沉默Salusin-β对糖尿病大鼠内皮功能障碍的影响及机制研究

Effect and mechanism of silencing Salusin-βon endothelial dysfunction in diabetes rats

目的分析短发夹RNA(shRNA)沉默心血管调节肽(Salusin-β)对糖尿病(DM)大鼠内皮功能障碍的影响及其潜在的机制。方法52只SD大鼠随机抽取12只为正常对照组(NC组),其余大鼠采用喂食高糖高脂饲料和腹腔注射链脲佐菌素的方法制备DM模型。将36只建模成功的大鼠分为DM组、Ad-Scr shRNA组和Ad-Salusin-βshRNA组,每组12只。Ad-Scr shRNA组大鼠尾静脉注射腺病毒空载体(Ad-scramble shRNA),Ad-Salusin-βshRNA组大鼠尾静脉注射编码Salusin-βshRNA的腺病毒载体(Ad-Salusin-βshRNA)。每2周注射1次,NC组和DM组注射等量生理盐水。4周后,检测大鼠空腹血糖(FBG)和血清Salusin-β水平以及胸主动脉血管舒张功能;酶联免疫吸附试验检测大鼠胸主动脉肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6、IL-1β、丙二醛(MDA)、超氧化物歧化酶(SOD)水平;二氢乙锭(DHE)染色检测胸主动脉中活性氧(ROS)水平;苏木精-伊红染色观察大鼠胸主动脉组织病理学变化;荧光定量PCR检测大鼠胸主动脉Salusin-β和还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶2(NOX2)mRNA表达水平;Western blot法检测大鼠胸主动脉NOX2、核因子κB p65(NF-κB p65)蛋白表达。结果与NC组相比,DM组大鼠FBG、血清Salusin-β水平升高,胸主动脉TNF-α、IL-6、IL-1β、MDA、ROS水平升高,胸主动脉内膜-中膜厚度增厚,Salusin-βmRNA、NOX2 mRNA和蛋白、细胞核NF-κB p65蛋白水平升高,Ach诱发的内皮依赖性血管舒张功能下降,胸主动脉SOD和细胞质NF-κB p65蛋白表达水平降低(P<0.05)。与DM组和Ad-Scr shRNA组相比,Ad-Salusin-βshRNA组大鼠FBG和血清Salusin-β表达下降,胸主动脉TNF-α、IL-6、IL-1β、MDA、ROS表达下降,胸主动脉内膜-中膜厚度变薄,Salusin-βmRNA、NOX2 mRNA和蛋白、细胞核NF-κB p65蛋白表达水平降低,Ach诱发的内皮依赖性血管舒张功能升高,胸主动脉SOD和细胞质NF-κB p65蛋白表达水平升高(P<0.05)。结论shRNA沉默Salusin-β可减轻DM大鼠内皮功能障碍,其作用机制可能与抑制NOX2/ROS/NF-κB信号通路活化有关。

Objective To analyze the effect of short hairpin RNA(shRNA)silencing Salusin-βon endothelial dysfunction in diabetes mellitus(DM)rats and its potential mechanism.Methods Twelve of 52 SD rats were randomly selected as the normal control group(NC group).The rest of rats were fed high sugar and high fat diet and intraperitoneally injected streptozotocin 60 mg/kg to prepare DM model.Thirty-six rats were successfully modeled and were divided into the DM group,the Ad-Scr shRNA group and the Ad-Salusin-βshRNA group,with 12 rats in each group.Adenovirus empty vector encoding scramble shRNA(Ad-scramble shRNA)was injected into the tail vein of rats in the Ad-Scr shRNA group,and adenoviral vector encoding Salusin-βshRNA(Ad-Salusin-βshRNA)was injected into the tail vein of rats in the AdSalusin-βshRNA group.The drug was administered once every 2 weeks.The NC group and the DM group were injected with the same volume of normal saline.After 4 weeks,fasting blood glucose(FBG)and serum Salusin-βlevels and the vasodilation function of thoracic aorta were measured.Enzyme linked immunosorbent assay was used to detect levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,IL-1β,malondialdehyde(MDA)and superoxide dismutase(SOD)in thoracic aorta.Dihydroethidium(DHE)staining was used to detect the level of reactive oxygen species(ROS)in the thoracic aorta.Hematoxylin-eosin staining was used to observe histopathological changes of thoracic aorta.Fluorescence quantitative PCR was used to detect mRNA levels of Salusin-βand nicotinamide adenine dinucleotide phosphate oxidase 2(NOX2)in thoracic aorta.Western blot assay was used to detect expression levels of NOX2 and nuclear factor-κB p65(NF-κB p65)protein in thoracic aorta.Results Compared with the NC group,the FBG and serum Salusin-βwere increased,TNF-α,IL6,IL-1β,MDA and ROS in thoracic aorta were also increased,and intima-media thickness of the thoracic aorta was thickened in the DM group.Salusin-βmRNA,NOX2 mRNA and protein,nuclear NF-κB p65 protein levels were increased in the DM group.Ach induced endothelium dependent vasodilation,thoracic aorta SOD,cytoplasmic NF-κB p65 protein levels were decreased(P<0.05).Compared with the DM group and the Ad-Scr shRNA group,FBG and serum Salusin-βdecreased,thoracic aorta TNF-α,IL-6,IL-1β,MDA and ROS decreased,intima-media thickness of thoracic aorta became thinner in the Ad-Salusin-βshRNA group.Salusin-βmRNA,NOX2 mRNA and protein,nuclear NF-κB p65 protein levels were decreased in the Ad-Salusin-βshRNA group,and Ach induced endothelium dependent vasodilation,thoracic aorta SOD,cytoplasmic NF-κB p65 protein levels were increased(P<0.05).Conclusion shRNA silencing Salusin-βcan reduce endothelial dysfunction in DM rats,and its mechanism may be related to the inhibition of the activation of NOX2/ROS/NF-κB signaling pathway.