臺灣博碩士論文加值系統

肝臟纖維化是一個動態的過程，在多數慢性肝損傷病人身上，肝纖維化最終會導致肝硬化。但是，肝臟纖維化同時也是可逆的。避免肝臟星狀細胞的活化與發炎是治療肝纖維化很重要的對策。目前治療肝臟疾病的療法仍有許多未解之侷限。本論文中，我利用兩種方法來研究中藥在治療肝纖維化與肝臟星狀細胞活化上的可能性。首先，本團隊多年來致力於研究肝病常用中藥丹參。過去，團隊發現其中的水溶性成分中的純化物丹參酚酸B (salvianolic acid B, C36H30O16, 分子量 = 746) 有顯著抑制肝損傷與肝臟星狀細胞活化的效果。因此，除了丹參酚酸B，我企圖進一步探討脂溶性成分中的丹參酮IIA (tanshinone IIA, C19H18O3, MW = 294.34) 是否有能力抑制肝臟星狀細胞活化，並且了解其作用機轉。再者，我用另一種思維開發具有潛力的新藥。於預試驗中篩選上百種從中藥萃取之純化物，並發掘中藥蛇床子中的純化物蛇床子素具有顯著抑制肝臟星狀細胞活化效果。因此，我在本論文中深入探討蛇床子素在大鼠肝纖維化與肝臟星狀細胞活化之影響。
本研究使用脂多醣 (lipopolysaccharide, LPS, 100 ng/ml) 刺激大鼠星狀細胞株 (HSC-T6) 活化。丹參酮IIA (10 uM) 治療活化的HSC-T6 活化，並與丹參酚酸B (200 M) 相比較。所有的藥物濃度對細胞皆沒有毒性。丹參酮IIA明顯降低脂多醣誘導之NF-κB活性，NF-κB-p65的入核反應，以及ERK, JNK 和p38的磷酸化。此外，傷口癒合試驗與細胞遷移試驗皆證實，脂多醣誘導之HSC-T6的趨化性被丹參酮IIA所抑制。而與趨化性相關之基因表現，包括CCL2, CCL3, CCL5也都受到調控。另外，丹參酮IIA亦顯著抑制 LPS所刺激之發炎反應與纖維化機轉相關基因表現如IL-1b，TNF-a，iNOS，ICAM-1，和 IL-6。至於，肝臟星狀細胞活化時所分泌之纖維蛋白 a-SMA的蛋白質及基因表現，丹參酮IIA皆有效調降。
針對蛇床子素(osthole, C15H16O3, 分子量 = 244.29) 的研究，則是以硫代乙醯胺 (Thioacetamide，TAA) 誘發大鼠肝臟纖維化來評估蛇床子素療肝損傷的能力。將28隻大鼠隨機分成三個組別：控制組，TAA組及蛇床子素 (10 mg/kg) 治療TAA組。蛇床子素明顯降低TAA所誘發AST 和ALT，改善肝臟受損組織與肝臟纖維化情形，並且減少膠原蛋白與-SMA蛋白沉積。此外，蛇床子素有效抑制了與纖維化有關的基因表現與p65的入核表現。TAA所引起的氧化反應被蛇床子素改善之外，發炎反應相關基因表現與細胞激素生成也都得到改善。在細胞機轉方面，我同時探討了蛇床子素對大鼠及人類的肝臟星狀細胞 (HSC-T6 和 LX-2) 的抑制作用。蛇床子素 (10 ug/ml) 有效降低TGF-b1誘導之細胞遷移與侵犯反應。此外，蛇床子素也減少TNF- 刺激的NF-κB活性。蛇床子素對TGF-b1或是 ET-1所誘發的肝臟星狀細胞收縮性皆有效緩解。本論文證實丹參酮IIA具有降低脂多醣誘發之肝臟星狀細胞活化。此外，也發現蛇床子素對TAA所引起大鼠肝損傷有改善，並且可以抑制肝臟星狀細胞活化。
本研究結果顯示，中藥丹參中的脂溶性純化物丹參酮IIA具有抑制大鼠肝臟星狀細胞活化的能力，而中藥蛇床子中的蛇床子素可改善大鼠肝損傷並且抑制大鼠肝臟星狀細胞活化。本論文結果可以提供中醫用藥的佐證，並且給予中醫藥於科學研究方向之參考。

Hepatic fibrosis is a dynamic process which ultimately leads to cirrhosis in many patients with chronic hepatic injury. However, progressive fibrosis is a reversible scarring response. Preventing activation and inflammation of hepatic stellate cells (HSCs) is the major strategy to treat hepatic fibrosis. Clinical therapies for hepatic diseases remain unsatisfactory. In the present studies, two approaches were used to investigate the potentials of Traditional Chinese Medicines (TCM) in treating hepatic fibrosis and HSC activation. Previously, our group has taken years to study Salvia miltiorrhiza Bae. (S. miltiorrhiza) which is a common herb used in the prescriptions of treating hepatic disease. It was found that Sal B exerts inhibitory ability in HSC activation and hepatic injury. I attempted to explore whether the Tanshinone IIA (Tan IIA, C19H18O3, MW = 294.34), one of lipophilic diterpene, in S. miltiorrhiza could inhibit HSC activation. Another approach in our lab was that we screened hundreds pure compounds isolated from herbs, we discovered that osthole, an active component contained in the fruit of Cnidium monnieri (L.) Cusson (C.monnieri (L.) Cusson), could inhibit HSC activation effectively in preliminary screening. Therefore, I further conducted both in vitro and in vivo studies to investigate the therapeutic effects of osthole on rat liver fibrosis and HSC activation.
We used lipopolysaccharide (LPS) (100 ng/ml) to stimulate rat hepatic stellate cells (HSC-T6). Tan IIA (10 uM) was used to inhibit LPS-induced HSC activation and compared with Sal B (200 uM). All concentrations of Tan IIA (1 to 10 uM) and Sal B (200 uM) showed no cytotoxicity against HSC-T6 cells. LPS stimulated NF-κB luciferase activities, nuclear translocation of NF-κB-p65, and phosphorylations of ERK, JNK and p38, were suppressed by Tan IIA. In addition, Tan IIA significantly inhibited LPS-induced HSCs chemotaxis, in both wound-healing and trans-well invasion assays. Moreover, Tan IIA attenuated LPS-induced mRNA expressions of CCL2, CCL3, CCL5. Furthermore, Tan IIA also attenuated LPS-induced fibrosis-related mRNA expressions, including IL-1, TNF-, iNOS, ICAM-1, and IL-6. In addition, the protein and gene expression of a-SMA were both downregulated by Tan IIA in HSC-T6 cells.
For the investigation of osthole (C15H16O3, MW = 244.29), I established the thioacetamide (TAA)-model of Sprague-Dawley (SD) rats to induce hepatic fibrosis. Rats were divided into three groups: control, TAA, and TAA + osthole (10 mg/kg). In vivo, osthole significantly reduced liver injury by diminishing levels of plasma AST and ALT, improving histological architecture, decreasing collagen and a-SMA accumulation, and improving hepatic fibrosis scores. Additionally, osthole reduced the expression of fibrosis-related genes significantly. Osthole also suppressed the production of fibrosis-related cytokines and chemokines. Moreover, nuclear translocation of p65 was significantly suppressed in osthole-treated liver. Osthole also ameliorated TAA-induced injury through reducing cellular oxidation. Osthole showed inhibitory effects on inflammation-related genes and chemokine production as well. In vitro, we assessed osthole effects in activated HSCs (HSC-T6 and LX-2). Osthole (10 ug/ml) attenuated TGF-b1-induced migration and invasion in HSCs. Furthermore, osthole decreased TNF-a-triggered NF-κB activities significantly. Besides, osthole attenuated TGF-b1- or ET-1-induced HSC contractility. Our results demonstrated that Tan IIA decreased LPS-induced HSC activation. In addition, osthole improved TAA-caused liver injury, fibrogenesis and inflammation in rats, and significantly suppressed HSCs activation in vitro.
According to the study results, Tan IIA from S. miltiorrhiza could inhibit HSC activation, and osthole from C.monnieri Cusson could improve rat liver injury and also attenuate HSC activation. This thesis provides the evidences for using S. miltiorrhiza and C.monnieri (L.) Cusson in the clinical and the academic of TCM.
 

誌謝 i
中文摘要 ii
Abstract iv
Index vi
Index of Table ix
Index of Figure ix
Abbreviations 1
Chapter 1 Introduction 3
1.1 Background 4
1.2 Overview of the liver 4
1.2.1 Anatomy of the liver 4
1.3 Hepatic fibrosis and HSCs 5
1.3.1 General aspect of hepatic fibrosis and cirrhosis 5
1.3.2 Activation of HSCs in the hepatic disease 5
1.3.3 Stimuli and cytokines associated with HSCs 6
1.3.4 Therapeutic approaches to the treatment of hepatic fibrosis 7
1.4 Approaches to study hepatic fibrosis 8
1.4.1 In vitro models of hepatic fibrosis 8
1.4.2 In vivo models of hepatic fibrosis 9
1.5 Treatment of hepatic fibrosis in traditional Chinese medicine (TCM) 10
1.5.1 Hepatic fibrosis in TCM 10
1.5.2 Different approaches to studying TCM in hepatic fibrosis. 11
1.6 Rationale and aim of the study 14
Chapter 2 Materials and Methods 15
2.1 Materials (by alphabetical order) 16
2.1.1 Instruments 16
2.1.2 Chemicals and reagents 16
2.1.4 Experimental kit 17
2.1.5 Antibodies and conditions 18
2.1.6 Softwares 18
2.2 Methods 19
2.2.1 Cell culture and Hepato-fibrotic rats 19
2.2.2 Preparation of drugs and chemicals 20
2.2.3 Cell viability assay (MTT assay) 20
2.2.4 Immunofluorescent staining 20
2.2.5 Reporter gene assay (Luciferase assay) 20
2.2.6 Western blotting analysis 22
2.2.7 Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis 23
2.2.8 Wound-healing assay 24
2.2.9 Trans-well invasion assay 25
2.2.11 Histological examination and immunohistochemistry 25
2.2.12 Measurement of oxidative products in liver tissues 25
2.2.13 ELISA for cytokines 26
2.2.14 Contraction assay 26
2.2.15 Statistical analysis 27
Chapter 3 28
Results 28
3.1 Inhibitory Effect of Tanshinone IIA on Rat Hepatic Stellate Cells 29
3.1.1 Tanshinone IIA has no cytotoxicity in HSC-T6 cells 29
3.1.2 Tanshinone IIA attenuated LPS-induced NF-B activation in HSC-T6 cells 29
3.1.3 LPS-stimulated phosphorylations of mitogen-activated protein kinases (MAPKs) were inhibited by Tan IIA in HSC-T6 cells 30
3.1.4 Tan IIA suppressed LPS-stimulated chemotaxis of HSC-T6 cells 30
3.1.5 Tan IIA inhibited LPS-induced fibrogenic -SMA production in HSC-T6 cells 31
3.2 Osthole ameliorates hepatic fibrosis and inhibits hepatic stellate cell activation 31
3.2.1 Osthole protected the rat liver against TAA-stimulated injury 31
3.2.2 Osthole attenuated TAA-induced rat liver fibrosis 32
3.2.3 Osthole suppressed TAA-induced oxidation and inflammation in rat liver 33
3.2.4 Osthole downregulated chemotaxis and contraction of activated HSCs 34
Chapter 4 Discussion 35
4.1 Tan IIA from S. miltiorrhiza inhibited HSC activation and liver diseases 36
4.1.1 TLR signaling and HSC activation 36
4.1.2 Tan IIA inhibits LPS-induced mechanism 36
4.1.3 Tan IIA shows inhibitory effects in different pathologic studies 37
4.1.4 Tan IIA inhibits HSC migration and related signaling transduction 37
4.1.5 Tan IIA exerts anti-inflammatory effect in activated HSCs 37
4.2 Osthole against liver fibrosis and HSC activation 38
4.2.1 Osthole attenuated distinct pathologies in in vivo models 38
4.2.2 Osthole exerts anti-inflammatory effect in TAA-injected rats 39
4.2.3 The rat model of TAA-induced liver injury 39
4.2.4 Dosing of osthole for gavage feeding 40
4.2.5 Osthole inhibits migration, invasion, and contraction in activated HSCs 41
4.3 The challenge of applying Tan IIA and osthole in the clinic 41
Chapter 5 Conclusion 43
References 76
Appendix 90

Index of Table
Table 1. The percentage of each cell population in the liver. 45
Table 2. The top 10 Chinese herbal formula prescribed for chronic hepatitis in Taiwan during 2002 45
Table 3. Forward and reverse primers used in qRT-PCR. 46

Index of Figure
Figure 1. Hepatic stellate cell activation 47
Figure 2. Chemical structures of Sal B (A), Tan IIA (B), and osthole (C). 48
Figure 3. Cell viability and proliferation of HSC-T6 cells treated with Tan IIA and Sal B. 49
Figure 4. Tan IIA decreased LPS-induced NF-B activity in HSC-T6 cells. 50
Figure 5. Tan IIA decreased LPS-induced p65 translocation in HSC-T6 cells detecting by Western blotting assay. 51
Figure 6. Tan IIA decreased LPS-induced p65 translocation in HSC-T6 cells detecting by immunofluorescent staining assay. 52
Figure 7. Tan IIA decreased LPS-induced inflammatory gene expression in HSC-T6 cells. 54
Figure 8. Tan IIA attenuated LPS-stimulated MAPKs (pERK1/2, pJNK1/2 and p38) signaling 55
Figure 8. Tan IIA attenuated LPS-stimulated MAPKs (pERK1/2, pJNK1/2 and p38) signaling 56
Figure 9. Tan IIA attenuated LPS-stimulated mRNA expressions of CCL2, CCL3 and CCL5 57
Figure 10. Tan IIA inhibited LPS-induced HSC-T6 migration 58
Figure 11. Tan IIA inhibited LPS-induced HSC-T6 invasion 59
Figure 12. Tan IIA inhibited LPS-induced -SMA production in HSC-T6 60
Figure 13. General profiles in control rats and TAA-induced fibrotic rats with or without osthole treatment. 61
Figure 13. General profiles in control rats and TAA-induced fibrotic rats with or without osthole treatment. 62
Figure 14. Osthole attenuated liver injury and fibrogenesis in the TAA rat model 64
Figure 14. Osthole attenuated liver injury and fibrogenesis in the TAA rat model 65
Figure 15. Osthole attenuated production of -SMA in the TAA rat model 66
Figure 16. Osthole attenuated fibrogenesis-related gene expression and cytokine level in the TAA rat model 67
Figure 17. Osthole suppressed translocation of inflammatory and oxidative protiens in TAA-injected rat liver 68
Figure 18. Osthole suppressed oxidative products in TAA-injected rat liver 69
Figure 19. Osthole suppressed of inflammation-related gene expression and cytokine level in TAA-injected rat liver 70
Figure 20. Cell viability of osthole in HSC-T6 and LX-2 cells. 71
Figure 21. Osthole downregulated HSC migration. 72
Figure 22. Osthole downregulated HSC invasion. 73
Figure 23. Osthole downregulated NF-B activity in HSCs. 74
Figure 24. Osthole downregulated HSC contraction. 75

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