羧甲司坦调控HDAC2活性逆转COPD激素抵抗作用的表观遗传学机制研究

COPD, which has corticosteroid resistance, is largely caused by inactivation of histone deacetylase 2 (HDAC2). To find the drug targets for HDAC2 is still today's research focus. Of epigenetics development for COPD research provides a new idea. Our present study demonstrated that thiol derivative-carbocysteine, which has mucoactive, antioxidant and anti-inflammatory properties has an effects on the HDAC2 expression and activities. However, the manner in which regulated HDAC2 expression and activities by carbocysteine in our experimental model and whether it targets a histone or non-histone remains to be elucidated. In this study, we will explore 1) whether exist the relationship between the carbocystein and HDAC2 expression and activities; 2) whether SUMO modification involving in HDAC2，if so, its molecular mechanisms and the role of carbocysteine. 3) Where are potential modification sites, using high content analysis platform, immunofluorescence, siRNA, ChIP, and other technical means at the molecular, cellular level and whole animal level. The implementation of this project not only to expand the understanding of the relationship between the carbocysteine and GC resistance, but also to rice the theory of epigenetic regulatory mechanisms involving in histone modifications, and provide us to better carry out the study of GC resistance mechanism.

HDAC2表达及活性下降与COPD患者对激素治疗抵抗密切相关。基于HDAC2调控机制探寻逆转COPD激素抵抗的药靶仍然是人们关注的热点。小分子巯基衍生物-羧甲司坦具有祛痰、抗炎及抗氧化作用。循证医学发现，羧甲司坦能显著降低COPD患者急性加重率，提高患者的生活质量。本课题组发现，其能逆转COPD模型HDAC2表达及活性下降，增强GC治疗敏感性。但是，羧甲司坦以何种方式调控HDAC2，其调控作用是否靶向组蛋白尚不清楚。本课题拟运用高内涵分析平台、悬浮芯片、siRNA干扰、ChIP等技术手段，1）探讨羧甲司坦与炎症因子、HDAC2之间的关系；2）明确羧甲司坦对HDAC2的SUMO修饰的调控作用及对组蛋白乙酰化的影响；3）明确HDAC2的SUMO化修饰靶位。本项目的实施不仅拓展人们对羧甲司坦与激素抵抗性COPD之间关系的认识，更可以丰富表观遗传调控理论，为激素抵抗性COPD的治疗提供创新理论。

糖皮质激素（GC）在COPD治疗中常出现抵抗或失敏现象。研究表明GC抵抗与HDAC2表达和活性密切相关。羧甲司坦（S-CMC）为临床常用祛痰药具有抗氧化和抗炎能力，是否可以调控HDAC2改善GC敏感性尚未见报道。.基于GC抵抗的COPD动物模型，采用ELISA、组化及免疫组化染色、Western blot、白细胞分类计数、离体肌张力实验及肺功能检测等技术，观察第6-12周COPD模型大鼠各项病理指标探讨S-CMC是否对GC抵抗作用具有改善作用。结果表明：联用S-CMC能有效地改善GC抵抗现象；S-CMC也可逆转模型动物肺组织中HDAC2表达和活性的下调。.S-CMC改善GC敏感性机制研究发现：1）S-CMC可呈剂量依赖地从效能（Imax）和效价（IC50）两方面改善DEX治疗的敏感性；2）单用S-CMC可逆转香烟烟雾提取液（CSE）引起的HDAC2下调，而联用DEX逆转HDAC2下调的效果更为显著；3）慢病毒转染shRNA沉默HDAC2蛋白后，S-CMC增强DEX敏感性的作用消失；4）S-CMC调控HDAC2的作用可被巯基还原剂DTT增强，被巯基氧化剂diamide抑制；5）SPR和酶功能学实验发现，S-CMC与HDAC2无直接相互作用，GSH与HDAC2有直接相互作用；6）ChIP技术证实，S-CMC可上调HDAC2对组蛋白H4的去乙酰化功能，抑制IL-8的基因转录，此作用可被谷胱甘肽合成酶抑制剂BSO阻断。结果表明，S-CMC是以巯基/谷胱甘肽依赖方式上调HDAC2表达与活性而增强GC敏感性。 .运用SUMO化预测软件、定点突变和Co-IP技术确证：生理条件下，HDAC2与SUMO1存在相互作用；HDAC2的SUMO化修饰主要是由SUMO1亚型完成。而SUMO1修饰位点可能为K51和K462位点，其中K51位点的SUMO1修饰可以调节HDAC2的去乙酰化功能。氧化应激状态下HDAC2的SUMO1修饰增强，而S-CMC能够调控HDAC2发生去SUMO化，恢复HDAC2的去乙酰化功能，从而增强GC的敏感性。.综上，S-CMC通过上调HDAC2表达和活性改善氧化应激引起的GC抵抗现象，研究结果为临床治疗COPD提供新的理论依据。