一磷酸腺苷激活依赖性蛋白激酶（AMPK）调控巨噬细胞分化从而调控心脏纤维化的作用和机制的研究

Cardiac fibrosis is the critical pathological process during hypertension-induced heart injury. Abnormal activation of inflammatory cells, such as macrophages, mediate this process, and different subtypes of macrophages play different roles during this pathological process. AMPK signaling pathway showed strong anti-inflammation and anti-fibrosis functions, but the detailed mechanisms are still not clear. Previous works from us proved that, whole body knockout of AMPK enhanced the cardiac fibrosis in AngII-infused mice. Thus we hypothesized that, AMPK regulates macrophage polarization by affecting its downstream transcription factors such as STAT1/3, so that protect heart from fibrosis. In this study, we will carry out mice bone marrow transplantation from AMPK knockout donors, in order to clarify the influence of macrophage AMPK on AngII-induced cardiac fibrosis in vivo; we will also carry out ex vivo tests on cultured macrophages, by using ELISA, flow cytometry, co-culture with cardiac fibroblasts, and other methods, to identify the features of macrophage polarization after activating or inhibiting AMPK, and to identify the feature changes of macrophage polarization after changing the expression of transcription factors such as STAT1/3, so that clarify the detailed mechanisms by which AMPK regulates macrophage polarization. Results from this study will uncover new mechanisms of how AMPK regulates macrophage polarization so that affects cardiac fibrosis, and will help to improve the clinical strategies for related cardiac diseases.

心脏纤维化是高血压引起心脏损伤的重要病理过程，炎症细胞如巨噬细胞的异常活化介导该过程，分化的巨噬细胞在其中起不同的作用。AMPK及其信号通路具有抗炎、抗纤维化的作用，但具体机制尚不清楚。我们的前期实验证实，AMPK全身敲除加重了小鼠灌注AngII后的心脏纤维化程度。由此我们提出假说：AMPK通过调节下游转录因子，如STAT1/3，影响巨噬细胞分化，从而抗纤维化。本项目将使用AMPK敲除骨髓的移植技术，在体验证巨噬细胞中的AMPK对AngII灌注引起的心脏纤维化的影响；在离体巨噬细胞上，使用ELISA、流式细胞、与成纤维细胞共培养等方法，通过改变AMPK活性，检测巨噬细胞的分化情况，再调节STAT1/3等转录因子的表达，检测巨噬细胞分化的改变，从而明确AMPK调控巨噬细胞分化的具体分子机制。本项目的结果将阐明AMPK调控巨噬细胞分化、影响心脏纤维化的新机制，帮助改善相关疾病的治疗策略。

心脏纤维化是高血压引起心脏损伤的重要病理过程，炎症细胞如巨噬细胞的异常活化介导该过程，分化的巨噬细胞在其中起不同的作用。AMPK及其信号通路具有抗炎、抗纤维化的作用，但具体机制尚不清楚。AMPK通路被证实能够调节巨噬细胞在主动脉瘤病变过程中的作用；我们的前期实验则证实，AMPK全身敲除加重了小鼠灌注AngII后的心脏纤维化程度。由此我们提出假说：AMPK通过调节下游转录因子，如STAT1/3，影响巨噬细胞分化，从而抗纤维化。本项目通过使用特异性敲除骨髓来源的单核细胞中AMPKα1的小鼠，在体证实，在血管紧张素（AngII）灌注引起的心脏纤维化过程中，敲除骨髓来源巨噬细胞中的AMPK，使得心室更加狭窄、心脏更加肥大、心脏中的炎性细胞浸润增多、心脏纤维化程度更高，同时循环血中巨噬细胞M1：M2型的比例更高，提示AMPK能够促进巨噬细胞分化为M2型、从而保护高血压应激条件下的心脏；在离体培养的野生型原代巨噬细胞上，分别使用AngII和AICAR刺激细胞，能够时间依赖性地增加AMPKα及STAT3的磷酸化水平，AICAR刺激会显著降低巨噬细胞炎性因子的表达水平，提示AMPK-STAT3信号通路参与调控高血压应激条件下的巨噬细胞功能和表型。上述结果证实了AMPK调控巨噬细胞分化、减轻高血压诱导的心脏纤维化的作用和机制，为改善治疗相关疾病的策略提供了新的依据。