生长分化因子-15在急性心肌梗死中对巨噬细胞极化的影响及其调控机制研究

Classically (M1) and alternatively (M2) activated macrophage subsets play different roles in the left ventricular remodeling after myocardial infarction (MI).M1 macrophages dominated on day 1-3 post-MI, whereas M2 macrophages represented the predominant macrophage subset after 5 days pf MI. It was called macrophage polarization. Biphasic regulation of macrophage sub-population is involved in MI with M1 macrophages peaking at the inflammatory stage followed by a latent appearance of M2 macrophages paralleling infarct repair and scar formation. Growth differentiation factor (GDF)-15, is a member of the transforming growth factor-βsuperfamily. GDF-15–deficient mice show increased infarct size and cardiomyocyte apoptosis after myocardial ischemia/reperfusion, ablation of GDF-15 in mice contributes to an increased frequency of cardiac rupture after myocardial infarction. Clinically, high circulating GDF-15 concentrations are associated with increased risks of mortality, recurrent myocardial infarction and adverse events in patients with AMI. Our previous study showed that AMI patients with ST-segment resolution ＜50%,GDF-15 level was positively related to IL-6, IL-8 and MCP-1(pro-inflammatory cytokines) concentration and inversely to the IL-10 concentration(anti-inflammatory cytokines). The precise regulation mechanism underlying M1/M2 polarization during MI is unknown. We hypothesized that high concentration of GDF-15 could inhibit M1 and active M2 in the early stage of MI, and then GDF-15 degraded gradually with no effect on the M1/M2 polarization in the late stage. MI was induced in wild type (WT) and GDF-15 deficient (GDF-15−/−) mice by left anterior descending coronary artery ligation，with extraneous GDF-15 or saline on 6 hours and 3 days after the surgery. Production of M1 macrophages including signature cytokines including IL-12, IL23and M2 signature cytokines including (IL-10、Arg-1) was demonstrated by immunohistochemistry, flow cytometry, quantitative real-time PCR, and ELISA assays. Cardiac function, ventricular dilatation and fibrosis were measured. Moreover, activation of the signal transducer and activator of transcription（STAT） 1, STAT3,STAT6 signaling pathway were measured at mRNA and protein levels by Western-Blot, real time Transcription-Polymerase Chain Reaction, Phospho antibody array and confocal microscopy. We hypothesize that GDF-15 may exert a protective effect against MI in vitro and in vivo. Exogenous GDF-15 may represent a new interventional target for treatment of post-infarct remodeling and subsequent heart failure.

巨噬细胞（MФ）亚型（M1/M2）在心肌梗死（MI）后的损伤修复中发挥重要作用。我们的前期工作发现生长分化因子（GDF）-15可以诱导巨噬细胞向M2型极化，既往研究报道该因子由缺血心肌细胞和极化的MФ共同分泌的炎症调节因子，可以在MI后的重构中发挥保护作用。为了探讨GDF-15是否通过影响MI后抑制M1/促进M2分化、进而改善心室重构这一调控机制。本课题拟体外给予不同浓度GDF-15刺激MФ极化，分析MФ极化方向，并通过观察通路中信号转导子及转录激活子(STAT)- 1、3、6的总蛋白、磷酸化蛋白的表达水平和在细胞中的定位，揭示GDF-15对MФ极化影响的可能机制；利用GDF-15基因敲除小鼠和野生型小鼠，结扎前降支观察二者在MI后MФ极化的差别，验证GDF-15对MФ极化的影响和可能的机制，并外源性给予GDF-15，证实GDF-15保护作用。研究成果可望为改善MI后心室重构提供新思路

巨噬细胞（MФ）亚型（M1/M2）在心肌梗死（MI）后的损伤修复中发挥重要作用。既往研究报道GDF-15由缺血心肌细胞和极化的MФ共同分泌的炎症调节因子，并可以可以在MI后的重构中发挥保护作用。拟探讨GDF-15是否通过影响MI后抑制M1/促进M2分化、进而改善心室重构这一调控机制。分别加入GDF15 10ng/ml、20ng/ml、50 ng/ml，并以LPS 100ng/ml+IFNg 20ng/ml刺激巨噬细胞向M1转化，用rPCR测定巨噬细胞M1极化方向产生的细胞因子IL-12、IL-6、iNOS、TNF-α以及M2极化产生的细胞因子IL-10、Ym-1表达水平未发现显著变化；进一步利用荧光微球吞噬实验和细菌吞噬实验检测不同浓度GDF-15l对于巨噬细胞吞噬功能的未产生影响。GDF15 20 ng/ml预处理巨噬细胞后，oxLDL诱导巨噬细胞的表达TNF-α增加。腺病毒感染过表达GDF-15，加入oxLDL 50 ug/ml,过表达GDF-15不影响巨噬细胞吞噬oxLDL，通过转录组测序分析差异表达基因过表达GDF-15主要对oxLDL诱导巨噬细胞的功能发挥抑炎的作用，可能的功能包括：下调B细胞的激活以及诱导凋亡，上调电压门控通路的活性、上皮管形态生成以及NF-κB级联反应。在临床患者中，测定心肌梗死不同阶段的患者血浆GDF-15 ,ICTP, PINP, PIIINP浓度，GDF-15水平在冠心病组、陈旧心肌梗死组和陈旧心肌梗死心力衰竭组逐渐升高，并与胶原降解的指标ICTP and PIIINP 正相关(r = 0.302, P < 0.001 & r = 0.206, P = 0.006)。GDF-15 与超声心动图舒张指标正相关E/Em and left atrial pressure (r = 0.349 and r = 0.358, P < 0.01) 与收缩功能指标负相关LVEF and Sm (r = -0. 623 and r = -0.365, P < 0.01)。.GDF-15 是心肌梗死后不良重构的保护性因子，并与心室不良重构有关。本研究发现，GDF-15对巨噬细胞的极化功能、吞噬功能均未发现有显著影响，但GDF-15与ox-LDL诱导的动脉粥样的硬化过程巨噬细胞的炎症表达发挥了作用。