miR375/ERα正反馈环对RASD1的调控在脑缺血再灌注损伤的作用及毛蕊异黄酮的干预机制

The mechanisms underlying cerebral ischemia/reperfusion injury are rather complicated, such as energy metabolism dysfunction of brain tissues following cerebral ischemic injury, platelet activation, massive free radicals producing, etc. Over the past few years，programmed cell death on the damage of cerebral tissues after ischemia has becomes a focus. Some studies found the protective effect of estrogen on focal cerebral ischemia/reperfusion injury by the estrogen receptor α (ERα). Furthermore, some studies identified miR-375 as the first miRNA with the capacity to enhance ERα signaling in ERα(+) cells. Their findings define a forward feedback pathway in control of ERα expression and miRNA target prediction identified RASD1 as a potential miR-375 target. Our previous studies suggested that low concentrations of calycosin(<16 μM) had stimulatory effects on the proliferation of ERα-positive cells in vitro and in vivo, and found that the stimulatory effects might relate to the level of ERα and the activation of extracellular signal-regulated kinase1/2(ERK1/2) MAPK. On the other hand, our finding illustrated calycosin had shown neuroprotective effects in cerebral ischemia /reperfusion rats, and the molecular mechanisms may correlate with the positive feedback between ER-α and miR-375. Furthermore, our previous studies confirmed that low concentrations of calycosin upregulated the level of miR-375 and downregulated the level of RASD1 protein in neurons. Based on the above information, we hypothesize that the positive feedback loop of miRNA-375/ERα and RASD1 may also be involved in the proliferative effect induced by calycosin in ERα-positive cells. By using cell counting, MTT assay, flow cytometry, luciferase activity assay, focal cerebral ischemia-reperfusion model, Western blot, qPCR,and immunohistochemistry, we will identify an upregulated or downregulated miRNA-375 in ERα-positive neurons that could interfere with ERα-MAPK, Akt signaling pathways through the regulation of its target, RASD1, in vitro and in vivo. Furthermore, we focus on the effect of calycosin on the positive feedback loop of miRNA-375/ERα and level of RASD1. Accordingly, further research should be carried out to elucidate the relationship among calycosin, RASD1, and the positive feedback loop of miRNA-375/ERα, which may provide experimental foundation for calycosin's future clinical use for focal cerebral ischemia/reperfusion injury.

脑缺血再灌注损伤的机制相当复杂,通过何种途径保护缺血性损伤神经元是研究热点，而雌激素对神经元的保护作用是通过ERα实现的。研究提出miR-375 与ERα 构成正反馈环路，下调RASD1 水平，促进ERα阳性细胞增殖。课题组研究表明毛蕊异黄酮通过ERK1/2 MAPK通路抑制细胞凋亡，并对缺血再灌注损伤有明显的保护作用，而这与其改变miR-375 、ERα水平有关。预实验显示毛蕊异黄酮可上调神经元细胞miRNA-375水平，下调RASD1 蛋白表达。据此，我们推测毛蕊异黄酮通过干预miR-375/ERα的正反馈环路，影响RASD1 表达来发挥对脑缺血再灌注损伤的保护作用。本项目拟通过细胞生物学、分子生物学、基因敲除动物模型等技术，观察miR-375、ERα 、RASD1 在体内外的信号级联，论证毛蕊异黄酮对正反馈环路的作用及下游信号通路的影响，为植物雌激素防治脑缺血再灌注损伤提供新思路。

脑缺血再灌注损伤的机制相当复杂,通过何种途径保护缺血性损伤神经元是研究热点，而雌激素对神经元的保护作用是通过ERα 实现的。研究提出miR-375与ERα构成正反馈环路，下调RASD1水平，促进ERα阳性细胞增殖。课题组研究表明毛蕊异黄酮通过ERK1/2 MAPK通路抑制细胞凋亡，并对缺血再灌注损伤有明显的保护作用，而这与其改变miR-375、ERα水平有关。本项目通过细胞生物学、分子生物学、基因敲除动物模型等技术，观察miR-375、ERα、RASD1在体内外的信号级联，论证毛蕊异黄酮对正反馈环路的作用及下游信号通路的影响，为植物雌激素防治脑缺血再灌注损伤提供新思路。本课题实验结果显示毛蕊异黄酮通过上调miR-375/ERα的正反馈环路，下调RASD1表达，影响MAPK、PI3K/Akt等信号通路蛋白磷酸化水平，从而发挥对脑缺血再灌注损伤的保护作用，而在体内实验中药物也能对MCAO模型发挥神经保护作用，其机制与体外实验相同。另一方面，低浓度异黄酮类植物雌激素可促进ERα阳性细胞CNE2细胞增殖，减少其细胞凋亡，其药物的作用机制与ERα-miR375-PTEN -ERK1/2-bcl-2信号通路有关。此外，课题组发现植物雌激素肉桂酸能够抑制CNE2细胞增殖，诱导CNE2细胞凋亡，通过增加CNE2细胞中KLF6、Fas-L、Bax、P53和caspase-3蛋白表达，降低Bcl-2和cyclin D1蛋白表达。本项目的实验数据可为众多的异黄酮类植物雌激素治疗神经性疾病和抗肿瘤提供新的思路，以及为不同药物寻找各自特异性的作用靶点。