Nstpbp168是由植物分離出來的純化合物，在我們先前的研究發現它是一種新穎的AMPK活化劑。而本次研究目主要是探討Nstpbp168在氧化壓力及脂質毒性下對胰島素分泌細胞的活存率與活性氧化物產生是否是有正面影響。在氧化壓力的刺激下，胰島素分泌細胞RINm5F曝露於含有40 μM過氧化氫溶液，而後投予100 μM Nstpbp168培養十八小時，計數細胞的存活率與活性氧化物生成量。此外，在脂質毒性刺激方面，則是將RINm5F及肝臟細胞HepG2培養於含有100 μM Nstpbp168 及palmitate/BSA混合物的培養液，進而以MTT檢測其細胞存活率及DCFH-DA染劑觀察細胞內超氧物質含量的螢光圖像。研究的結果顯示，Nstpbp168可以在過氧化氫誘導之氧化壓力下呈現劑量依存的方式防止細胞死亡。此外，在NBT檢測的結果發現細胞經由Nstpbp168處理後能有效抑制過氧化氫所誘導出活性氧化物，並證明Nstpbp168對於AMPK的活化呈現劑量依存的關係。AMPK的拮抗劑Compound C可明顯阻止Nstpbp168的保護作用在過氧化氫對於胰島素分泌細胞產生的傷害。此外，Nstpbp168具有防止palmitae引起之細胞死亡，推論是藉由促進AMPK活化以減少活性氧的產生。綜以上所述，Nstpbp168對於細胞具有保護作用，可有效防止氧化性壓力及脂質累積之毒性所造成的傷害，而這些保護的功能可能均是藉由促進AMPK活化的機制所達成。

Nstpbp168, a pure compound isolated from natural product has been shown to be a novel AMPK activator in our previous study. The aim of this study was to assess the possible beneficial effect of Nstpbp168 on cell survival and ROS production in insulin secreting cell under oxidative stress/lipotoxicity. In the part of oxidative stress stimulation, RINm5F was first exposed to 40 μM hydrogen peroxide and then incubated in medium w/o 100 μM Nstpbp168 for following cell viability, ROS level and related signal transduction measurement, respectively. Besides, in the part of lipotoxicity, RINm5F and the liver cell, HepG2, were treated w/o 100 μM Nstpbp168 and exposed to palmitate/BSA mixture to employ the MTT/DCFH-DA assessment. The present results showed that Nstpbp168 could prevent cell death from H2O2-induced oxidative stress dose-dependently along with lowering H2O2-induced ROS production. Meanwhile, Nstpbp168 treatment also activated AMPK dose-dependently. Compound C, a selective AMPK antagonist, could significantly block the protective effect of Nstpbp168 on H2O2-induced damage in insulin secreting cell. Moreover, Nstpbp168 had the potential to prevent cell death from palmitae-induced injury, which might be through promoting AMPK activity as well as reduced the ROS production. Taken together, it is suggested that Nstpbp168 might have a potential protective effect on ROS/lipotoxicity-induced cell damage through AMPK-mediated pathway in insulin secreting cells.

Contents

Abbreviations………………………………………………………...1
Chinese abstract……………………………………………………...2

Abstract ………………………………………………………………4

Chapter1. Introduction………………………………………………5
1.1 Current status of diabetes 6
1.2 β-Cell and the development of diabetes 6
1.3 Reactive oxygen species (ROS) and β-Cell dysfunction 8
1.4 AMP-activated protein kinase (AMPK) pathway 9
1.5 AMPK activation and diabetes 11
1.6 AMPK activator Nstpbp168 13

Chapter 2. Materials and Methods…………………………………16
2.1 Reagents 16
2.2 Cell Culture 16
2.3 Palmitate/BSA complex preparation 17
2.4 Cell viability assay 17
2.5 Western blotting 18
2.6 Determination of reactive oxygen species (ROS) 19
2.7 Evaluation of ROS production by DCFH-DA 19
2.8 Glucose stimulated insulin secretion (GSIS) 20
2.9 Statistical Analysis 20

Chapter 3. Results…………………………………………………….21
3.1 The effects of Nstpbp168 on cell survival 21
3.2 Scavenging capacity of Nstpbp168 on H2O2-induced ROS22
3.3 The anti-oxidant protective ability of Nstpbp168 is through activating AMPK 22
3.4 Nstpbp168 prevented Palmitate-induced lipotoxicity 24
3.5 Inhibition of palmitate-induced ROS production by Nstpbp168 26
3.6 Effects of Nstpbp168 on glucose- stimulated insulin secretion in mouse islets. 27

Chapter 4. Discussion…………………………………………………. 28
4.1 Nstpbp168 has antioxidant potential 28
4.2 Activated by Nstpbp168 can reduce the oxidative-induced cellular damage 29
4.3 Different level of palmitate-induced AMPK activation and cellular damage in pancreatic cells and liver cells 31
4.4 AMPK can be activated under H2O2/Palmitate stimulation 33
4.5 Activation of AMPK is essential in early onset of diabetes 34
4.6 Conclusion 35

References………………………………………………………………36

Figures………………………………………………………………….46
Figure 1. Nstpbp168 could suppress H2O2-induced cytotoxicity in a dose-dependent manner. 46
Figure 2. Suppressive effect of Nstpbp168 on the production of H2O2-induced ROS. 48
Figure 3. The protective effect of Nstpbp168 on H2O2-induced cell damage through activation of AMPK. 49
Figure 4. The effect of Nstpbp168 on palmitate-induced lipotoxicity in RINm5F. 51
Figure 5. The effect of Nstpbp168 on AMPK activation in palmitate treated RINm5F. 53
Figure 6. The protective effect of Nstpbp168 on HepG2 cells with or without palmitate treatment. 54
Figure 7. The effect of Nstpbp168 on AMPK expression in palmitate-treated HepG2 cells. 55
Figure 8. Imagination of DCFH-DA stained ROS in palmitate treated RINm5f cells with or without drug co-administration. 56
Figure 9. Effects of Nstpbp168 on intracellular ROS scavenging in HepG2. 58
Figure 10. Effects of Nstpbp168 on glucose-stimulated insulin secretion in mouse islets. 60

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