TyrRs/PARP-1共同激活调控M2巨噬细胞活化在腹膜透析相关纤维化中的作用及机制

Recently studies show that alternatively activated macrophages (M2 cells) play critical roles in tissue remodeling. Our previous study found that the number and percentage of M2 cells were significantly increased in the peritoneum of peritoneal dialysis (PD) related fibrosis. At the same time, the activity of arginase 1 also increased and the levels of functional cytokines of M2, such as interleukin-10, translated growth factor-β, were highly increased. On the other way, we found high glucose increased ADP-ribose polymerase-1 (PARP-1) mRNA expression by peritoneum and high glucose induced EMT and the accumulation of ECM in peritoneal mesothelial cells. Moreover, down-regulate PARP-1 by PARP-1siRNA or PJ34 (inhibitor of PARP-1) may reverse EMT and the accumulation of extracellular matrix (ECM) induced by high glucose in peritoneal mesothelial cells (PMCs). A research published on Nature proved that Human tyrosyl tRNA synthetase (TyrRs) is a potent PARP-1 activating effector target for resveratrol. In summary, we put forward our question: If Tyr-TRN acting as a signal mediator synergisticly reacts with PARP-1 to play roles on the differentiation of M2 cells? We are going to test our ideas by following investigations: 1. We will establish model of M2 cells and upregulate or downregulate the expression of TyrRs, PARP-1, then investigate the effect of regulation of the differentiation of M2 cells. 2. The interaction of TyrRs with PARP-1 and downstream signal protein of P50, P65 will be tested by Immuno-precipitation (IP). 3. A TyrRS mutant (TyrRS-dNLS), with reduced nuclear localization and TyrRS-Y341A mutation, with breaking the Y341 OH--H-bond to a backbone carbonyl oxygen will be established, and then the effect of those two mutation on the expression of PARP-1 and differentiation of M2 cells will be studied. 4. We will establish the PD related peritoneal fibrosis animal model, and then upregulate or downregulate the expression of TyrRs, PARP-1 to investigate the degree of peritoneal fibrosis, epithelial to mesenchymal transition (EMT) of PMCs, the differentiation of M2. In a word, this study will illuminate the mechanism of M2 cells in PD related fibrosis, so that to provide evidence for prevention and treatment of PD related fibrosis.

我们已经证明替代活化巨噬细胞（M2）在腹膜透析相关纤维化（PDF）中起关键作用。那么，M2在PDF中的作用机制是什么？新近证实酪氨酰tRNA合成酶（TyrRs）除发挥蛋白质管家作用外，还作为一种信号介质，在组织重塑中发挥重要作用。我们预实验发现M2内高表达TyrRs，免疫共沉淀示TyrRs与其共同效应靶点多聚ADP核糖聚合酶1（PARP-1）在腹膜M2细胞核中相互结合。由此我们提出：TyrRs入核与PARP-1共同激活可能是M2活化及促PDF的新机制。本研究拟在细胞及动物模型中采用CoIP、腺病毒突变体、siRNA干扰、超声微泡转化等技术，旨在回答TyrRs/PARP-1共同激活是否为M2活化及促腹膜纤维化作用的关键机制。本研究将通过体内外实验，探索纤维化腹膜中M2细胞的作用机制，为PDF防治开辟新思路。

腹膜透析相关纤维化（PDF）是腹膜透析最重要并发症之一，影响病人长期存活。巨噬细胞（Mφ）根据活化途径可分为M1和M2细胞。我们前期已经证明M2细胞在PDF中起关键作用。然而M2在PDF中的作用机制目前尚不清楚，新近证实酪氨酰tRNA合成酶（TyrRs）除发挥蛋白质管家作用外，还作为一种信号介质，在组织重塑中发挥重要作用。我们预实验发现M2内高表达TyrRs，免疫共沉淀示TyrRs与其共同效应靶点多聚ADP核糖聚合酶1（PARP-1）在腹膜M2细胞核中相互结合。因此我们提出假说：TyrRs入核与PARP-1共同激活可能是M2活化及促PDF的新机制。.本项目计划通过在细胞模型中观察从M0（初始巨噬细胞）向M2活化时TyrRs/PARP-1的激活状态、相互作用，并在PDF动物模型中验证TyrRs/PARP-1通路促纤维化的作用机制。.课题基本按照计划，进行了细胞实验、动物实验：（1）体外建立M2细胞模型，观察TyrRs的核定位、与PARP-1的相互作用：小鼠永生化J774A.1细胞株致在IL-4和IL-13刺激24小时诱导成M2细胞。免疫共沉淀显示TyrRs与PARP-1在M2细胞中相互结合。（2）PDF动物模型中分析TyrRs/PARP-1通路在PD相关腹膜纤维化中的作用机制：SD雄性大鼠，随机分为对照组和模型组，每日腹腔内注射4.25%腹膜透析液（100mL/kg），共4周，建立PDF动物模型。研究结果发现，在PD相关性纤维化大鼠中，腹膜M2细胞内表达TyrR与PARP-1均表达增加。（3）分析巨噬细胞在高糖环境中（模拟巨噬细胞在PD中的细胞模型）向M2细胞活化的情况：结果显示M2巨噬细胞（CD206阳性细胞）则呈浓度梯度性和时间性依耐性激活。M2细胞功能性酶精氨酸酶1（Arg1）、功能性细胞因子TGF-β水平在不同糖浓度中呈浓度梯度增高。（4）该项目并分析了腹膜透析患者腹腔M2细胞的活化情况：分析隔夜使用1.5%和2.5%葡萄糖浓度透析病人腹腔M1和M2巨噬细胞比例。结果显示M2巨噬细胞在2.5%糖浓度透析液中显著升高。.上述结果初步证明TyrRs/PARP-1通路激活在腹膜M2细胞活化中的重要作用，阐明了TyrRs/PARP-1调控腹膜M2细胞活化在PDF中的新机制。为通过阻断TyrRs/PARP-1轴防治PDF提供潜在靶点。