四氫生物蝶呤調控內皮細胞糖代謝及促進糖尿病小鼠傷口愈合的作用
發(fā)布時間：2018-09-10 15:00
【摘要】：四氫生物蝶呤(BH4)是內皮細胞中內皮型一氧化氮合酶（eNOS）合成NO活性的關鍵輔助因子，在調控eNOS合成NO及減少過氧化物（SO）產(chǎn)生、修復糖尿病性內皮細胞功能受損具有重要的作用。BH4已被認為是治療糖尿病血管疾病的合理靶點。但其調節(jié)內皮細胞功能的更進一步作用機制仍不清楚。 過去我們實驗研究顯示，BH4的合成可介導NO生物活性，來正反饋調節(jié)PI3K/Akt信號通路促進腫瘤的血管生成。PI3K/Akt途徑作為胰島素調控內皮細胞葡萄糖代謝的主要信號轉導途徑，為BH4調控內皮細胞的糖代謝提供可能機制。新生血管的重塑作為傷口愈合的重要過程，為BH4促進糖尿病傷口愈合提供可能。 本研究為驗證BH4對葡萄糖代謝的調控及其機制開展了系列細胞實驗。首先，在低糖(5.5mM)條件，發(fā)現(xiàn)墨蝶呤(Sep)合成來源的BH4能正調控BAEC葡萄糖代謝相關蛋白。通過對BAEC胞內乳酸及乳酸代謝相關蛋白的檢測，發(fā)現(xiàn)BH4的合成可能更多地促進葡萄糖進入三羧酸循環(huán)。在人臍帶靜脈內皮細胞(HUVEC)及牛動脈內皮細胞(BAEC)中，BH4的合成對Akt及Erk1/2信號通路均具有時間依賴性的活化作用。接著，通過加入PI3K/Akt和Erk1/2的活化抑制劑LY294002、PD98059處理BAEC30min，顯示：BH4調控內皮細胞糖代謝的作用是通過或部分通過介導PI3K/Akt信號上調的；且PI3K/Akt與Erk1/2通路間可能存在“交叉會談”，Erk1/2磷酸化的抑制一定程度上上調Akt的磷酸化。最后，加入墨蝶呤還原酶及eNOS的抑制劑SSZ、L-NAME處理BAEC48h，顯示：在低糖環(huán)境下，BH4的合成對Akt及葡萄糖代謝的正調控作用，是由NO介導的；但在高糖(30mM)環(huán)境，Sep介導的Akt與葡萄糖代謝作用并不依賴于NO。在低氧(1%O2)環(huán)境下，發(fā)現(xiàn)BH4的合成仍能上調Akt與葡萄糖代謝。 綜合上述研究結果，Sep來源的BH4能通過介導NO正調控PI3K/Akt信號通路，來上調內皮細胞葡萄糖的代謝；但在高糖環(huán)境下，這一過程可能并不依賴于NO介導。 本研究為驗證BH4對糖尿病傷口愈合的促進作用進行了動物實驗。實驗結果顯示，比較同一天的相對傷口閉合面積，實驗組(Sep處理)相較于對照組(DMSO處理)，在正常小鼠傷口愈合的第6天及第9天，有顯著性的促進作用；在糖尿病小鼠中，在第9天時有顯著性促進作用。
[Abstract]:Tetrahydrobiopterin (BH4) is a key cofactor in the synthesis of NO by endothelial nitric oxide synthase (eNOS) in endothelial cells. It regulates the synthesis of NO by eNOS and reduces the production of (SO). Repair of diabetic endothelial cell dysfunction. BH4 has been considered as a reasonable target for the treatment of diabetic vascular diseases. However, the further mechanism of its regulation of endothelial cell function is still unclear. In the past, our experimental studies have shown that the synthesis of BH4 can mediate the biological activity of NO, which can positively and feedback regulate the PI3K/Akt signaling pathway to promote tumor angiogenesis. PI3K / Akt pathway is the main signal transduction pathway of insulin regulating glucose metabolism in endothelial cells. It provides a possible mechanism for BH4 to regulate glucose metabolism in endothelial cells. As an important process of wound healing, neovascularization provides the possibility for BH4 to promote diabetic wound healing. A series of cell experiments were carried out to verify the regulation and mechanism of BH4 on glucose metabolism. Firstly, under the condition of low glucose (5.5mM), it was found that BH4, a source of mopterin (Sep) synthesis, could regulate the glucose metabolism related proteins of BAEC. Through the detection of intracellular lactate and lactate metabolism-related proteins in BAEC, it was found that the synthesis of BH4 might promote glucose to enter the tricarboxylic acid cycle. The synthesis of BH4 in human umbilical vein endothelial cells (HUVEC) and bovine arterial endothelial cells (BAEC) has a time-dependent activation on Akt and Erk1/2 signaling pathways. Then, the treatment of BAEC30min, with PI3K/Akt and Erk1/2 activation inhibitor LY294002,PD98059 showed that the role of BH4 in regulating glucose metabolism in endothelial cells was up-regulated by or in part mediated by PI3K/Akt signal. Furthermore, the inhibition of Erk 1 / 2 phosphorylation in PI3K/Akt and Erk1/2 pathway may up-regulate the phosphorylation of Akt to some extent. Finally, the treatment of BAEC48h, with mopterin reductase and SSZ,L-NAME, an inhibitor of eNOS, showed that the positive regulation of Akt and glucose metabolism induced by BH4 was mediated by NO. However, the metabolism of Akt and glucose mediated by Akt in high glucose (30mM) environment is independent of NO.. Under hypoxia (1%O2), it was found that the synthesis of BH4 could still up-regulate the metabolism of Akt and glucose. Based on the above results, BH4 from Sep can up-regulate glucose metabolism in endothelial cells by mediating NO regulating PI3K/Akt signaling pathway, but this process may not depend on NO mediated in high glucose environment. The aim of this study was to test the effect of BH4 on diabetic wound healing. The results showed that compared with the control group (DMSO), the experimental group (Sep treatment) significantly promoted the wound healing on the 6th and 9th day of wound healing in normal mice, and in diabetic mice, compared with the control group (DMSO treatment), the relative wound closed area on the same day was significantly increased. On the 9th day, there was a significant promoting effect.
【學位授予單位】：華僑大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R965
本文編號：2234793
[Abstract]:Tetrahydrobiopterin (BH4) is a key cofactor in the synthesis of NO by endothelial nitric oxide synthase (eNOS) in endothelial cells. It regulates the synthesis of NO by eNOS and reduces the production of (SO). Repair of diabetic endothelial cell dysfunction. BH4 has been considered as a reasonable target for the treatment of diabetic vascular diseases. However, the further mechanism of its regulation of endothelial cell function is still unclear. In the past, our experimental studies have shown that the synthesis of BH4 can mediate the biological activity of NO, which can positively and feedback regulate the PI3K/Akt signaling pathway to promote tumor angiogenesis. PI3K / Akt pathway is the main signal transduction pathway of insulin regulating glucose metabolism in endothelial cells. It provides a possible mechanism for BH4 to regulate glucose metabolism in endothelial cells. As an important process of wound healing, neovascularization provides the possibility for BH4 to promote diabetic wound healing. A series of cell experiments were carried out to verify the regulation and mechanism of BH4 on glucose metabolism. Firstly, under the condition of low glucose (5.5mM), it was found that BH4, a source of mopterin (Sep) synthesis, could regulate the glucose metabolism related proteins of BAEC. Through the detection of intracellular lactate and lactate metabolism-related proteins in BAEC, it was found that the synthesis of BH4 might promote glucose to enter the tricarboxylic acid cycle. The synthesis of BH4 in human umbilical vein endothelial cells (HUVEC) and bovine arterial endothelial cells (BAEC) has a time-dependent activation on Akt and Erk1/2 signaling pathways. Then, the treatment of BAEC30min, with PI3K/Akt and Erk1/2 activation inhibitor LY294002,PD98059 showed that the role of BH4 in regulating glucose metabolism in endothelial cells was up-regulated by or in part mediated by PI3K/Akt signal. Furthermore, the inhibition of Erk 1 / 2 phosphorylation in PI3K/Akt and Erk1/2 pathway may up-regulate the phosphorylation of Akt to some extent. Finally, the treatment of BAEC48h, with mopterin reductase and SSZ,L-NAME, an inhibitor of eNOS, showed that the positive regulation of Akt and glucose metabolism induced by BH4 was mediated by NO. However, the metabolism of Akt and glucose mediated by Akt in high glucose (30mM) environment is independent of NO.. Under hypoxia (1%O2), it was found that the synthesis of BH4 could still up-regulate the metabolism of Akt and glucose. Based on the above results, BH4 from Sep can up-regulate glucose metabolism in endothelial cells by mediating NO regulating PI3K/Akt signaling pathway, but this process may not depend on NO mediated in high glucose environment. The aim of this study was to test the effect of BH4 on diabetic wound healing. The results showed that compared with the control group (DMSO), the experimental group (Sep treatment) significantly promoted the wound healing on the 6th and 9th day of wound healing in normal mice, and in diabetic mice, compared with the control group (DMSO treatment), the relative wound closed area on the same day was significantly increased. On the 9th day, there was a significant promoting effect.
【學位授予單位】：華僑大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R965
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