益气温经法调控骨特异性转录因子Runx2乙酰化修饰水平治疗原发I型骨质疏松的机制研究

Over the years, the Reinforcing QI Warming Meridians Therapy has been employed in the clinical treatment of primary type I osteoporosis with significant clinical effect. This therapy could up-regulate Runx2 expression levels by inhibiting ubiquitination degradation of Runx2. Our previous studies have shown that HDAC4 was down-regulated after treatment through the Reinforcing QI Warming Meridians Therapy suggesting that this therapy could affect Runx2 expression level by regulating Runx2 acetylation modifications, but the specific regulatory mechanism of Runx2 acetylation modifications remains unclear. In order to clarify the mechanism of Runx2 acetylation modification by the Reinforcing QI Warming Meridians Therapy in primary type I osteoporosis, this study will use acetylation proteomics to quantify the acetylation sites and the expression levels of Runx2, HDAC4, SIRT1, EP300 and KAT2B before and after the use of Reinforcing QI Warming Meridians Therapy or Caltrate D combined with Rocaltrol Pulse Therapy, and between primary type I osteoporosis group and normal controls group. Then verify the results by ELISA and plot the Runx2 acetylation modification atlas. The study aims to clarify the mechanism of Runx2 acetylation modification and the role of Runx2-related acetyltransferase and deacetylase in the regulation of Runx2 acetylation by the Reinforcing QI Warming Meridians Therapy. This study may provide new evidence for the study on prevention and treatment of primary type I osteoporosis.

益气温经法治疗原发I型骨质疏松疗效显著，其可抑制Runx2泛素化降解从而上调Runx2。研究表明组蛋白去乙酰化酶HDAC4通过去除Runx2乙酰化修饰使其更易发生泛素化，而下调Runx2。我们近期研究发现，益气温经法治疗后HDAC4下调，提示该法可通过调控Runx2乙酰化修饰，影响Runx2表达水平，但该法调控Runx2乙酰化修饰的具体机制尚不明确。本项目拟采用乙酰化修饰定量蛋白质组学技术对原发I型骨质疏松组与绝经后骨量正常组、益气温经法组治疗前后和基础用药组治疗前后血清中Runx2及HDAC4，SIRT1，EP300和KAT2B等调控酶的乙酰化位点及表达量进行鉴定和定量，绘制图谱，并对其进行验证。联合Runx2下游靶蛋白表达和疗效评价，本研究将明确Runx2乙酰化修饰调控的机制及Runx2相关乙酰化酶和去乙酰化酶的作用，为益气温经法治疗原发I型骨质疏松的机制及中医药现代化研究提供新依据。

益气温经法治疗原发I型骨质疏松疗效显著，其可抑制Runx2泛素化降解从而上调Runx2。以往研究显示组蛋白去乙酰化酶HDAC4通过去除Runx2乙酰化修饰使其更易发生泛素化，而下调Runx2，结合前期研究结果，益气温经法治疗骨质疏松后HDAC4下调，提示该法可通过调控Runx2乙酰化修饰，影响Runx2表达水平，为进一步明确Runx2乙酰化修饰在益气温经法在治疗骨质疏松中的作用和可能机制。本研究中我们采用乙酰化修饰定量蛋白质组学技术对原发I型骨质疏松组与绝经后骨量正常组、益气温经法组治疗前后和基础用药组治疗前后血清中Runx2及和其相关的HDAC4，SIRT1，EP300和KAT2B等调控酶的乙酰化位点及表达量进行鉴定和定量。血清蛋白质组学共鉴定到蛋白569个，其中原发I型骨质疏松相关差异表达蛋白55个，上调19，下调36;益气温经法代表发强骨饮干预相关差异表达蛋白49个，上调18，下调31。血清乙酰化修饰组学总鉴定到112个乙酰化修饰蛋白,统计结果表明，69.37%蛋白质上分布2个及以上修饰位点,其中P02768蛋白质上含有多达50个修饰位点,以表达倍数>1.2倍且P value<0.05为标准，得到与原发I型骨质疏松症相关的上调、下调修饰肽段数目分别为25和6；得到与益气温经法代表发强骨饮干预相关的上调、下调修饰肽段数目分别为18和10。通过以上数据绘制了骨质疏松症疾病的乙酰化修饰变化图谱和益气温经法治疗骨质疏松症的乙酰化修饰图谱。但是研究中并未成功鉴定到Runx2及和其相关的HDAC4，SIRT1，EP300和KAT2B等调控酶及其乙酰化修饰位点信息，通过进一步的生物信息学分析发现鉴定到的GAPDH,H3F3B,SOD2,ALB,APOA1和APOA2等乙酰化蛋白与Runx2，HDAC4，SIRT1，EP300和KAT2B均在共同表达,相互作用等方面关联，其相关性有待进一步研究。本研究未成功鉴定Runx2，HDAC4，SIRT1，EP300和KAT2B可能与其在细胞核中表达，血液中丰度低，加之翻译后修饰对蛋白丰度要求更高有一定关系，尝试骨质疏松成骨细胞模型有望为Runx2等乙酰化修饰鉴定提供有力帮助，后期将补充原计划的验证工作。本研究目前研究结果为益气温经法治疗原发I型骨质疏松的乙酰化翻译后修饰机制及中药复方治疗骨质疏松症的现代化研究提供了一定的新依据。