12-脂氧合酶基因过表达对大鼠颈动脉内皮功能的影响及机制

Effects of 12-lipoxygenase overexpression on endothelial function in rat carotid artery balloon injury model

目的研究12-脂氧合酶(12-lipoxygenase,12-LO)过表达对内皮功能的影响,并探讨其可能的机制。方法 SD大鼠分为假手术组(sham)、球囊损伤组(balloon injury,BI)、球囊损伤+空载组(balloon injury+lentivirus-GFP,BI+Lv-GFP)、球囊损伤+过表达组(balloon injury+lentivirus-12-LO,BI+Lv-12-LO)。除sham组外,各组行颈动脉球囊损伤术,BI+Lv-GFP组与BI+Lv-12-LO组术后转染Lv-GFP、Lv-12-LO。HE染色观察内膜增生情况,Western blot检测12-LO转染情况。定量PCR、免疫组化及Western blot检测内皮型一氧化氮合酶(endothelial nitric oxide synthase,e NOS)、细胞间黏附分子-1(intracellular adhesion molecule-1,ICAM-1)的表达。结果 Lv-12-LO在血管壁成功转染并表达(P<0.01)。与sham组比较,BI组内中膜厚度(intima-media thickness,IMT)显著升高(P<0.01),ICAM-1mRNA(P=0.001)及蛋白水平(P=0.038)表达增加,e NOS mRNA(P=0.012)及蛋白水平(P=0.009)表达降低。与BI+Lv-GFP组比较,BI+Lv-12-LO组IMT升高(P=0.001),ICAM-1 mRNA(P=0.009)及蛋白水平(P=0.002)进一步升高,e NOS mRNA(P=0.004)及蛋白水平(P=0.020)进一步降低。免疫组化结果显示,球囊损伤后ICAM-1在血管壁中表达增加(P=0.007),e NOS表达减少(P=0.001);与BI+Lv-GFP组相比,BI+Lv-12-LO组ICAM-1表达进一步增加(P<0.01),e NOS表达进一步减少(P<0.01)。结论 12-LO过表达可能通过影响内皮因子以及炎症因子的释放而加重血管内皮功能的损伤。

Objective To determine the effect of 12-lipoxygenase （12-LO） over-expression on endothelial function and investigate its underlying mechanisms. Methods Sprague Dawley rats were randomly divided into sham operation group, balloon injury group （BI）, balloon injury ＋ Lentivirus-GFP group （BI ＋Lv-GFP）, balloon injury ＋ Lentivirus-12-LO group （BI ＋ Lv-12-LO）. Except the rats of sham operation group, all the other rats were inflicted with carotid artery balloon injury operation. BI ＋ Lv-GFP group and BI ＋ Lv-12-LO group were followed by being transfected with lentivirus expressing GFP or 12-LO into the injured artery. In 12 d after transfection, all rats were sacrificed. Intimal hyperplasia was observed using HE staining. The expression of 12-LO was detected by Western blotting. The expression levels of endothelial nitric oxide synthase （eNOS） and intracellular adhesion molecule-1 （ ICAM-1 ） were detected by real time-qPCR, immunohistochemistry and Western blotting. Results Lv-12-LO was successfully transfected and expressed in the vascular wall （ P 〈 0.01 ）. Compared with the sham operation group, the intima-media thickness （IMT） was increased （P 〈0.01 ）. the exoression of ICAM-1 at mRNA （P =0.001 and protein （P = 0. 038） levels was elevated, and the expression of eNOS mRNA （P = 0. 012） and protein （P = 0. 009） was decreased in the BI group. Compared with the BI ＋ Lv-GFP group, the IMT was increased （P = 0. 001 ）, the expression of ICAM-1 mRNA （ P = 0. 009） and protein （ P = 0. 002 ） was significantly increased, and the expression of eNOS mRNA （ P = 0. 004 ） and protein （ P = 0. 020 ） were significantly decreased of BI ＋ Lv-12-LO group. Immunohistochemical assay showed the positive rate of ICAM-1 was higher （P = 0. 007）, meanwhile the eNOS positive rate was lower in the BI group than the sham operation group （P =0. 001 ）. Compared with the BI ＋ Lv-GFP group, the ICAM-1 positive rate was significantly increased （P 〈0.01 ）, and the eNOS positive rate was significantly decreased （P 〈0.01 ） in the BI ＋ Lv-12-LO group. Conclusion 12-LO over-expression aggravates the damage of vascular endothelial function by affecting the expression of endothelial factors and inflammatory factors.