睾酮调控线粒体融合素2基因抑制泡沫细胞形成的实验研究

Accumulation of foam cells in atherosclerotic lesions is the hallmark of early-stage atherosclerosis. The macrophages and vascular smooth muscle cells(VSMC) can become foam cells as a result of a disturbed balance between the uptake of cholesterol from lipoproteins and cholesterol efflux. A number of recent cross-sectional studies support the idea that physiological level of androgens may be protective against atherosclerosis, because men with low testosterone levels contributes to the onset, progression, or both of cardiovascular diseases(CVD) and CVD risk factors such as obesity, type 2 diabetes, and hypertension. However, the role of testosterone and its underlying cellular and molecular mechanisms in foam cells formation are poorly understood. We recently found that, in cultured macrophages and VSMC from male wild-type mice that express the androgen receptor, testosterone inhibited the formation of foam cells induced by ox-LDL. Moreover, the expression of the scavenger receptor CD36 in the cultured VSMC was significantly suppressed, while the expression of the ATP binding cassette(ABC) transporters ABCA1 was significantly increased by testosterone. Mitofusin 2(Mfn2) is an essential transmembrane GTPase embedded in the mitochondrial outer membrane, and also localized on the endoplasmic reticulum, which mediates mitochondrial fusion and tethers the endoplasmic reticulum to mitochondria to form interorganellar bridges. Mfn2 plays a central role in mitochondrial metabolism and may be associated with obesity, early-onset stroke and apoptosis. Furthermore, our recent studies have shown that the expression of Mfn2 was reduced in foam cells harvested from ApoE-/- mice arteries. The over-expression of Mfn2 reduced foam cells formation originating from VSMC induced by ox-LDL, effects which were found to be dependent on the binding of Mfn2 to Ras and the subsequent inhibition of PI3K/Akt and MAPK(ERK1/2, p38MAPK) pathways, with subsequent transcription factor PPARγ activation. Most recently, our studies have demonstrated that Mfn2 expression levels can be up-regulated by testosterone with a slightly dose-dependent manner. The present study will examine the specific effect and underlying celluar and molecular mechanisms of Mfn2 mediates inhibitory effects of testosterone on the foam cells formation in vitro and in vivo. Cultures of macrophages and VSMC in vitro with the up- or down-expression of Mfn2 treated with physiological concentrations of testosterone were employed. The formation of foam cells, the expression of signal transduction pathways related protein(PI3K/Akt、MARKs、PPARγ) and cholesterol transport related protein(CD36、SR-A、ABCA1、ABCG1) will be assessed. Furhermore, we will study the effect of testosterone on high cholesterol diet induced atherosclerosis in castrated male mice. This project with great scientific theory value and clinical application prospect will provide a new potential target for atherosclerosis.

我们前期研究发现：1）模拟生理浓度睾酮可抑制胆固醇在巨噬细胞和血管平滑肌细胞（VSMC）内蓄积，与其下调清道夫受体CD36和上调ATP结合盒转运子ABCA1表达有关，但其分子机制尚不清楚；2）线粒体融合素2（Mfn2）基因在ApoE-/-小鼠动脉粥样硬化（AS）斑块的泡沫细胞内表达显著降低，适度上调Mfn2表达则通过抑制PI3K-Akt和ERK1/2、p38MAPK信号通路激活PPARγ，促进胆固醇外排，减少泡沫细胞的形成；3）睾酮可上调Mfn2表达。由此我们推测，睾酮通过调控Mfn2基因进而影响下游信号通路活性而抑制泡沫细胞形成。本实验利用携带Mfn2基因的重组腺病毒和RNA干扰等技术，分别从细胞和动物水平深入研究Mfn2基因在睾酮抑制泡沫细胞形成中的作用，希望阐明睾酮通过调控Mfn2抑制泡沫细胞形成的主要分子机制。可为防治AS提供新的干预靶点和手段，具有较大的科学理论价值和临床应用前景

泡沫细胞形成是AS早期的标志性事件，研究泡沫细胞形成的调控机制，可为AS防治提供新的干预靶点。流行病学报道，生理浓度的睾酮具有抗AS作用，但其分子机制尚不十分清楚。我们前期发现，线粒体融合素2基因（mitofusin2，Mfn2）是PI3K/Akt及MAPKs信号通路中的重要调控因子，可抑制VSMC转化为泡沫细胞；睾酮可抑制泡沫细胞形成。本课题建立ox-LDL诱导的平滑肌细胞和巨噬细胞源性泡沫细胞模型以及去势大鼠模型，化学合成mfn2-siRNA，并利用免疫印迹法、免疫组化等方法研究：1）生理剂量睾酮对去势大鼠主动脉VSMC线粒体损伤的保护作用；2）睾酮对ox-LDL诱导大鼠VSMC表型转化和增殖的抑制作用；3）睾酮对ox-LDL诱导大鼠VSMC转化为泡沫细胞的抑制作用；4）mfn2在睾酮抑制平滑肌源性泡沫细胞形成过程中的作用；5）睾酮对ox-LDL诱导小鼠巨噬细胞转化为泡沫细胞的抑制作用。目前取得下列研究结果：1）生理剂量睾酮可维持去势大鼠VSMC的线粒体形态和膜电位水平，增加细胞ATP含量，降低氧化应激水平，对去势导致的VSMC线粒体损伤具有保护作用。睾酮上调Mfn2表达是其可能机制；2）睾酮可抑制ox-LDL 诱导的大鼠VSMC由收缩型向合成型转化及细胞增殖，可能与其上调Mfn2抑制ERK1/2信号通路有关；3）睾酮以雄激素受体依赖的方式，抑制ox-LDL诱导的大鼠VSMC泡沫化过程，其作用与睾酮上调Mfn2表达进而调节CD36和ABCA1表达有关；4）mfn2是睾酮发挥抑制大鼠VSMC泡沫化作用的一个靶基因。睾酮通过调节mfn2及其下游CD36和ABCA1表达，抑制ox-LDL诱导下VSMC转变为泡沫细胞。5）睾酮可抑制ox-LDL诱导的巨噬细胞源性泡沫细胞形成，其作用可能与睾酮上调Mfn2表达进而调节CD36和ABCA1表达有关。通过本课题研究，进一步确定mfn2基因是泡沫细胞形成过程中的重要调控点，同时初步阐述了睾酮抗AS的若干机制。睾酮作为内源性生物活性物质可上调mfn2基因表达，在泡沫细胞形成前期，睾酮对VSMC线粒体损伤具有保护作用，对VSMC表型转化和细胞增殖具有抑制作用；在泡沫细胞形成过程中，睾酮通过mfn2调节CD36和ABCA1表达，直接抑制泡沫细胞形成。