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GRB2基因沉默介导PI3K/AKT/NF-κB信号通路对动脉粥样硬化大鼠脂质沉积和炎性浸润的影响
作者:申童1  喻青松2  董飒英1 
单位:1. 乐普(北京)医疗器械股份有限公司 研发部, 北京 102200;
2. 北京化工大学 生命学科学与技术学院, 北京 100029
关键词:动脉粥样硬化 生长因子受体结合蛋白2 磷脂酰肌醇3-激酶/蛋白激酶B/核因子κB信号通路 脂质沉积 炎性浸润 
分类号:R543.5
出版年·卷·期(页码):2019·38·第五期(792-799)
摘要:

目的:探讨生长因子受体结合蛋白2(GRB2)基因沉默介导磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/核因子κB(NF-κB)信号通路对动脉粥样硬化(AS)大鼠脂质沉积和炎性浸润的调控机制。方法:生理盐水组作为对照,构建AS大鼠模型并且将模型大鼠分为如下4组:阴性对照组(转染GRB2 shRNA阴性对照序列)、GRB2 shRNA组(转染GRB2 shRNA序列)、通路抑制剂组(PI3K/AKT通路抑制剂处理)、联合组(GRB2 shRNA序列联合PI3K/AKT通路抑制剂共同处理)。定量即时聚合酶链反应(qRT-PCR)以及蛋白质印迹法检测各组大鼠主动脉PI3KR1、AKT2、NF-κB p65 mRNA和蛋白的表达。HE染色观察各组大鼠主动脉组织形态。油红O染色对各组大鼠动脉脂质沉积进行定量。酶联免疫吸附法(ELISA)测定各组大鼠外周血肿瘤坏死因子-α(TNF-α)、C-反应蛋白(CRP)、脂蛋白相关磷脂酶A2(Lp-PLA2)水平。结果:qRT-PCR、蛋白质印迹法实验结果显示:与生理盐水组相比,PI3K/AKT信号通路在AS大鼠中显著活化(P<0.05);与阴性对照组相比,大鼠经GRB2沉默或PI3K/AKT通路抑制剂处理后,PI3KR1、NF-κB p65蛋白及mRNA,AKT mRNA和p-AKT表达显著下调(均P<0.05)。HE染色结果显示:阴性对照组动脉组织中呈现AS病理表现,沉默GRB2或抑制PI3K/AKT信号通路活性后病理状况改善,联合组改善更明显。油红O染色显示:与阴性对照组相比,GRB2 shRNA组、通路抑制剂组及联合组脂质沉积面积显著减少(均P<0.05)。ELISA结果显示:与阴性对照组相比,单独沉默GRB2、抑制PI3K/AKT通路或两者联合处理后,大鼠外周血TNF-α、CRP、Lp-PLA2表达显著下调(均P<0.05)。结论:下调GRB2能够抑制PI3K/AKT/NF-κB通路,减少AS大鼠主动脉的脂质沉积和炎性浸润,在AS中起保护作用。

Objective: To investigate the mechanism of growth factor receptor bound protein 2(GRB2) gene silencing on lipid deposition and inflammatory infiltration in atherosclerosis(AS) rats through the regulation of PI3K/AKT/NF-κB signaling pathway. Methods: Normal saline group was set as control group, AS model rats were constructed and divided into four groups:negative control group(transfected with negative control sequence of GRB2 shRNA), GRB2 shRNA group(transfected with GRB2 shRNA sequence), pathway inhibitor group(treated with PI3K/AKT pathway inhibitor), combined group(treated with GRB2 shRNA sequence combined with PI3K/AKT pathway inhibitor). The expression of PI3KR1, AKT2 and NF-κB p65 in aorta of rats in each group was detected by quantitative real time polymerase chain reaction(qRT-PCR) and Western blotting. The morphology of aorta was observed by HE staining. Oil red O staining was used to quantify lipid deposition in the arteries of rats in each group. Enzyme-linked immunosorbent assay(ELISA) was utilized to determine the level of tumor necrosis factor α (TNF-α), C-reactive protein(CRP), lipoprorein-assoeiated phosPhohPaseA2(Lp-PLA2) in peripheral blood of rats in each group. Results: qRT-PCR and Western blotting experiments showed that PI3K/AKT signaling pathway was significantly activated in AS rats compared with saline group(P<0.05). Compared with the negative control group, the expression of PI3KR1, NF-κB p65, AKT decreased significantly after GRB2 silencing or PI3K/AKT pathway inhibition(all P<0.05). The results of HE staining indicated that AS pathological state was obvious in negative control group, the pathological condition was improved after GRB2 silencing or PI3K/AKT signaling pathway inhibition, combined group showed more obvious improvement. Oil red O staining showed that compared with negative control group, the area of lipid deposition in GRB2 shRNA group, pathway inhibitor group and combined group decreased significantly (all P<0.05). ELISA results indicated that compared with negative control group, silencing of GRB2, inhibition of PI3K/AKT pathway or combined with the above two treatments could significantly down-regulate the levels of TNF-α, CRP and Lp-PLA2 in priipheral blood in each group(all P<0.05). Conclusion: Down-regulation of GRB2 can lessen the lipid deposition and inflammatory infiltration of AS model rats through inhibiting PI3K/AKT/NF-κB signaling pathway, GRB2 gene silencing exerts protective effects in AS.

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