鼠李糖乳桿菌作為益生菌對抗 SARS-CoV-2 膜糖蛋白誘導的炎症

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姓名

甘鄔洋(Uuganbayar Gankhuyag) 查詢紙本館藏

畢業系所

生醫科學與工程學系

論文名稱

鼠李糖乳桿菌作為益生菌對抗 SARS-CoV-2 膜糖蛋白誘導的炎症
(Lactobacillus rhamnosus as a probiotic bacterium against SARS-CoV-2 membrane glycoprotein-induced inflammation)

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摘要(中)

表面醣蛋白為新型冠狀病毒最豐富之蛋白，但該蛋白對於引發新冠肺炎角色還未完全
明確，對比於綠螢光蛋白，在實驗小鼠鼻腔中點該表面醣蛋白可以在肺部沖洗液中引
發介白素六(IL-6) 、一種被當作免疫風暴指標之蛋白的表現，但在磷酸二酯酶(PDE4B)
基因缺陷小鼠中卻無法引發 IL-6 之表現，可見 PDE4B 在 IL-6 訊號傳遞中扮演著重要的
角色，鼠李糖乳桿菌 EH8 亞種 (L. rhamnosus) 之菌絲體發酵產物-丁酸，可以降低PDE4B
之表現量以及 IL-6 在巨噬細胞中的分泌，餵食實驗小鼠菌絲體可以增加共生菌
L. rhamnosus 的數量，小鼠在供給 L. rhamnosus 和菌絲體兩個禮拜後可以降低表面醣
蛋白引發之 PDE4B 表現和 IL-6 分泌，在實驗小鼠餵食游離脂肪酸受體 2(Ffar2) 之抑制
劑 GLPG-0974 後，益生菌效果都會被停止，Ffar2 在腸道-肺部連結可以負調節PDE4B-IL-6
之訊號傳遞將可以做為發展益生菌治療新冠肺炎引發免疫風暴中的一個目標。我與 Pham
Minh Tan 一起進行了我的碩士研究工作，他是博士。我們實驗室的學生。這項研究
工作提交給營養生物化學雜誌正在審查中。作為本文的合著者，我的主要貢獻包括在
人物圖例中的描述。

摘要(英)

The most abundant protein of SARS-CoV-2 is membrane glycoprotein, but the role of
this protein in COVID-19 is unclear. The intranasal administration of membrane
glycoprotein significantly increased IL-6 in BALF in mice compared to green
fluorescent protein (GFP)-inoculated mice. Asignificant decrease was observed in IL-6
levels effected by membrane glycoprotein in PDE4B knockout mice, showing that
PDE4B is essential to the process of IL-6 signaling. In macrophages, butyric acid is
produced by L.rhamnosus EH8 strain fermentation, which inhibits the PDE4B and
affects the release of IL-6. Mice fed mycelia had higher relative abundances of
commensal L. rhamnosus. Feeding L. rhamnosus with mycelia for two weeks
significantly reduced PDE4Band IL-6 in mice. In mice treated with GLPG-0974, the
probiotic effect of L. rhamnosus with mycelia was decreased. In COVID-19, Ffar2
may activate the gut-lung axis for down-regulating the PDE4B-IL-6 signaling.
Treatment of cytokine storms with probiotics may be applicable to this target. I
conducted my master researcher work withPham Minh Tan who is a PhD student in
our lab. This research work submitted to the Journal of Nutritional Biochemistry under
review. As a co-author for this paper, my major contributions including described in
figure legends.

關鍵字(中)

★ 免疫風暴
★ 鼠李糖乳桿菌
★ 介白素 6
★ 磷酸二酯酶 4B
★ 新型冠狀病毒
★ 表面醣蛋白

關鍵字(英)

★ Cytokine storm
★ Lactobacillus rhamnosus
★ SARS-CoV-2
★ Il-6
★ PDE4B
★ membrane glycoprotein

論文目次

Table of Contents
Acknowledgements ................................................................................................................... 1 Abstract...................................................................................................................................... 2 List of Figures ............................................................................................................................ 5 List of Tables.............................................................................................................................. 5 Abbreviation List ....................................................................................................................... 6 Chapter 1 Introduction ............................................................................................................... 1
1.1 SARS-CoV-2 ................................................................................................................... 1 1.2 Cytokine storm (IL-6) by SARS-Cov-2........................................................................... 2 1.3 PDE4 ................................................................................................................................ 3 1.4 Role of gut bacteria in Covid-19...................................................................................... 4 1.5 Lactobacillus rhamnosus.................................................................................................. 5 1.6 Literature review .............................................................................................................. 6
1.6.1 SARS-CoV-2 to cytokine storm .................................................................................. 6
1.6.2 Probiotic bacteria are reducing IL-6 cytokine .......................................................... 7
Chapter 2 Research objectives ................................................................................................... 8 Chapter 3 Materials and Methods ............................................................................................ 10 3.1 Materials ........................................................................................................................ 10 3.1.1 Experimental equipment, reagents and primers ......................................................... 10 3.2 Methods.......................................................................................................................... 12 3.2.1 Ethics statement .......................................................................................................... 12 3.2.2 Bacterial identification. (16s gene sequencing) ...................................................... 12 3.2.3 Fungi identification. (ITS gene sequencing) ............................................................ 13 3.2.4 Purification and expression of SARS-CoV-2 membrane glycoproteins. .................... 13 3.2.5 The Detection of IL-6 in BALF ................................................................................ 14
III
3.2.6 Quantitative reverse transcription polymerase chain reaction ............................... 14
3.2.7 Mycelium preparation.............................................................................................. 15
3.2.8 Fermentation of mycelium of L. rhamnosus EH8 ....................................................... 15
3.2.9 Detection of L. rhamnosus .......................................................................................... 15
3.2.10 Cell culture........................................................................................................... 16
3.2.11 A high performance liquid chromatography (HPLC) method for detecting butyric
acid ...........................................................................................................16
3.2.12 Inhibition of Ffar2................................................................................................ 17
3.2.13 Statistical analysis ............................................................................................... 17
Chapter 4 Results ..................................................................................................................... 18
4.1 The membrane glycoprotein of SARS-CoV-2 induces IL-6 via PDE4B ...................... 18
4.2 SARS-CoV-2 membrane glycoprotein-induced IL-6 is diminished by mycelium fermentation of L. rhamnosus EH8 ............................................................................... 20
4.3 As a result of mycelium fermentation of L.rhamnosus EH8, butyric acid was produced and macrophage IL-6 secretion and PDE4B expression were reduced......................... 22
4.4 L. rhamnosus EH8 mycelium fermentation reduces PDE4B and IL-6 by regulating Ffar2............................................................................................................................... 24
4.5 SARS-CoV-2 membrane glycoprotein increased PDE4B and IL-6 production in macrophage cells............................................................................................................ 25
Chapter 5 Discussion ............................................................................................................... 26
Chapter 6 Conclusions ............................................................................................................. 27
Future work.............................................................................................................................. 28
References ............................................................................................................................... 29

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指導教授

黃俊銘(Chun-Ming Huang)

審核日期

2021-7-30

推文