重组高密度脂蛋白激活PI3K/Akt信号通路调控心肌自噬保护心功能的分子机制研究

HDL is an independent protective factor of atherosclerotic disease. HDL connected sphingosine-1-phosphate (HDL-S1P) is documented to have amazing cardiovascular protection. HDL-S1P is reported to intervene in cardiomyocyte survival. Autophagy is one pathway of cell death. PI3K/Akt signalling pathway had been confirmed playing an important role in autophagy. During the process of happening and developing of load-induced heart failure, cardiomyocyte autophagy promotes myocardial hypertrophy transforming to myocardial dysfunction, in which cardiomyocyte loss induced by autophagy is key point. There are no related report about the correlation among S1P, autophagy and heart failure. Based on our new findings that reconstituted HDL (rHDL) with S1P can protect heart function in load-induced heart failure, we hypothesis that rHDL protect heart function possibly via PI3K/Akt signaling pathway suppressing excessive cardiomyocyte autophagy activated by load-induced heart failure. In the present study, cultured neonatal rat cardiomyocytes and adult mice are performed to prepared in vitro and in vivo, and were interfered with rHDL. RT-PCR and Western blot were performed to detect autophagy related genes and protein, p-Akt and p-mTOR. Furthermore, S1P receptors will be identified by S1P receptor siRNA. All of these will help us try to demonstrate the mechanism that rHDL via PI3K/Akt signaling pathway suppressing autophagy to protect heart function. Our work will discover a new mechanism about the cardiovascular protection of HDL and provide new possible method for improving load-induced heart failure in clinic.

HDL是独立的心血管保护因素，与HDL结合的S1P调节了心肌细胞的存活。自噬是细胞死亡的方式之一，PI3K/Akt信号通路是调控自噬的经典途径。随着压力负荷持续，心肌细胞过度自噬导致细胞丢失，心功能进一步恶化。目前尚未见有关S1P、心肌细胞自噬及心功能之间的研究报道。结合我们前期研究发现：包含S1P成分的重组HDL（rHDL）能够保护压力负荷HF的心功能，我们提出假设：rHDL可能通过PI3K/Akt信号通路调控心肌细胞自噬保护压力负荷HF的心功能。本研究通过体内及体外实验，观察rHDL对压力负荷HF心肌细胞自噬和心功能的影响；结合信号通路特异性抑制剂，及siRNA干扰技术，运用RT-PCR、Western blot等方法检测自噬相关蛋白和信号通路相关蛋白的表达，并确定参与其中的S1P受体亚型。从而深入探讨rHDL保护压力负荷HF心功能的分子机制，为心力衰竭的治疗提供新视角。

目的：探讨了1-磷酸鞘氨醇（S1P）及其受体在抑制心肌细胞自噬、改善心肌细胞肥大、保护心脏功能的分子机制。.内容：（1）探讨S1P移植心肌细胞自噬的机制，建立心肌细胞自噬模型，通过测定心肌细胞内自噬小体数量、自噬相关蛋白、重要的自噬功能基因表达及Akt、mTOR磷酸化的水平，验证S1P抑制心肌自噬的机制。（2）明确介导S1P抑制心肌自噬的S1PR类型，应用siRNA技术分别敲低三种S1P受体（S1PR）的表达，用Western blot 方法检测自噬相关蛋白LC3-II/I及P62的表达，并检测Akt、mTOR磷酸化的水平。（3）通过建立S1PR1敲低的小鼠心脏模型，检测心功能指标、自噬指标及PI3K/Akt 信号通路中的相关蛋白变化，验证S1P是通过S1PR1受体介导的PI3K信号通路在心肌自噬和心功能保护中发挥作用。.结果：（1）S1P干预后饥饿诱导的心肌细胞自噬及PE诱导的心肌细胞肥大明显减弱，表现为心肌细胞内自噬小体形成减少、自噬相关蛋白LC3Ⅱ/Ⅰ表达减少，心肌细胞相对面积减小。（2）体外S1PR1敲低后心肌细胞自噬及肥大程度较对照组、S1PR2及S1PR3敲低组明显加重，明确S1PR1是介导S1P抑制心肌自噬的受体类型，并通过PI3K信号通路调控自噬相关基因和蛋白。（3）体内敲除心肌细胞S1PR1后，压力负荷诱导的心肌自噬、心肌肥厚及心脏功能降低均较对照组明显加重，提示S1P是通过抑制心肌自噬保护心功能。.科学意义：首次提出探讨了S1P是通过PI3K/Akt 信号通路调控自噬相关基因和蛋白的表达，从而抑制压力负荷所致心肌细胞自噬，保护心功能；利用siRNA技术明确了S1P1型受体S1PR1介导了S1P对心肌细胞自噬的抑制作用；应用生物工程方法体内敲低S1PR1后，进一步证实心肌自噬增加、心肌肥厚及心功能减低加重。为深入了解HDL-S1P抑制压力负荷心力衰竭诱导的心肌细胞自噬的分子机制提供理论依据，为临床上压力负荷心力衰竭心功能的保护治疗提供新的视角。

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