Perlecan蛋白在子痫前期小鼠肾脏中的变化及作用机制研究

The Study of Protein Perlecan′s Changes and its Mechanism in the Kidney of Preeclampsia Mouse

目的:探讨Perlecan蛋白在子痫前期(PE)小鼠肾脏中的变化及其作用机制。方法:构建正常孕鼠、PE样小鼠模型(动物水平)及HSPG2特异性肾敲低小鼠模型并分离肾小球内皮细胞(细胞水平),对比观察小鼠肾脏基底膜负电荷改变、Perlecan蛋白及其降解酶Hpa含量变化,同时检测PI3K/AKT/mTOR信号通路相关蛋白以及自噬蛋白标志物的含量。结果:(1)成功构建了PE小鼠和HSPG2条件肾敲低小鼠模型。(2)相对于正常孕鼠,PE小鼠胶体铁染色显示负电荷基本消失,肾小球电荷屏障被破坏,蛋白免疫印迹结果显示PE小鼠肾小球中Perlecan蛋白表达(0.255±0.028)较正常孕鼠(0.509±0.022)显著降低;而Hpa表达(0.519±0.041)较正常孕鼠(0.260±0.063)显著升高(P均<0.05)。(3)与正常孕鼠相比,PE小鼠PI3K/AKT/mTOR信号通路相关蛋白PI3K(0.146±0.018 vs 0.448±0.048)、p-AKT(0.031±0.008 vs 0.286±0.030)和p-mTOR(0.179±0.029 vs0.364±0.025)表达显著下调,自噬相关标志物LC3-I/LC3-Ⅱ(0.546±0.060 vs 0.244±0.060)和Beclin-1(0.527±0.029 vs 0.191±0.044)表达显著升高,差异均有统计学意义(P均<0.05);PI3K/AKT/mTOR通路药理激活剂740 Y-P和MHY1485可以有效地弥补Perlecan敲低引起的自噬增加。结论:PE小鼠Perlecan蛋白下降除外引起肾小球电荷屏障损伤,还可能通过抑制PI3K/AKT/mTOR信号通路来促进细胞自噬引起肾脏血管内皮细胞损伤。

Objective:To evaluate the change of Perlecan protein in kidney of preeclampsia mice and its mechanism.Methods:Normal pregnant mouse,preeclampsia mouse model(animal level),HSPG2 conditional kidney knockout mouse model were constructed.The glomerular endothelial cells(cell level)were isolated.The changes of negative charge of basement membrane in mice kidneys,the content of perlecan protein and its degradation enzyme Hpa were observed.The contents of PI3K/AKT/mTOR signaling pathway related proteins and autophagy protein markers were detected.Results:(1)The preeclampsia mouse model and HSPG2 conditional kidney knockout mouse model were constructed successfully.(2)Compared with normal pregnant mice,colloidal iron staining showed that the negative charge disappeared and the glomerular charge barrier was destroyed in PEmouse model.Western blotting showed that the expression of perlecan protein in glomeruli of PE mouse model(0.255±0.028)was higher than that of normal pregnant mice(0.509±0.022).The expression of Hpa(0.519±0.041)was higher than that of normal pregnant mice(0.260±0.063)(P<0.05).(3)Compared with normal pregnant mouse,the expression of PI3K/AKT/mTOR signaling pathway related proteins in PE mouse model including PI3K(0.146±0.018 vs 0.448±0.048)、p-AKT(0.031±0.008 vs 0.286±0.030)and p-mTOR(0.179±0.029 vs 0.364±0.025)were significantly decreased whereas the autophagy biomarkers including LC3-Ⅰ/LC3-Ⅱ(0.546±0.060 vs 0.244±0.060)and Beclin-1(0.527±0.029 vs 0.191±0.044)increased(P<0.05).The pharmacological activators of PI3K/AKT/mTOR pathway including 740Y-P and MHY1485 can effectively compensate for the increased autophagy induced by Perlecan knockdown.Conclusions:The decrease of perlecan protein in PE mice not only causes the damage of glomerular charge barrier,but also promotes autophagy by inhibiting PI3K/AKT/mTOR signaling pathway.