ROS激活PLCγ-PDGF-D/PDGFR-β环路致复苏后成纤维细胞活化的机制研究

Post-resuscitation myocardial dysfunction is one of the main causes of early death after restoration of spontaneous circulation. Both existing data and our preliminary study demonstrated that early level of reactive oxygen species was negatively correlated with cardiac function after resuscitation. And there was asynchrony between ROS change and cardiac outcome, which existed diastolic heart failure and cardiac fibrosis in the inner layers of ventricular wall at 3 days post-resuscitation. However it is still unclear about the mechanism how ROS induced cardiac fibrosis. In the study, we hypothesize that ROS-mediated the feedback loop between phospholipase c gamma and platelet derived growth factor/platelet derived growth factor receptor-β signaling (PLCγ-PDGF-D/PDGFR-β) regulate cardiac fibrosis after resuscitation. The current study aims to investigate how ROS-mediated PLCγ-PDGF-D/PDGFR-β loop induces cardiac fibroblasts activation and changes cell niche using inhibitor and sgRNA interference of PLCγ or PDGFR-β both in a rat model of cardiopulmonary resuscitation and in vitro model of oxygen-glucose deprivation/oxygen-glucose restoration. This study will provide important insights into the pathophysiological mechanism of post-resuscitation myocardial dysfunction, and will render novel experimental evidence to attenuate post-resuscitation cardiac dysfunction targeting for inhibition of cardiac fibroblasts activation.

复苏后心功能障碍是自主循环恢复早期死亡的主要原因之一。已有研究及前期工作证实，早期活性氧簇（ROS）水平与复苏后心功能呈负相关，ROS变化与心功能转归不同步，复苏后3天存在舒张性心衰伴室壁内层纤维化。ROS与复苏后心脏纤维化的关系，目前尚不清。本项目提出磷酯酶γ-血小板源性生长因子-D/血小板源性生长因子-β环路（PLCγ-PDGF-D/PDGFR-β）介导ROS增高导致复苏后心脏纤维化的假说。本项目拟采用大鼠心肺复苏模型，同时体外建立氧糖剥夺/氧糖恢复模型，应用拮抗剂及sgRNA沉默抑制PLCγ或PDGFR-β，从基因和信号传导水平研究ROS激活心肌成纤维细胞PLCγ-PDGF-D/PDGFR-β，细胞活化并改变微环境。本研究为阐述复苏后心功能障碍的病理生理机制提供新的方向，并为靶向抑制成纤维细胞活化改善复苏后心功能提供一定的理论和实验依据。

复苏后心功能障碍是心脏骤停患者自主循环恢复后早期的主要死因之一。研究已证实，复苏后４ｈ出现严重心肌收缩和舒张功能不全，随后心功能逐步改善，复苏后3天内心肌收缩功能有恢复，而复苏后3天仍存在舒张性心衰伴室壁间质增生。复苏后早期活性氧簇（ROS）水平与复苏后心功能及生存时间呈负相关，但ROS与复苏后心肌胶原增生的关系，值得进一步研究。本项目提出了ROS激活磷脂酶Cγ-血小板源性生长因子-D/血小板源性生长因子-β信号（PLCγ-PDGF-D/ PDGFR-β）导致复苏后心脏纤维增生的科研设想。研究采用大鼠心肺复苏模型，同时体外建立成纤维细胞氧糖剥夺/氧糖恢复模型，应用拮抗剂及携带PDGFR-β 基因的重组腺病毒抑制或过表达PDGFR-β，或拮抗剂及携带PLCγ基因的重组腺病毒抑制或过表达PLCγ，从基因和蛋白水平评价心脏及心肌成纤维细胞PLCγ-PDGF-D/PDGFR-β信号系统，促进成纤维细胞活化，胶原增生和微环境改变的机制。结果发现，心肺复苏后大鼠血清和成纤维细胞培养基中PDGF-D 增加，PDGF-D/PDGFR-β信号通路活化，Collagen Ⅰ、SMA、Vimentin、PDGFR-β等细胞外基质蛋白表达增加，天狼猩红染色测心肌胶原容积分数显著增高。抑制或过表达PLCγ或PDGFR-β，上述指标明显减少或增高。外源性给予H2O2，建立成纤维细胞氧化应激模型，检测丙二醛（MDA）、氧化型／还原型谷胱甘肽（GSH）、超氧化物歧化酶（SOD）等表达水平增高，培养基PDGF-D表达增高，细胞外基质蛋白表达增加。应用ROS抑制剂， PDGF-D/PDGFR-β表达水平以及纤维化指标下调。本研究为阐述复苏后心功能障碍的病理生理机制提供新的方向，并为靶向抑制成纤维细胞活化改善复苏后心功能提供一定的理论和实验依据。