热休克蛋白27通过调控CBP抑制巨噬细胞过度炎症反应的分子机制研究

As one of the transcriptional cofactors, CBP mediates the activation of many transcription factors and implicates in different cellular processes, including regulation of the cell cycle, differentiation, tumorigenesis, and apoptosis. It has been reported that CBP was involved in the release of late inflammatory cytokines HMGB1. However, it is unclear how CBP participates in regulating innate immune and inflammation. We found that LPS stimulation resulted to HSP27 phosphorylation. Phosphorylated HSP27 upregulated CBP and promoted early pro-inflammatory cytokine release. And after that, HSP27 dephosphorylated and combined with CBP to inhibit its translocation to nucleus, and then prevent HMGB1 translocating from nucleus to plasma in late period of LPS stimulation. If HSP27 phosphorylation was inhibited, early proinflammatory cytokine release reduced, but late proinflammatory cytokine HMGB1 release increased. The further study demonstrated if HSP27 phosphorylation was inhibited, CBP down-regulated during the early period of LPS stimulation, whereas CBP up-regulated in the late phage of LPS stimulation. We also found that p53 was involved in regulating CBP expression. These results suggested that HSP27 might inhibit excessive inflammatory response through promoting CBP expression at the early of LPS stimulation and preventing its entrance to nucleus in the late period of LPS stimulation. Thus, in this program, we will perform following investigation. (1) The effect of LPS-induced HSP27 phosphorylation on CBP expression and macrophage activation during the early and late period of infection. (2) The molecular mechanism by which HSP27 affects CBP expression through regulating p53 transcription activity. (3) The molecular mechanism by which HSP27 interacts with CBP and regulates the translocation of CBP from the cytoplasm to the nucleus. (4) The effect of CBP inhibition on the animal model of sepsis. These researches will help us to find the novel mechanism by which HSP27 regulates the innate immune and maintains homeostasis.

CBP作为转录辅激活因子介导转录因子激活，但在炎症调控中作用不明。我们的研究表明，LPS刺激单核/巨噬细胞可致HSP27磷酸化，磷酸化的HSP27通过p53促进CBP上调和早期促炎因子释放；但在LPS刺激后期，HSP27去磷酸化后与CBP结合抑制其入核，减少致死性细胞因子HMGB1释放。如抑制HSP27磷酸化，在LPS刺激早期减少CBP表达和炎症因子释放，在后期则导致CBP上调和HMGB1释放。提示细菌感染时HSP27发生磷酸化和非磷酸化转化，通过在感染早期和晚期对CBP的不同调控，控制过度炎症反应。本项目拟研究：LPS所致HSP27磷酸化在感染早期和晚期对CBP表达及巨噬细胞激活的影响；HSP27通过调控p53影响CBP表达的机制；HSP27与CBP结合调控其入核并调控HMGB1出核的机制；LPS刺激后期抑制CBP对脓毒症动物模型的影响。阐明CBP参与HSP27维持炎症反应稳态的机制。

脓毒症是严重的全身性炎症反应性疾病，以过度的炎症反应激活以及炎症因子风暴产生为特征，其临床病例的死亡率较高，目前难以治疗。CBP作为转录辅激活因子在细胞核中介导多种转录因子活性，参与多种生理和病理生理过程，可促进炎症因子的释放，但CBP在炎症反应中的详细作用还不清楚。本课题组过去曾在HSP27的抗炎作用方面进行了研究，其研究结果提示了HSP27的抗炎作用，在本项目中，我们发现了CBP在介导HSP27抗炎作用上的新功能，并发现CBP作为靶点在治疗全身炎症反应性疾病（如脓毒症）中的作用。以下是本项目的研究结果。我们的研究表明，LPS刺激单核/巨噬细胞可致HSP27磷酸化，磷酸化的HSP27通过p53的激活促进CBP上调和炎症因子的释放；而在细胞质中的非磷酸化HSP27促进CBP的泛素化降解，减少促炎因子的释放。因此HSP27通过自身的磷酸化和非磷酸化的转变，经CBP介导，调控单核/巨噬细胞的炎症因子释放。如抑制HSP27磷酸化，在LPS刺激早期减少CBP表达和炎症因子释放；但在LPS刺激后期，HSP27 磷酸化后入核与CBP结合，抑制其乙酰转移酶活性，进而抑制致死性晚期细胞因子HMGB1的乙酰化、出核以及释放；另外在LPS刺激后期，磷酸化的HSP27减少ROS的过度产出，进而抑制LPS所致CBP上调和HMGB1释放。动物实验结果显示，在LPS所致内毒素血症和CLP手术所致脓毒症小鼠模型中，造模8小时后，经腹腔给动物注射CBP溴区抑制剂SGC-CBP30或CBP的新型抑制剂吲哚布洛芬，小鼠的死亡率显著下降，提示CBP可能是治疗脓毒症（特别是脓毒症后期）的新靶点，通过抑制CBP乙酰转移酶活性，可以在治疗脓毒症中发挥重要作用。综上所述，本项目揭示了CBP在HSP27调控单核/巨噬细胞过度炎症反应中的重要作用，调控细胞中CBP水平或CBP的乙酰转移酶活性可以控维持炎症反应程度。CBP的抑制剂具有有效治疗脓毒症的潜在作用，可在今后治疗脓毒症的药物研发中发挥重要作用。