不同含量的锌对糖尿病肾病的作用及相关机制研究

发布时间：2018-06-08 10:14

本文选题：锌 + 糖尿病肾病　；参考：《吉林大学》2015年博士论文

【摘要】：糖尿病（diabetes mellitus，DM）是由葡萄糖产生和代谢异常导致内环境紊乱而引发的多因素的慢性代谢障碍性疾病。它的主要特点是由于胰岛的产生、分泌异常或二者皆可和胰岛素抵抗、炎症、氧化应激等造成的血糖和血脂代谢失衡。随着经济的迅猛发展、生活标准的提高、饮食和生活方式的改变和老龄化，糖尿病已经成为世界范围内的重要的公共健康问题，是继恶性肿瘤和心脑血管疾病之后的第三大威胁公共健康的疾病。据统计糖尿病的全球患病人数在2000年已经达到1.51亿人，预计2030年在年龄大于20岁的人群中患病人数将达到5.52亿。糖尿病及其并发症的治疗给个人、家庭及社会都带来了沉重的经济负担，如何预防糖尿病和控制糖尿病并发症的发生发展，已经成为医学界迫于被解决的热点问题。糖尿病可以影响机体的多种器官系统，，如肾脏系统、神经系统和眼睛等，随着时间的进展都可以导致严重的糖尿病并发症。作为糖尿病微血管疾病之一的糖尿病肾病，不仅是糖尿病最常见的并发症之一，也是糖尿病患者死亡的最主要病因之一。尽管控制血糖、血压、降低脂质水平和抑制肾素血管紧张素系统被用于治疗糖尿病，但糖尿病肾病的发生和发展仍是不可预防的。糖尿病肾病的主要组织学特点是肾小球肥大、肾小球基底膜增厚、细胞外基质增多和最终的肾小球球性硬化。而氧化应激介导糖尿病对肾脏组织功能的不利影响；高血糖诱导的活性氧自由基干扰微血管和促进细胞外基质积聚。所以，如何调节氧化酶活性和提高抗氧化能力被认为是预防和延缓糖尿病肾病的发生和发展的重要手段之一。锌作为人体的必需微量元素之一，参与多种生理过程。同时，锌是蛋白的必要组成部分，主要参与维持细胞结构和细胞膜的稳定性。锌的功能包括可以维持人类基因组中2000多种锌依赖的转录因子的细胞结构稳定，并且对超过300多种锌金属酶的生物活性起着关键作用。在糖尿病状态下，常伴有锌缺乏。除了饮食过程中锌摄入不足，糖尿病患者吸收和/或重吸收锌受损和尿锌排泄增加，都可以增加内源性锌的丢失。锌不仅在胰岛素作用和碳水化合物代谢中起着重要的作用，而且在机体的抗氧化机制中也有着不可或缺的作用。目前，氧化应激已经被认为在糖尿病及其并发症的发病机制中至关重要。锌是超氧化物歧化酶等关键的抗氧化酶的重要组成部分，锌缺乏导致抗氧化酶的抗氧化能力下降，从而导致机体内氧化应激增加。但是不同含量的锌对糖尿病肾病的作用及其可能的具体机制尚不明确，为此我们进行了以下研究。我们给予8周龄的FVB雄性小鼠小剂量STZ（50mg/kg）连续腹腔注射5天，1周后通过小鼠尾静脉检测血糖，以血糖水平大于16.7mmol/L定为成功建立1型糖尿病动物模型。成模后，我们给予美国Research Diets公司的商品化不同含量锌的成品饲料饲养糖尿病和对照组小鼠3个月，通过体内锌含量检测证实成功建立不同锌含量的1型糖尿病动物模型。然后，我们通过Western blot，病理组织学检查等实验方法检测肾功能、肾脏病理改变、氧化损伤、炎症损伤和纤维化损伤，探讨不同含量的锌饲养对糖尿病小鼠诱导肾脏损伤的作用。随后，我们通过检测Nrf2及其下游抗氧化因子的表达水平，探讨不同含量的锌对糖尿病诱导肾脏损伤作用的可能机制。最后，我们检测AKT、GSK-3β及Fyn水平，进一步探讨锌对AKT及其下游相关代谢信号的激活作用，从而探讨锌、AKT和Nrf2之间可能的关系。结果如下： 1.不同含量的锌饲养糖尿病小鼠3个月，糖尿病各组小鼠间的血糖、体重和肾重/体重比值水平差异无显著性改变。 2.在糖尿病各组中，低锌干预的糖尿病组小鼠的死亡率、ACR值和尿素氮值显著升高；正常锌和高锌干预可以改善上述症状，尤其是高锌干预。 3.与其相应对照组比较，糖尿病组中肾脏氧化损伤指标SOD1和SOD2的表达下降，炎症指标PAI-1和TNF-α、纤维化指标CTGF和TGF-β1的表达增加；在糖尿病各组中，低锌干预可以使上述损伤进一步加重，而正常锌和高锌干预后，尤其是高锌干预，可以改善上述肾脏损伤。 4.通过PAS染色发现与其相应对照组比较，糖尿病组出现肾小球肥大、细胞外基质增多和肾小管间质损伤；通过透射电镜检查发现与其相应对照组比较，糖尿病组表现为肾小球基底膜增厚、上皮足突节段融合和肾小管上皮细胞中线粒体肿胀；低锌干预有进一步加重上述损伤的趋势，而正常锌和高锌干预可以部分改善肾脏的病理损伤。 5.与其相应对照组比较，糖尿病组肾脏中Nrf2及其下游抗氧化因子的表达下降；在糖尿病各组中，低锌干预可以使其表达进一步下降，而正常锌和高锌干预，可以诱导Nrf2及其下游抗氧化因子的表达上调，尤其是高锌干预。 6.与其相应对照组比较，糖尿病组肾脏中AKT和GSK-3β的磷酸化水平下降；在糖尿病各组中，低锌干预可以使其磷酸化水平进一步下降，而正常锌和高锌干预，可以调节AKT和GSK-3β的磷酸化水平升高，尤其是高锌干预。 7.与其相应对照组比较，糖尿病组肾脏中Fyn核转位增加，导致Nrf2从核内转出增加，最终Nrf2降解增加；在糖尿病各组中，低锌干预可以使Fyn核转位进一步增加，而正常锌和高锌干预，Fyn核转位减少，从而上调Nrf2的表达，尤其是高锌干预。通过以上实验结果，我们得出结论：低锌干预一定程度上加重了糖尿病诱导的肾脏损伤；而给予正常锌和高锌干预可以对糖尿病诱导肾脏损伤起到部分的保护作用，尤其是高锌干预；这种保护作用部分是通过诱导Nrf2的表达和功能而实现的。而可能的调节机制之一是锌部分通过活化AKT降低GSK-3β的活性来抑制Fyn核转位，从而导致Nrf2在细胞核内积聚而增加其下游抗氧化因子NQO1、SOD等的表达部分实现其抗氧化损伤的作用，最终对糖尿病诱导肾脏损伤起到一定程度的保护作用。
[Abstract]:Diabetes mellitus (DM) is a multi factor chronic metabolic disorder caused by the disturbance of glucose production and metabolism, which is mainly characterized by the production of islets, abnormal secretion of insulin, and the imbalance of blood glucose and lipid metabolism caused by insulin resistance, inflammation, oxidative stress and so on. With the rapid development of economy, the improvement of standard of living, the change of diet and lifestyle and aging, diabetes has become an important public health problem in the world. It is the third major threat to public health after malignant tumor and cardiovascular disease. According to statistics, the number of diabetic global diseases has reached 1. in 2000. 5 billion 100 million people are expected to reach 552 million in the population older than 20 years of age in 2030. The treatment of diabetes and its complications has brought a heavy economic burden to individuals, families and society. How to prevent diabetes and control the development of diabetes complications has become a hot issue in the medical field.
Diabetes can affect a variety of organ systems, such as the kidney system, the nervous system, and the eyes, which can lead to serious diabetic complications as time progresses. As one of the diabetic microvascular diseases, diabetic nephropathy is not only one of the most common complications of diabetes, but also the most important disease of diabetic patients. One of the reasons. Although controlling blood sugar, blood pressure, lowering lipid levels and inhibiting the renin angiotensin system are used in the treatment of diabetes, the occurrence and development of diabetic nephropathy are still not preventable. The main histologic features of diabetic nephropathy are glomerular hypertrophy, thickening of the glomerular basement membrane, increased extracellular matrix and final glomeruli. And oxidative stress mediates the adverse effects of diabetes on the function of the kidneys; hyperglycemia induced reactive oxygen free radicals interferes with microvessels and promotes the accumulation of extracellular matrix. Therefore, how to regulate oxidase activity and increase antioxidant capacity is considered as an important means to prevent and delay the occurrence and development of hyperglycuria nephropathy One of.
Zinc is one of the essential trace elements of the human body and participates in a variety of physiological processes. At the same time, zinc is a necessary component of the protein. It is mainly involved in maintaining the stability of the cell structure and cell membrane. The function of zinc includes the stability of the cell structure of more than 2000 zinc dependent transcription factors in the human genome, and more than 300 kinds of zinc gold. The biological activity of the enzyme plays a key role.
In diabetes, zinc deficiency is often accompanied by deficiency of zinc. In addition to insufficient zinc intake during diet, the loss of endogenous zinc can be increased in diabetic patients with impaired absorption and / or reabsorption of zinc and increased urine zinc excretion. Zinc not only plays an important role in insulin and carbohydrate metabolism, but also in the body's antioxidant mechanism. At present, oxidative stress has been considered important in the pathogenesis of diabetes and its complications. Zinc is an important component of the key antioxidant enzymes such as superoxide dismutase. Zinc deficiency leads to the decline in antioxidant capacity of antioxidant enzymes, resulting in increased oxidative stress in the body. The role of zinc in diabetic nephropathy and its possible mechanism are not clear.
We gave 8 weeks old FVB male mice with small dose STZ (50mg/kg) for 5 days of continuous intraperitoneal injection. After 1 weeks, the blood glucose was detected by the tail vein of mice. The model of type 1 diabetes mellitus was established successfully with the blood glucose level greater than 16.7mmol/L. After 3 months of disease and control mice, the zinc content of type 1 diabetic animal models with different zinc content was successfully established. Then, we examined renal function, pathological changes of kidney, oxidative damage, inflammatory injury and fibrosis by Western blot and histopathological examination. The effect of diabetic mice induced renal injury. Then, by detecting the expression level of Nrf2 and its downstream antioxidant factors, we explored the possible mechanism of different levels of zinc on diabetic induced renal injury. Finally, we detected the levels of AKT, GSK-3 beta and Fyn, and further explored the activation of zinc on AKT and its downstream related metabolic signals. To explore the possible relationship between zinc, AKT and Nrf2. The results are as follows:
1. there was no significant difference in blood sugar, body weight and kidney weight / body weight ratio among diabetic mice in different concentrations of zinc for 3 months.
2. in the diabetic group, the mortality, ACR and urea nitrogen in the low zinc intervention diabetic group increased significantly, and the intervention of normal zinc and high zinc could improve the above symptoms, especially the high zinc intervention.
3. compared with the corresponding control group, the expression of renal oxidative damage index SOD1 and SOD2 decreased in the diabetic group, the expression of inflammatory markers PAI-1 and TNF- a, the expression of CTGF and TGF- beta 1 in the fibrosis index increased. In the diabetic group, the low zinc intervention could further aggravate the above damage, and the prognosis of normal zinc and high zinc, especially the high zinc intervention, could be used in diabetic group. To improve the injury of the kidneys.
4. compared with the corresponding control group, the diabetic group showed the glomerular hypertrophy, the increase of extracellular matrix and the renal tubulointerstitial damage in the diabetic group. Through the transmission electron microscopy, the diabetic group showed the thickening of the glomerular basement membrane, the fusion of the epithelial podocyte segment and the mitochondria in the renal tubular epithelial cells compared with the corresponding control group. Swelling, low zinc intervention further aggravated the above damage trend, while normal zinc and high zinc intervention can partially improve the pathological damage of kidney.
5. compared with the corresponding control group, the expression of Nrf2 and its downstream antioxidant factors in the kidneys of the diabetic group decreased, and the low zinc intervention could further decrease the expression in the diabetic group, while the intervention of normal zinc and high zinc could induce the up regulation of Nrf2 and its downstream antioxidant factors, especially the high zinc intervention.
6. compared with the corresponding control group, the level of phosphorylation of AKT and GSK-3 beta in the kidneys of the diabetic group decreased, and the low zinc intervention could further decrease the phosphorylation level in the diabetic group, while the normal zinc and the high zinc intervention could regulate the increase of the phosphorylation level of AKT and GSK-3 beta, especially the high zinc intervention.
7. compared with the corresponding control group, the Fyn nucleus transposition in the kidneys of the diabetic group increased, resulting in the increase of Nrf2 from the nucleus and the increase of Nrf2 degradation. In all diabetic groups, the low zinc intervention could further increase the transposition of the Fyn nucleus, while the normal zinc and high zinc intervention, the Fyn nuclear transposition decreased, so that the expression of Nrf2 was up-regulated, especially the high zinc intervention.
Through the experimental results, we conclude that low zinc intervention aggravates the renal injury induced by diabetes to some extent, and that the intervention of normal zinc and high zinc can partly protect the kidney injury induced by diabetes, especially the high zinc intervention; this protection is partly by inducing the expression and function of Nrf2. One of the possible regulatory mechanisms is that one of the possible mechanisms of regulation is that the zinc part reduces the GSK-3 beta activity by activating AKT to inhibit the transposition of the Fyn nucleus, resulting in the accumulation of Nrf2 in the nucleus and increasing its downstream antioxidant factor NQO1, SOD and other expressions to achieve its antioxidant damage, which ultimately induces a certain degree of renal injury induced by diabetes. The protective effect.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R587.2

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