缺氧诱导NHE1参与calpain介导ABCA1降解及胆固醇逆转运障碍

ABCA1 is thought to be the gatekeeper in reverse cholesterol transport and be beneficial to antiatherosclosis. Severe hypoxia exists in atherosclerotic plaque，accompanying with compromised reverse cholesterol transport, but its mechanisms remain to be elucidated. Calpain mediates ABCA1 degradation.Our previous study demonstrated that hypoxia increased activity of NHE1 and calpain. Thus，we hypothesize that hypoxic induction of NHE1 expression is involved in the ABCA1 degradation, reverse cholesterol transport and atherogenesis. To address the issue, firstly,we examine the relationship of hypoxia and NHE1 expression in atherosclerotic plaques and then the effects of NHE1 expression and activity on foam cell formation and cholesterol efflux. Secondly, the ABCA1 degradation and calpain activity is to explore after knockdown of NHE1 by small inferring RNA. Thirdly, the interaction and subcellular localization of NHE1, calpain and ABCA1 is investigated using coimmunoprecipitation or immunofluorescence and then ABCA1 degradation and calpain activity are detected, after the treatment with NHE1 inhibitor, calcium chelating agent and Ezrin knockdown. This study will be to further insight the novel molecular mechanism whereby hypoxia and pH regulation participate in atherogenesis due to compromised reverse cholesterol transport. NHE1 as well as calpain might represent a potential target for the treatment and prevention of atherosclerosis and its related diseases.the finding from this study will add new experimental evidence for the development of NHE1-related drugs.

ABCA1是胆固醇逆转运(RCT)的守门者，具有抗动脉粥样硬化（AS）作用。斑块缺氧广泛存在，伴随RCT功能紊乱，但机制不明。研究证实缺氧诱导NHE1及 calpain活性增加，calpain介导ABCA1降解。故推测缺氧诱导NHE1表达，调节calpain活性，参与ABCA1降解、RCT紊乱及AS形成。为此，首先明确斑块缺氧及NHE1表达，探讨缺氧巨嗜细胞NHE1表达及活性对泡沫细胞形成及胆固醇外流的影响。其次，研究siRNA下调NHE1表达对calpain活性及ABCA1降解影响。最后，从pH、Ca++及Ezrin角度探讨calpain活性改变对ABCA1降解的机制，明确缺氧条件下NHE1、calpain及ABCA1相互作用及细胞内共定位改变。本研究可望阐明缺氧及pH调控通过RCT改变参与AS斑块形成新机制，防治AS及相关疾病提供新思路，开发NHE1相关药物奠定实验基础。

按照项目书2010.01-2012.12研究计划,本课题组经过4年努力，顺利完成课题预期目标。发表论文8篇，其中medline收录1篇，中文核心期刊8篇，完成1名研究生培养，在读研究生2名。整个项目基本按照计划进行研究，并根据实验需要适当增加缺氧对细胞膜ABCA1蛋白降解及在体实验, 取得了预期实验结果。我们实验结果表明：动脉粥样硬化斑块NHE1表达升高，虽ABCA1亦有表达，但NHE1表达上调部位ABCA1表达少；缺氧上调RAW264.7细胞NHE1表达、细胞内钙离子浓度([Ca2+]i )及calpain活性，下调细胞总及细胞膜ABCA1蛋白；上调或下调NHE1表达及活性，分别上调及下调细胞内钙离子浓度([Ca2+]i )及calpain活性，下调或上调ABCA1表达；在此基础上给予钙离子螯合剂及calpain抑制剂干预，相应影响ABCA1表达。敲除Ezrin后，可以抑制ABCA1降解。相应结果发表在《细胞与分子免疫学杂志》、《中国病理生理杂志》、《中国动脉硬化杂志》、《中国现代医学杂志》等期刊中，尚有多篇文章审稿中，尚有结果正在整理准备投稿。项目培养研究生1名，本科生1名（获本科优秀毕业论文，本校生物技术200余篇中4篇之一），在读研究生2名。