从SDF-1/CXCR4信号通路调节心脏干细胞自噬研究鹿红方修复受损心肌的机制

Cardiac stem cells possess great potential on cardiac repair. The source and function of cardiac stem cells are associated with kidney essence in Traditional Chinese Medicine theories. Our previous study has proven that Luhong formula with the major action of supplementing the kidney, replenishing essence and quickening the blood promoted cardiac homing of cardiac stem cells and enhanced cell survival in infarcted myocardium via SDF-1/CXCR4 signaling pathway. It is newly reported that the autophagy can be induced by SDF-1/CXCR4 signaling pathway. Hence we hypothesized that Luhong formula can enhance cardiac stem cell survival under oxidative stress through autophagy induced by SDF-1/CXCR4 signaling pathway. Here, we plan to test our hypothesis in an oxidative stress model for cardiac stem cells and a murine acute myocardial infarction model. The aim of this protocol is to unveil the mechanism of Luhong formula’s cell protecting efficacy, to detect the target on which Luhong formula improves the survival and performance of cardiac stem cells in infarcted myocardium，to provide evidence and strategy for using Chinese medicine with the effect of supplementing the kidney, replenishing essence and quickening the blood as an adjuvant therapy for cardiac repair.

心脏干细胞移植修复损伤心肌具有广阔的应用前景。干细胞来源和功能与中医藏象理论中肾精有较多共性。前期我们证实具有补肾填精活血功效的鹿红方可通过调控SDF-1/CXCR4信号通路促进心脏干细胞向受损心肌归巢，并提高心脏干细胞在心梗区域的抗凋亡能力。结合“SDF-1/CXCR4信号通路可诱导细胞自噬”的新认识，我们推测鹿红方可通过SDF-1/CXCR4信号通路诱导心脏干细胞自噬，增强心脏干细胞在氧化应激下的抗凋亡能力，从而改善其在心肌损伤区域的存活和修复损伤心肌的能力。拟制备心脏干细胞氧化应激模型和小鼠心梗模型，从整体和细胞水平研究鹿红方通过SDF-1/CXCR4信号通路诱导心脏干细胞自噬，阐释鹿红方提高氧化应激下心脏干细胞抗凋亡能力的机制，明确鹿红方改善心脏干细胞修复受损心肌的能效，为补肾填精活血中药辅助细胞疗法修复受损心肌提供依据和思路。

心脏干细胞移植修复损伤心肌具有广阔的应用前景。干细胞来源和功能与中医藏象理论中肾精有较多共性。前期我们证实具有补肾填精活血功效的鹿红方可通过调控SDF-1/CXCR4信号通路促进心脏干细胞向受损心肌归巢，并提高心脏干细胞在心梗区域的抗凋亡能力。结合“SDF-1/CXCR4信号通路可诱导细胞自噬”的新认识，我们推测鹿红方可通过SDF-1/CXCR4信号通路诱导心脏干细胞自噬，增强心脏干细胞在氧化应激下的抗凋亡能力，从而改善其在心肌损伤区域的存活和修复损伤心肌的能力。在本项目中，我们通过制备心脏干细胞氧化应激模型和小鼠心梗模型，从整体和细胞水平研究鹿红方通过SDF-1/CXCR4信号通路诱导心脏干细胞自噬，并提高心脏干细胞在缺血缺氧环境的抗凋亡能力。流式细胞分析结果显示，鹿红方组细胞凋亡比例最低；Western blot检测结果显示，鹿红方显著降低凋亡蛋白Bax的表达，促进保护性Bcl-2的表达；且上述效应能被自噬抑制剂3-MA及SDF-1/CXCR4信号通路拮抗剂AMD3100阻断；机制研究显示，鹿红方能显著促进心脏干细胞SDF-1/CXCR4的表达，并因此介导自噬关键蛋白beclin-1和LC3-II的分泌，从而控制适当自噬。动物模型的数据显示，鹿红方干预的心脏干细胞移植能显著改善心梗模型小鼠的LVEF及FS，但对LVDs及LVDd的改变没有统计学意义。TTC染色结果证实鹿红方干预的心脏干细胞可显著减少梗死面积。TUNEL染色显示，鹿红方干预的心脏干细胞移植使凋亡细胞显著减少。通过上述结果，我们证明了鹿红方预处理能增强心脏干细胞在缺血缺氧环境下的抗凋亡能力，我们阐释了鹿红方抗凋亡能力的机制，即通过促进SDF-1/CXCR4信号通路的表达，从而调控自噬。同时本项目明确了鹿红方改善心脏干细胞修复受损心肌的能效，为补肾填精活血中药辅助细胞疗法修复受损心肌提供了依据和思路。