四氢生物蝶呤调控内皮细胞糖代谢及促进糖尿病小鼠伤口愈合的作用
发布时间：2018-09-10 15:00
【摘要】：四氢生物蝶呤(BH4)是内皮细胞中内皮型一氧化氮合酶（eNOS）合成NO活性的关键辅助因子，在调控eNOS合成NO及减少过氧化物（SO）产生、修复糖尿病性内皮细胞功能受损具有重要的作用。BH4已被认为是治疗糖尿病血管疾病的合理靶点。但其调节内皮细胞功能的更进一步作用机制仍不清楚。 过去我们实验研究显示，BH4的合成可介导NO生物活性，来正反馈调节PI3K/Akt信号通路促进肿瘤的血管生成。PI3K/Akt途径作为胰岛素调控内皮细胞葡萄糖代谢的主要信号转导途径，为BH4调控内皮细胞的糖代谢提供可能机制。新生血管的重塑作为伤口愈合的重要过程，为BH4促进糖尿病伤口愈合提供可能。 本研究为验证BH4对葡萄糖代谢的调控及其机制开展了系列细胞实验。首先，在低糖(5.5mM)条件，发现墨蝶呤(Sep)合成来源的BH4能正调控BAEC葡萄糖代谢相关蛋白。通过对BAEC胞内乳酸及乳酸代谢相关蛋白的检测，发现BH4的合成可能更多地促进葡萄糖进入三羧酸循环。在人脐带静脉内皮细胞(HUVEC)及牛动脉内皮细胞(BAEC)中，BH4的合成对Akt及Erk1/2信号通路均具有时间依赖性的活化作用。接着，通过加入PI3K/Akt和Erk1/2的活化抑制剂LY294002、PD98059处理BAEC30min，显示：BH4调控内皮细胞糖代谢的作用是通过或部分通过介导PI3K/Akt信号上调的；且PI3K/Akt与Erk1/2通路间可能存在“交叉会谈”，Erk1/2磷酸化的抑制一定程度上上调Akt的磷酸化。最后，加入墨蝶呤还原酶及eNOS的抑制剂SSZ、L-NAME处理BAEC48h，显示：在低糖环境下，BH4的合成对Akt及葡萄糖代谢的正调控作用，是由NO介导的；但在高糖(30mM)环境，Sep介导的Akt与葡萄糖代谢作用并不依赖于NO。在低氧(1%O2)环境下，发现BH4的合成仍能上调Akt与葡萄糖代谢。 综合上述研究结果，Sep来源的BH4能通过介导NO正调控PI3K/Akt信号通路，来上调内皮细胞葡萄糖的代谢；但在高糖环境下，这一过程可能并不依赖于NO介导。 本研究为验证BH4对糖尿病伤口愈合的促进作用进行了动物实验。实验结果显示，比较同一天的相对伤口闭合面积，实验组(Sep处理)相较于对照组(DMSO处理)，在正常小鼠伤口愈合的第6天及第9天，有显著性的促进作用；在糖尿病小鼠中，在第9天时有显著性促进作用。
[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.
【学位授予单位】：华侨大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965
本文编号：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.
【学位授予单位】：华侨大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965
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1 杨巍;罗春元;于春雷;王莉;许丽芳;李一;;不同剂量STZ诱导小鼠糖尿病模型的发病机制[J];吉林大学学报(医学版);2006年03期
2 许樟荣;;我国糖尿病流行病学研究概况[J];总装备部医学学报;2007年01期
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