高磷下调SIRT1诱导内皮细胞早衰在尿毒症加速性动脉粥样硬化中作用的研究

The mechanism of uremic accelerated atherosclerosis(AS) is not clear.Our previous study found uremia could induce senescence of aortic endothelial cells,and it is well known that endothelial dysfunction caused by senescence is initial for AS,while downregulation of the anti-aging gene SIRT1 may be related to that.Our previous study also found the hyperphosphatemia accompanied by uremia might induce the elevating expression of phosphate transporters(PiT-1/2) on the plasm membrane of endothelial cells and further phosphate influx,in which the decreasing expression of Klotho involved. Phosphate influx may induce further phosphorylation modification of specified sites of SIRT1 transcription activating pathway ,and eventually downregulation of SIRT1 expression.So we speculate that downregulation of SIRT1 expression by high-phosphate is the leading cause of endothelial senescence and further accelerated AS.In this research,we plan to establish the high phosphate culture system of rat aortic endothelial cell and the uremic hyperphosphatemic accelerated AS model of ApoE knocked-out mice,and detect the senescent probes and factors of endothelial cells,the expression of PiT-1/2,phosphate influx,and the phosphorylation modification level of specified sites of SIRT1 transcription activating pathway and their expression.And observe the influence on endothelial senescence and AS by RNAi,adenovirus transfection and Klotho intervention to the phosphate transporters and SIRT1 transcription activating pathway.We hope to explore the mechanism of endothelial senescence induced by high-phosphate,and find a new way to prevent uremic accelerated AS.

尿毒症加速性动脉粥样硬化（AS）的发生机制尚不清楚。前期研究发现尿毒症可诱导血管内皮细胞早衰，早衰导致的内皮功能紊乱则是AS的启动因素，而SIRT1的下调可能与内皮细胞早衰有关。尿毒症并发的高磷血症可诱导内皮细胞磷转运蛋白（PiT-1/2）表达上调及磷内流增加，Klotho表达降低参与其中。磷内流增加可能引起SIRT1转录激活通路相关位点磷酸化修饰进而导致其表达下调。因此推测高磷下调SIRT1表达可能是导致内皮细胞早衰及加速性AS的原因。本研究拟建立大鼠主动脉内皮细胞高磷培养体系以及ApoE-/-尿毒症高磷血症加速性AS小鼠模型，检测内皮细胞衰老指标、PiT-1/2表达及磷内流以及SIRT1转录激活通路表达及相关位点磷酸化水平，并以RNAi、腺病毒转染、Klotho干预等方式作用于PiT-1/2及SIRT1转录激活通路，观察其对内皮细胞早衰及AS的影响，为尿毒症加速性AS的防治提供新的思路

尿毒症加速性动脉粥样硬化（AS）的发生机制尚不清楚。前期研究发现尿毒症可诱导血管内皮细胞早衰，早衰导致的内皮功能紊乱则是AS的启动因素，而SIRT1的下调可能与内皮细胞早衰有关。尿毒症并发的高磷血症可诱导内皮细胞磷转运蛋白（PiT-1/2）表达上调及磷内流增加，Klotho表达降低参与其中。磷内流增加可能引起SIRT1转录激活通路相关位点磷酸化修饰进而导致其表达下调。因此推测高磷下调SIRT1表达可能是导致内皮细胞早衰及加速性AS的原因。本研究建立了大鼠主动脉内皮细胞高磷培养体系，研究发现高磷作用下PiT-1转录水平明显升高（P<0.01）而PiT-2转录水平轻度升高(P<0.05)，PiT-1表达明显升高(P<0.05)，PiT-2表达无明显变化。31P-NMR检测结果提示细胞内磷水平明显升高；高磷作用下与对照组相比，PGC-1α、FoxO1、SIRT1转录水平均下降，其中SIRT1从1h开始显著下降，FoxO1下降差异不显著，PGC-1α4h下降显著，p<0.05。PGC-1α、FoxO1、SIRT1表达变化不显著，而p- SIRT1、p-PGC-1α表达显著降低，其中p- SIRT1降低最为明显，而p- FoxO1表达无显著变化。Klotho干预对高磷环境下内皮细胞的结果显示磷内流显著降低，而PiT-1表达无显著变化。Klotho干预可显著上调PGC-1α、SIRT1转录，而对FoxO1转录无明显影响，但可显著上调SIRT1、PGC-1α、FoxO1的表达。综上所述，高磷诱导PiT-1表达上调，促进其向细胞内流，磷内流的增加可通过抑制SIRT1通路促进内皮细胞早衰而Klotho干预可显著抑制磷内流，从而诱导SIRT1通路激活，从而抑制内皮细胞早衰，最终抑制了加速性AS的发生。本研究证实了高磷下调SIRT1表达可能是导致内皮细胞早衰及加速性AS的原因，因此为尿毒症动脉粥样硬化的治疗提供了新的思路。