Toll样受体4表达调控在促红细胞生成素抗炎与抑制心肌纤维化中的作用与机制

As cardiac fibroblasts proliferation and collagen secretion is the major mechanism of myocardial remodeling, therapeutic targeting of it may be the novel opportunity for cardiovascular diseases. Recently, erythropoietin (EPO) treatment animal model exhibited less ventricular remodelling after coronary artery ligation and suppressed inflammatory cell infiltration and cytokine expression. However, the cellular mechanism underlying the effect of EPO are not yet understood. Toll-like receptor 4(TLR4) is one group of innate immune response recognization receptor which is engaged by pathogen-associated molecular patterns and can induce the expression of pro-inflammatory cytokines. Others have shown us that antagonism of TLR4 could attenuate fibrosis in liver, lung and kidney et al. Interestingly, there is also some evidence that PI3K/Akt, the cytoprotection pathway of EPO, is involved in the regulation of TLR4 expression. Our previous study have demonstrated that EPO attenuated cardiac fibroblasts proliferation. Based on these findings, we postulate that EPO may act in antagonism of myocardial fibrosis and antiinflammatory through downregulating TLR4 expression in cardiac fibroblasts. To test the hypothesis, cultured cardiac fibroblasts and rat model with abdominal aorta constriction were used to evaluate the effects of EPO on cardiac fibroblasts migration, proliferation, collagen, transforming growth factor-beta1(TGF-beta1) and pro-inflammatory cytokines secretion, we will try to elucidate the role of PI3K/Akt-TLR4- TGF-beta1/inflammatory cytokines signal pathway in EPO attenuating myocardial fibrosis. We expect our study will do some favors for prevention of myocardial remodeling, especially for providing a novel insight into the EPO cardioprotective effect , which would further the development of novel therapeutic strategies in cardiovascular diseases.

心脏成纤维细胞增殖及胶原蛋白分泌是心肌重构的主要机制，干预心脏成纤维细胞生物活性是治疗心肌重构的新靶点。TLR4是与炎症因子表达有关的免疫识别受体，而且拮抗TLR4能够抑制肝、肺、肾等器官纤维化，有研究表明PI3K/Akt信号通路参与了TLR4的表达调控。动物实验表明EPO可抑制心梗后的心肌重构，而且还能在多个组织器官中发挥抗炎作用，但其作用机制目前均不清楚。我们前期研究工作证实EPO能抑制心脏成纤维细胞增殖，因此我们猜想EPO可能通过调控TLR4表达发挥其抗炎与抑制心肌纤维化作用。本研究拟采用细胞培养和动物模型实验观察EPO对心脏成纤维细胞增殖、迁徙及细胞炎症因子分泌的影响；阐明EPO通过PI3K/Akt-TLR4- TGF-β1/炎症因子表达途径抑制心肌纤维化的作用机制。通过本课题研究，有望为心肌重构防治提供新靶点和新思路，为EPO临床应用于心血管疾病防治提供新的理论依据。

心肌纤维化与心肌重构是心脏疾病进展恶化的关键，炎症目前也被认为是参与其进展的重要因素。现在的研究发现：促红细胞生成素（EPO）在多个器官具有保护作用，能够抑制肝、肾等多个器官纤维化，也能在多个器官抑制炎症因子表达。而Toll样受体4（TLR4）作为免疫识别受体既具有促进炎症因子表达作用，又被报道具有促进纤维化作用。因此，本课题通过观察TLR4的表达变化探讨EPO抗炎与抗心肌纤维化的具体机制。通过EPO干预心肌成纤维细胞与EPO基因转染主动脉缩窄大鼠心脏模型实验，本课题组利用ELISA、流式细胞术及荧光共聚焦显微镜等观察心肌成纤维细胞增殖、迁徙、胶原蛋白与炎症因子表达、心肌重构及TLR4表达等指标，利用实时定量RT-PCR、Western blot等分子生物学技术阐明EPO通过TLR4途径抗炎与抗心肌纤维化的分子机制。本课题组还观察了心肌炎患者的炎症因子表达变化及EPO对血管内膜损伤后的炎症因子表达及血管重构的影响作用。结果发现：细胞与动物模型实验结果完全一致，EPO能够抑制心肌成纤维细胞增殖与迁徙，抑制胶原蛋白表达及心肌重构，抑制TLR4及其下游炎症因子TGF-β1, TNF-α, IL-1β, IL-6 、 IL-17A、MMP-2和MMP-9的表达。而且，分子生物学实验证实：EPO抗心肌纤维化的作用与其下调TLR4表达有关，TLR4再表达能逆转EPO的保护作用；而PI3/Akt信号通路又是EPO抑制TLR4表达的分子机制。本研究也发现，心肌炎患者中其炎症因子水平与其心功能情况呈负相关，EPO可通过抗炎机制拮抗内膜损伤后的颈动脉血管重构。总之，通过本课题研究，我们认为EPO对心血管系统具有保护作用，其机制与其兴奋PI3K/Akt信号通路，下调TLR4表达，进而抑制TGF-β1, TNF-α, IL-1β, IL-6 、 IL-17A、MMP-2和MMP-9等炎症因子表达有关。本课题研究成果可为今后临床规范使用EPO等药物提供临床依据，更可从抗炎角度为心肌重构与心血管纤维化等疾病防治提供新思路、新靶点和新方向。