DLEU1通过调控p38/JNK信号通路促进高血压大鼠血管平滑肌细胞增殖和血管重塑的实验研究

DLEU1 Promotes Vascular Smooth Muscle Cell Proliferation and Vascular Remodeling in Hypertensive Rats by Regulating p38/JNK Signaling Pathway

目的:探讨淋巴细胞白血病缺失基因1(DLEU1)促进高血压大鼠血管平滑肌细胞(VSMCs)增殖和血管重塑的机制及相关信号通路。方法:将12只DLEU1转基因大鼠(DTRs)随机分为DTRs组、p38组、JNK组,每组4只,分别经腹腔注射二甲基亚砜溶液、p38丝裂原活化蛋白激酶(MAPK)特异性抑制剂SB203580、JNK抑制剂SP600125,并以Wistar大鼠(4只)为对照组,采用苏木精-伊红(HE)染色确定主动脉、心脏和肾脏的形态结构。通过转染DLEU1过表达质粒,构建DLEU1过表达的VSMCs,同时给予SB203580、SP600125干预,通过溴脱氧核苷尿嘧啶(BrdU)实验、细胞周期及迁移实验比较不同干预细胞的生物活性的差异。结果:DTR组大鼠血管腔面积显著减小、中膜增厚,肾组织中可见小动脉增生,血管壁和肾小球增厚,p38及JNK抑制剂可减轻DLEU1介导的血管中膜增厚的效应,增加中膜/管腔面积比,肾脏小动脉的病理改变也有所改善。实时荧光定量聚合酶链式反应(RT-PCR)检测显示,转染过表达DLEU1质粒后,VSMCs中DLEU1的相对表达量显著提高(P<0.05),表明细胞模型构建成功。蛋白免疫印迹法(Western Blot)检测发现,磷酸化p38和JNK蛋白的表达上调。DLEU1过表达导致细胞增殖率显著增加。经p38和JNK抑制剂处理后,细胞增殖率均有所下降(P<0.05)。p38和JNK抑制剂可显著阻断细胞周期,处理后,G0/G1期细胞比例均有所上升。过表达DLEU1可显著提高细胞中基质金属蛋白酶(MMP)-2和MMP-9的蛋白表达水平,而阻断p38和JNK可部分消除DLEU1对MMP-2和MMP-9蛋白表达的促进作用。Western Blot检测显示,DLEU1过表达细胞骨桥蛋白(OPN)蛋白表达水平明显上调,平滑肌22α(SM-22α)蛋白表达显著下调,经p38抑制剂和JNK抑制剂干预后,OPN蛋白表达水平明显降低,SM-22α蛋白表达水平上调。结论:高血压时DLEU1介导的VSMCs增殖和表型改变需要p38/JNK通路介导。

Objective:To investigate the mechanism and related signal pathways of lymphocytic leukemia 1(DLEU1)for promoting the vascular smooth muscle cells(VSMCs)proliferation and vascular remodeling in hypertensive rats.Methods:Twelve DLEU1 transgenic rats(DTRs)were randomly divided into DTRs group,p38 group,and JNK group,with 4 rats in each group.The rats in three groups were injected intraperitoneally with dimethyl sulfoxide solution,p38 mitogen-activated protein kinase(MAPK)specific inhibitor SB203580,and JNK inhibitor SP600125,snccessively.Wistar rats(n=4)were used as blank control group.The morphological structures of aorta,heart,and kidney were determined by Hematoxylin-eosin(HE)staining.DLEU1 overexpression plasmid was transfected to construct DLEU1 overexpression VSMCs.At the same time,SB203580 and sp600125 were given to intervene,and the biological activities of different intervention cells were compared by bromodeoxyuridine(BrdU)assay,cell cycle assay,and migration assay.Results:In DTR group,the area of vascular lumen decreased significantly,the middle membrane thickened,the proliferation of arterioles,the thickening of vascular wall and glomerulus were observed in renal tissue.p38 and JNK inhibitors could reduce the effect of DLEU1 mediated vascular middle membrane thickening,increase the ratio of middle membrane/lumen area,and improve the pathological changes of renal arterioles.Reverse transcription-polymerase chain reaction(RT-PCR)detection showed that the relative expression of DLEU1 in VSMCs significantly increased after transfection of DLEU1 plasmid(P<0.05),indicating that the cell model was successfully constructed.Western Blot showed that the expression of phosphorylated p38 and JNK protein up-regulated.Overexpression of DLEU1 resulted in a significant increase in cell proliferation.After treatment with p38 and JNK inhibitors,the cell proliferation rate decreased(P<0.05).p38 and JNK inhibitors could significantly block the cell cycle.After treatment,the proportion of G0/G1 cells increased.Overexpression of DLEU1 could significantly increase the protein expression of matrix metalloproteinase(MMP)-2 and MMP-9,while blocking p38 and JNK could partially eliminate the promoting effect of DLEU1 on the protein expression of MMP-2 and MMP-9.Western Blot analysis showed that the expression level of osteopontin(OPN)protein in DLEU1 overexpression cells significantly up-regulated,SM-22αprotein expression significantly down regulated.Conclusion:The proliferation and phenotypic changes of VSMCs mediated by DLEU1 in hypertension need to be mediated by p38/JNK pathway.