基于TLR4通路探讨肥大心肌细胞来源外泌体经Hsp90调节心肌炎症反应的机制

Cardiac hypertrophy is a complication of several cardiovascular diseases, which seriously threaten the health of people. Our group has confirmed important role of exosome-mediated inflammatory response in the occurrence and development of cardiac hypertrophy, but the underlying mechanisms remain poorly understood. Recently, emerging data have indicated that Heat shock protein 90 (Hsp90) derived from various cell types is divided into two categories: exosomal Hsp90 and exosome surface Hsp90. In a preliminary experiment we further found that the expression of Hsp90 was significantly enhanced in hypertrophic cardiac myocyte-derived exosomes. Many studies have also shown that Hsp90 regulated Toll-like receptors 4 (TLR4) signaling pathway, whereas the latter is closely related to cardiac inflammatory response. Therefore, we propose a new mechanism of regulation of cardiac inflammatory response. We suppose that hypertrophic cardiac myocyte-derived exosomes transport Hsp90, which was capable of activating TLR4 signaling pathway in macrophages and contribute to inflammatory response in a TLR4 mediated manner. The aim of this study is to detect the expression and distribution of Hsp90 in exosomes secreted from hypertrophic cardiac myocytes using transmission electron microscope, flow cytometry, western blot and real-time PCR methods. Also, the effect of Hsp90 presented in exosomes on cardiac inflammation and its possible mechanism will be investigated at whole body, organ and cellular levels. This work has the potential to bring new ideas for the successful prevention and treatment of cardiac hypertrophy.

心肌肥大是多种心血管疾病并发症，严重危害人类健康。本课题组已证实，外泌体介导的炎症反应在心肌肥大发生发展中作用关键，但机制不详。资料表明，多种细胞来源Hsp90分布于外泌体内及其膜表面上。在预实验中，我们也发现：肥大心肌细胞来源外泌体富含Hsp90。众多研究还表明，Hsp90可调控TLR4信号通路，而后者与心肌炎症反应关系密切。因此，我们推测“肥大心肌细胞来源外泌体经携带的Hsp9参与调节TLR4信号通路，进而影响心肌炎症反应”是肥大心肌炎症反应调节的新机制。本项目拟应用透射电子显微镜、流式细胞仪、Western blot、Real-time PCR等技术，分析肥大心肌细胞来源外泌体携带的Hsp90的表达、分布特征，进一步从整体-器官-细胞层次探讨肥大心肌细胞来源外泌体携带的Hsp90对TLR4介导的心肌炎症反应的影响及分子机制。此研究将为心肌肥大的防治提供新思路。

背景：心肌肥大是多种心血管疾病并发症，严重危害人类健康。外泌体介导的炎症反应在心肌肥大发生发展中起重要作用，但机制不详。.研究内容：本项目应用采用透射电子显微镜、流式细胞仪技术、real-time PCR、Western blot等方法分析肥大心肌细胞来源外泌体的数量、分布特征；并采用基因测序、ELISA、real-time PCR等技术分析肥大心肌细胞来源外泌体对心肌炎症反应的影响，进一步从整体-器官-细胞层次探讨肥大心肌细胞来源外泌体调节心肌炎症反应的分子机制。.主要结果：（1）肥大心肌细胞来源外泌体的数量、分布特征：Western blot结果发现，提取的外泌体可检测到标志蛋白CD63和CD81的表达，提示成功分离获得外泌体。电镜结果显示，与正常心肌细胞相比，肥大心肌细胞来源外泌体数量明显增加。AChE活性分析进一步证实AngII促进肥大心肌细胞分泌外泌体。与正常心肌细胞相比，肥大心肌细胞来源外泌体中Hsp90的表达水平显著上调。（2）肥大心肌细胞来源外泌体对心肌炎症反应的影响：与正常心肌细胞来源外泌体比较，肥大心肌细胞来源外泌体促进RAW264.7细胞分泌IL-6和IL-8，提示肥大心肌细胞来源外泌体介导了巨噬细胞的免疫炎症反应。（3）肥大心肌细胞来源外泌体调节心肌炎症反应的分子机制：敲除AGO2表达能显著抑制外泌体介导的RAW264.7细胞炎症反应，提示外泌体可能通过转运非编码RNA参与RAW264.7细胞分泌IL-6和IL-8。采用miRNA高通量测序技术分析外泌体中miRNAs的表达。与正常心肌细胞来源外泌体相比，肥大心肌细胞来源外泌体中7个miRNAs差异表达，其中，4个miRNAs表达上调，3个miRNAs表达下调。差异表达miRNA靶基因预测，获得5477个靶基因。KEGG通路分析发现，MAPK信号通路和PI3K-Akt信号通路等参与肥大心肌炎症反应。进一步研究发现，外泌体通过转运miR-155调节MAPK信号通路调节肥大心肌炎症反应。同时，外泌体中Hsp90经TLR4信号通路也参与调节了心肌炎症。.关键数据及科学意义：肥大心机细胞来源外泌体通过携带转运miR-155调节MAPK信号通路，同时携带转运Hsp90调节TLR4信号通路，参与肥大心肌炎症反应的调节。此研究将为心肌肥大的防治提供新思路。