Discussion 22
Chapter 3- Repurposing INCI-registered compounds as skin prebiotics for probiotic Staphylococcus epidermidis against UV-B 26
3.1. Introduction 26
3.2. Material and methods 29
3.2.1. Ethics statement: 29
3.2.2. Bacterial fermentation: 29
3.2.3. Electricity detection: 30
3.2.4. Cyclic voltammetry: 30
3.2.5. Extraction of bacterial RNA: 31
3.2.6. Real-time qPCR (RT-qPCR): 31
3.2.7. GC-MS analysis: 32
3.3.8. UV-B exposure: 32
3.2.8. Western blotting: 33
3.2.9. Hematoxylin and Eosin (H&E) staining: 33
3.2.10. Minimum bactericidal concentration (MBC) 34
3.2.11. Statistical analysis: 34
3.3. Results 34
3.3.1. INCI-registered compounds function as skin probiotics for induction of SCFA production and bacterial electricity 34
3.3.2. Topical application of S. epidermidis plus LCC reduced UV-B-induced the formation of 4-hydroxynonenal (4-HNE) and cyclobutane pyrimidine dimer (CPD) 37
3.3.3. The S. epidermidis S2 isolate with low expression of pdh and pta genes was poorly electrogenic 39
3.3.4. LCC plus S. epidermidis S2 isolate did not confer protection against UV-B-induced skin injuries 40
Chapter 4- Electrogenic skin Staphylococcus epidermidis protects against ultraviolet damage 46
4.1. Introduction 46
4.2. Materials and method 49
4.2.1. Ethics statement 49
4.2.2. Bacterial culture and fermentation 50
4.2.3. Electricity detection in vitro 50
4.2.4. Cyclic voltammetry 51
4.2.5. Ferrozine assay 51
4.2.6. UV-B exposure 52
4.2.7. Iron calorimetric assay and labile iron staining 52
4.2.8. Western blotting 53
4.2.9. Intracellular ROS detection 54
4.2.10. Extraction of bacterial RNA 54
4.2.11. Real-time PCR 55
4.2.12. Mass spectrometry 55
4.2.13. Statistical analysis 56
4.3. Results 56
4.3.1. S. epidermidis produced electricity via glycerol fermentation 56
4.3.2. Electrons generated by S. epidermidis engage in the redox cycling of iron 59
4.3.3. S. epidermidis fermentation mitigated UV-B induced labile irons in skin 61
4.3.4. S. epidermidis fermentation suppresses UV-B-induced 4-HNE (4-hydroxy-2-nonenal) and cyclobutene pyrimidine dimer (CPD) 62
4.3.5. Cyclophilin A is an essential mediator of S. epidermidis’s electrogenic inhibition of 4-HNE production 64
4.4. Discussions 67
Chapter 5- Conclusion and outlook 78
Vita 81
Publications 82
Bibliography 83

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指導教授

黃俊銘(Chun-Ming Huang)

審核日期

2020-12-30

推文