迷走神经调节心梗后M2巨噬细胞活化及其通路

Following myocardial infarction (MI), circulating blood monocytes migrate into the infarcted myocardium, differentiate into macrophages, and aggravate inflammatory response. Macrophage is a primary responder cell type that is involved in the regulation of post-MI wound healing, secreting cytokines and growth factors, and the angiogenic response at multiple levels. Macrophage activation can be a heterogeneous process induced by cytokines, resulting in the generation of different classes of cells that exhibit diverse immunological functions- - classical (M1) and alternative (M2). M1 macrophages promote inflammation and extracellular matrix (ECM) destruction. M2 macrophages facilitate anti-inflammation, ECM reconstruction, cell proliferation and angiogenesis. IL-4 elicits the alternative activation of macrophages that involves stimulation of the cytokine receptors. Deletion of the the IL4 receptors in mice abrogates the ability of macrophages to acquire the specialized phenotypes. The PI3K/Akt/mTOR/p70S6k pathway has an established role in restricting proin?ammatory, and promoting anti-in?ammatory. Cot/tpl2 is a MAP3K responsible for the activation of MKK1-Erk1/2 pathway and the control of P-Ser473 Akt and P-Thr389 p70 S6k phosphorylation. Src homology 2-domain-containing inositol-5'-phosphatase (SHIP)-deficient murine macrophages are more sensitive to IL-4-mediated skewing to an alternatively activated phenotype. SHIP degradation was required to activate PI3K.Some researchers report that stimulation of vagal nerve and acetylcholine (ACh), as a transmitter of vagal nerve, can modulate the immune response and control inflammation through a 'nicotinic anti-inflammatory pathway' dependent on the α7-nicotinic acetylcholine receptor (α7-nAChR). In addition, in our previous work expound that stimulated vagal nerve and ACh can improve the cardiac function and decrease infarction size, as well as TNF-α production via muscarinic acetylcholine receptor-2 (M2R) and mitogen-activated protein kinase (MAPK). In our experiments, ACh increased expression and phosphorylation of mTOR in a dose-dependent manner. However, it is not clear that ACh improve myocardial inflammation through regulation of M2 macrophages polarization after MI. We hypothesize that ACh enhance alternatively activated macrophage through α7-nAChR and PI3K/Akt/mTOR/p70 S6K pathway to reduce the myocardial inflammation after MI. In this project, the MI model in mice and macrophage culture were established. Using α7-nAChR-knockout mice, siRNA technology and so on, cardiac function, macrophages polarization (M1/M2 ratio, IL-12/IL-10 ratio, NO/Arg-1 ratio, Ym1, Arg-1), IκB-NF-κB, Jak2/STAT3 and PI3K/Akt/mTOR/p70S6K pathway of macrophages were examined to disclose the mechanism of stimulated vagus and ACh that regulate M2 macrophages polarization to protect myocardium after MI, offering new strategy and promising approach for therapy of MI.

心肌梗死后，以巨噬细胞为主的炎性细胞浸润到受损局部，加重炎症反应和心肌损伤。活化为M2型的巨噬细胞可对抗炎症反应，促进血管新生和组织修复。研究发现刺激迷走神经可抑制M1表达多种致炎因子并有心肌保护作用。但尚无研究表明刺激迷走神经能否通过调节M2活化改善心梗后炎症反应而保护心肌。本课题拟运用Si RNA低表达α7-nAChR小鼠，建立心梗模型和体外巨噬细胞系培养，刺激迷走神经或给予ACh，检测心功能、心梗后巨噬细胞活化（M1/M2 ratio、IL-12/IL-10 ratio、NO/Arg-1 ratio）、胞浆酶活性（Cot/tpl2、SHIP）、IκB-NF-κB和Jak2/STAT3通路、PI3K/Akt/mTOR/p70S6K通路的变化；最终阐明迷走刺激调节心梗后巨噬细胞向M2活化的机制及主要靶点，为临床减轻心梗后炎症保护心肌提供新的理论依据和治疗靶点。

心肌梗死后，以巨噬细胞为主的炎性细胞浸润到受损局部，加重炎症反应和心肌损伤。活化为M2型的巨噬细胞可对抗炎症反应，促进血管新生和组织修复。研究发现刺激迷走神经可抑制M1表达多种致炎因子并有心肌保护作用。但尚无研究表明改善迷走神经活性能否通过调节巨噬细胞活化改善心梗后炎症反应而保护心肌。本课题选用昆明白小鼠，给予异丙肾上腺素（ISO）20mg/kg/d皮下注射2周建立心肌纤维化模型，干预组给予3mk/kg/d吡斯的明（PYR）灌胃，阳性药物对照组给予β-受体阻断剂普萘洛尔（PPN）30mg/kg/d灌胃，实验结束后行心动超声检测心功能，组织化学染色检测心肌纤维化程度，流式细胞仪检测心脏巨噬细胞的表型；发现吡斯的明可显著改善心功能和心自主神经活性、减轻心肌纤维化并减少心脏巨噬细胞的浸润和活化；这一结果表明吡斯的明可能通过改善心自主神经活性、减少巨噬细胞浸润和活化从而抑制心肌纤维化。为进一步探讨可能的机制，选用小鼠巨噬细胞系RAW267.4细胞为研究对象，应用LPS诱导巨噬细胞的经典活化（M1），给予乙酰胆碱（ACh）进行干预，发现ACh可抑制LPS诱导的巨噬细胞基质金属蛋白酶-9（MMP-9）mRNA、蛋白和酶活性的升高以及巨噬细胞细胞迁移，分别应用α7-nAChR阻断剂MLA、JAK2 阻断剂AG490、STAT3阻断剂stattic以及α7-nAChR SiRNA干预后可拮抗ACh对巨噬细胞活化的抑制作用；检测MMP-9生成、细胞迁移和多种信号分子的活化水平，发现ACh可通过α7-nAChR调节JAK2/STAT3和p38 MAPK/NF-κB通路进而抑制巨噬细胞MMP-9的生成和细胞迁移。