臺灣博碩士論文加值系統

幽門螺旋菌 (H. pylori) 是一種微好氧螺旋形狀的革蘭氏陰性菌，會存在於人類的胃以及十二指腸的表皮黏膜上。幽門螺旋菌會引起胃襯的慢性發炎並且與十二指腸、胃潰瘍和以及胃癌的發展有密切關係。幽門螺旋菌對於胃腸道潰瘍的發生有密切相關，一般認為滅除幽門螺旋菌可以使得胃發炎復原，亦可治療潰瘍性疾病。現有多種治療幽門螺旋菌感染的療法，最為普遍使用的是三合一療法，包含了兩種抗生素與一種胃酸分泌抑制劑。然而，在治療幽門螺旋菌感染上，抗生素抗藥性的增加與抗微生物劑的使用有關。因此，根治幽門螺旋菌過程中需要有輔助治療的方法。樟薄孔菌（Antrodia camphorata）是在台灣的一種稀有的藥材，其子果實的名稱是眾所周知的牛樟芝或樟芝。在台灣，牛樟芝對人體的神奇功效，被許多機構廣泛的調查與研究。在本篇論文中，從牛樟芝上分離出13種萃取物。在13種萃取物中，不論是在胃上皮細胞與小鼠巨噬細胞上，AC-3 (15α-Acetyldehydrosulphurenic acid)與AC-10 (Antcin C)對於幽門螺旋菌擁有較高的抑制效果。此外，對於NF-κB以及IL-8的抑制，也顯示出AC萃取物的抗發炎功效。期望此實驗結果可以為治療幽門螺旋菌感染帶來新的方法。

Helicobacter pylori is a Gram-negative, micro-aerophilic bacterium that inhabits various areas of the stomach and duodenum. It causes a chronic inflammation of the stomach lining and strongly linked to the development of duodenal and gastric ulcers and stomach cancer. It has been well confirmed that infection with H. pylori is an etiological agent of peptic ulcer, and eradication of this organism can not only make the associated gastritis regression but cure these diseases. There are several treatments of H. pylori infection, most common treatment is triple therapy, including two antibiotics and a proton pump inhibitor. However, the increasing of antibiotic resistant rate was associated with the usage of antimicrobial agents in the treatment of H. pylori infection. Thus, the complementary therapeutic methods in eradication of H. pylori were needed. Antrodia camphorata (A. camphorata) is a rare treasure in Taiwan, the fruiting body of A. camphorata is well known by named niu-chang-chih or jang-jy. The miraculous efficacy in human body had been investigated and researched by many organizations in Taiwan. In this study, thirteen compounds were isolated from A. camphorata. Among them, compound 15α-Acetyldehydrosulphurenic acid (number 3) and Antcin C (number 10) had higher inhibition effects on H. pylori-induced inflammatory responses in gastric epithelial cells and mouse macrophage RAW 264.7 cells than other compounds. In addition, these constituents also inhibited the IL-8 protein expression and NF-κB signal activation in H. pylori-infected gastric epithelial cells and it might contribute to the potential role of A. camphorata as an anti-inflammatory action. Results from our study might bring a new method for treating of H. pylori infection.

Content
Abstract III
中文摘要 (Abstract in Chinese) V
Introduction 1
Helicobacter pylori infection and inflammation responses 1
Treatments of H. pylori infection 2
Antrodia camphorata 5
Materials and methods 8
Bacterial strain and cell culture 8
Determination of anti-bacterial activity 8
Cell survival assay (MTT method) 9
Inhibition of H. pylori adherence to and invasion into epithelial cells 9
Transient transfection of NF-κB and IL-8 reporter genes 10
Measurement of interleukin-8 expression 11
Inhibition of macrophage activation 11
Western blot for assay of iNOS expression 12
Cell survival assay (MTT method) in macrophages 12
Statistical evaluation 13
Results 14
Inhibition of H. pylori growth 14
Treatment of A. camphorata constituents AC-3, AC-10, and AC-12 does influence epithelial cell viability 14
Inhibition of H. pylori adherence and invasion of gastric epithelial cells 15
AC-3 inhibit H. pylori-induced inflammation of gastric epithelial cells 15
Treatment of A. camphorata constituents AC-3, AC-10, and AC-12 does influence marcophages viability 17
A. camphorata constituents AC-3 and AC-10 inhibit LPS-mediated nitric oxide production in macrophages 17
Western blot of inhibition of iNOS production 18
Discussion and conclusion 19
References 22
Table 1 Molecular weight of A. camphorata constituents 30
Table 2 Inhibitory effects of A. camphorata constituents 31
Fig. 1. Chemiacl structures of A. camphorata constituents 1-11 32
Fig. 2. Effect of A. camphorata constituents (AC-3, AC-10, and AC-12) and DMSO control on gastric epithelial cell viability 35
Fig. 3. Effect of A. camphorata constituents (AC-3, AC-10, and AC-12) on H. pylori adhesion of gastric epithelial cells 36
Fig. 4. Effect of A. camphorata constituents (AC-3, AC-10, and AC-12) on H. pylori invasion of gastric epithelial cells 37
Fig. 5. Effect of A. camphorata constituents (AC-3, AC-10, and AC-12) on H. pylori-induced NF-κB activation in gastric epithelial cells 38
Fig. 6. Effect of A. camphorata constituents (AC-3, AC-10, and AC-12) on H. pylori-induced IL-8–luciferase activity in gastric epithelial cells. 39
Fig. 7. Effect of A. camphorata constituents (AC-3, AC-10, and AC-12) and DMSO control in RAW 264.7 cells 40
Fig. 8. Inhibition by A. camphorata constituents (AC-3, AC-10, and AC-12) and DMSO control of LPS-induced NO production in RAW 264.7 cells 41
Fig. 9. Effects of A. camphorata constituents (AC-3 and AC-10) on the iNOS protein expression in RAW 264.7 cells 42
Appendix 1 Therapy of H. pylori infection 43
Appendix 2 Drugs used in H. pylori therapy 44

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