神經病變是糖尿病常見的病徵之一，過去常認為周邊的神經病變為糖尿病的主要症狀。然而，越來越多文獻顯示中樞神經系統可能也參與糖尿病所造成的神經病變。 到目前為止沒有任何有效的治療方法可以預防或治療糖尿病的神經病變，控制血糖被認為或許是能預防糖尿病神經病變的最佳手段。鳶尾素(Irisin) 是透過第三型纖維連接蛋白(FNDC5)蛋白質水解後所產生的運動賀爾蒙，會在運動過後大量表現。Irisin 會刺激粒線體進行能量代謝，調控血糖值的穩定並參與抗發炎的作用。我們想探討是否Irisin的基因傳送可以透過對抗高血糖或是發炎反應來改善糖尿病所導致的神經病變 。在本篇研究中我們利用 Insulin2Akita 小鼠作為糖尿病的動物模型來探討糖尿病神經病變， Insulin2Akita 小鼠的 insulin 2 基因會有自發性的突變，造成胰島素的錯誤摺疊並且對胰臟的β 細胞產生毒性，導致β 細胞的減少和胰島素無法正常分泌。我們在Insulin2Akita 小鼠的黑質緻密部(substantia nigra)中發現氧化壓力的標誌8-OHdG 和鐵離子在免疫化學染色實驗裡比起C57BL/6小鼠有更顯著的上升，並且伴隨著促發炎因子(pro-inflammatory cytokine) IL-6和 IL-1β的提升和I-Ba1+ /CD11c+ 微膠質細胞(microglia)的活化。氧化壓力的增加在其他文獻中認為和鐵依賴性凋亡(ferroptosis)相關，ferroptosis是一種與細胞凋亡不同的新型細胞凋亡方式。 麩胱甘肽過氧化物酶4 (GPX4)的減少是ferroptosis的主要特徵，GPX4的表現量的減少會造成脂質的過氧化物累積並活化ferroptosis。我們發現Irisin的基因傳送可以恢復Insulin2Akita 小鼠的substantia nigra中GPX4的表現量和減少鐵離子的累積. 另外，在實驗中也可以看到神經發炎也參與了糖尿病神經病變，Irisin的基因療法在Insulin2Akita小鼠的substantia nigra中改善了microglia的活化和降低了pro-inflammatory cytokine表現，過量的microglia活化會產生一系列的促發炎媒介導致神經發炎並且對周遭的神經細胞產生危害。總而言之，我們在實驗中看到Irisin基因傳送緩解了Insulin2Akita小鼠substantia nigra中的氧化壓力從而改善ferroptosis和神經發炎。這些發現支持了Irisin可以被發展成透過緩解ferroptosis和對抗發炎反應改善糖尿病患者的神經病變的治療策略。

Diabetic neuropathy (DN) is a major complication of diabetes mellitus (DM). DN is usually considered a disease of the peripheral nervous system in the past. Nevertheless, more and more evidence has demonstrated that the central nervous system (CNS) is also involved in DN. Currently, there has no effective therapeutic method for preventing or treating DN. Management of blood glucose is regarded as the best approach for preventing the disease onset of DN. Irisin is a myokine through proteolytical cleavage from its precursor fibronectin type III domain containing 5 (FNDC5). Irisin is highly expressed in response to exercise, stimulates mitochondria increased energy expenditure, promotes glucose homeostasis, and exerts anti-inflammatory effects. We want to test whether Irisin gene delivery can ameliorate neuropathy through its anti-inflammation effect against hyperglycemia-induced DN. In this study, we utilized Insulin2Akita mice as our animal model of DM to investigate DN. Insulin2Akita mice have a spontaneous mutation in the insulin 2 gene leads to incorrect folding of the insulin protein producing toxicity in pancreatic β cells, reduced β cell mass, and reduced insulin secretion. It was observed that the oxidative stress marker 8-OHdG immunostaining and Fe3+ accumulation was significantly higher in the substantia nigra of Insulin2Akita mice than that in C57BL/6 mice. This was accompanied by elevated pro-inflammatory IL-6 and IL-1β expression and I-Ba1+ /CD11c+ microglia activation. Oxidative stress increase has been reported to link to ferroptosis. Ferroptosis is a novel form of nonapoptotic regulated cell death. Glutathione peroxidase 4 (GPX4) inactivation is the main characteristic of ferroptosis. Reduced GPX4 level leads to the accumulation of lipid peroxides and activates ferroptosis. It was found that Irisin gene delivery restored the GPX4 expression and reduced Fe3+ accumulation in the substantia nigra of Insulin2Akita mice. Furthermore, the data had shown that neuroinflammation was involved in DN. Irisin gene therapy alleviates microglia activation and descends pro-inflammation cytokines expression in the substantia nigra of Insulin2Akita mice. Excessively activated microglia produce several pro-inflammatory mediators, leading to neuroinflammation and damage to the surrounding neurons. In conclusion, we report that Irisin gene delivery in Insulin2Akita mice alleviates oxidative stress, thereby alleviating the ferroptosis and neuroinflammation in the substantia nigra of diabetic mice. These findings support that Irisin could be developed as a therapeutic method to improve DN through decreased activation of ferroptosis and anti-inflammation in substantia nigra in DM patients.

INDEX
1. 論文審定書 i
2. 中文摘要 ii
3. Abstract iv
4. Figure and Legend Index vii
5. Introduction 1
6. Specific aims 5
7. Materials and Methods 6
8. Results 13
9. Discussion 23
10. Figure and legends 26
11. References 44


FIGURE AND LEGEND INDEX
Figure 1. The effect of Irisin gene delivery on fasting blood glucose in Insulin2Akita mice. 26
Figure 2. The effect of Irisin gene delivery on nociceptive behaviors in Insulin2Akita mice. 28
Figure 3. The effect of Irisin gene delivery on behaviors in Insulin2Akita mice. 29
Figure 4. The effect of Irisin gene delivery on oxidative stress in substantia nigra of Insulin2Akita mice 30
Figure 5. The effect of Irisin gene delivery on pro-inflammatory cytokines in substantia nigra of Insulin2Akita mice. 31
Figure 6. The effect of Irisin gene delivery on astrocyte activation in substantia nigra of Insulin2Akita mice. 33
Figure 7. The effect of Irisin gene delivery on microglia activation in substantia nigra of Insulin2Akita mice. 35
Figure 8. The effect of Irisin gene delivery on Iron accumulation in substantia nigra of Insulin2Akita mice 37
Figure 9. The effect of Irisin on the regulator of lipid peroxidation in substantia nigra of Insulin2Akita mice. 38
Figure 10. The effect of Irisin on GDNF expression in substantia nigra of Insulin2Akita mice. 39
Figure 11 The effect of Irisin gene delivery on apoptosis in substantia nigra of Insulin2Akita mice. 40
Figure 12. The effect of Irisin gene delivery on dopaminergic neuron activity in substantia nigra of Insulin2Akita mice. 41
Figure 13. The effect of Irisin gene delivery on ɑ-synuclein accumulation in substantia nigra of Insulin2Akita mice. 42

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