建立新型的抗氧化小鼠模型以研究内源性抗氧化水平对Ⅱ型糖尿病和糖尿病肾病发展的影响

Extensive investigations have suggested oxidative stress is complicated in diabetes mellitus (DM) and diabetic nephropathy (DN) but most clinical trials have failed to support the putative role of antioxidant intervention. One critical problem is that the inclusion of patients without biochemical evidence of increased oxidative stress in clinical trials may dilute the possible positive outcome. I hypothesize that effective antioxidant therapy depends on baseline levels of endogenous antioxidant in DM and DN patients and a subgroup of the patients who have low cellular antioxidant levels would be susceptible to the DN and thus benefit greatly from antioxidant therapy. Another possible reason for the failure of clinical trials is that the tested antioxidants could not reach therapeutic levels in mitochondria. Mitochondrial dysfunction has been postulated as the primary initiating event in DN. Alpha-lipoic acid (LA), produced in mitochondria by lipoic acid synthase (Lias), is a potent antioxidant and a cofactor in mitochondrial enzyme complexes participating in energy production and has a high potential for mitochondrial protection. To test the hypotheses, three types of novel antioxidant mice exhibiting around 25%, 75% or 150% Lias gene expression of wild type has been specially created by genetic methods. These mice have correspondingly altered endogenous antioxidant baselines and oxidative stress marker levels that are also consistent, reliable and heritable. They have been crossed with Leprdb/db mice, a type 2 diabetic mouse model. The models allow us to precisely evaluate the impact of predisposed different baseline antioxidant levels on initiation and progression of DN (Aim 1). Additionally, these mice will be supplied with dietary LA to determine whether mitochondrial antioxidant levels will significantly increase and whether the disorders of DN will be attenuated after LA treatment (Aim 2). The project will provide valuable information regarding the impact of different antioxidant baseline levels on the initiation and development DN and effective antioxidant intervention using a group of unique antioxidant models, which will lead to a successful clinical trial by using antioxidant therapy in DN patients.

研究表明氧化应激参与糖尿病的发生和发展，但抗氧化治疗在多数临床试验中并未取得预想结果。其原因我们认为，内源性抗氧化基线水平决定抗氧化疗法的效果，只有低抗氧化水平患者才受益于该疗法。另一原因可能是抗氧化剂在线粒体内未达到有效浓度，而线粒体功能失调导致超氧阴离子自由基产生被认为是糖尿病肾病的主要始发因素。硫辛酸在线粒体中由硫辛酸合成酶催化产生，是一种强抗氧化剂并具有保护线粒体的潜力。为此我们特创建一组新颖的抗氧化小鼠模型，该模型约表达25%,75%或150%硫辛酸合成酶基因并有相应的内源性抗氧化基线水平；将它们与II型糖尿病模型小鼠交配。用新模型精确评估预先定量的抗氧化基线对糖尿病和糖尿病肾病发展的影响，并通过给予小鼠硫辛酸以确定其是否增加线粒体抗氧化水平，是否改善糖尿病肾病治疗效果。这一独特的抗氧化模型将为研究不同的抗氧化基础水平对疾病的影响和有效的抗氧化干预提供有价值的信息。

本课题针对机体抗氧化功能和氧化应激状态, 检查了最近我们新建立的一组新型的LiasLow/Low和LiasHigh/High抗氧化小鼠模型。这些小鼠与常用肥胖症与II型糖尿病模型Leprdb/db小鼠交配，以观察不同机体抗氧化能力对疾病的影响。 我们采用多种方法较为全面的检查机体抗氧化水平升高或降低对糖尿病、糖尿病肾病和脂肪肝的影响。 按照实验设计，我们发现，所检查的疾病大多数指标包括体重，血糖，血脂，机体糖耐量，炎性因子，肝肾功能与形态变化，胰岛的形态，血, 尿和组织中氧化应激和抗氧化标志物，与作为对照组的Lias+/+Leprdb/db小鼠相比，LiasHigh/High小鼠由于具有较高抗氧化能力，它们呈现明显较轻的疾病症状与病理变化，特别是它们肝中线粒体损伤明显减少，这证实我们的假说，即抗氧化功能和缓解疾病是主要通过保护线粒体而实现的。此外，在实验过程中，我们还对LiasLow/Low小鼠的存活期缩短现象用遗传学方法进行调查追究，并通过回交方法进行拯救，收到一定效果，从而揭示小鼠遗传背景对Lias基因敲除小鼠表现型的影响。总之，这项研究证实硫辛酸保护线粒体作为一种治疗机理和新的靶点，这个独特的模型将提供一个优良的临床前期研究平台，帮助开发新的抗氧化治疗策略和发现早期诊断标志物以期获得成功的临床试验。