Pcsk9基因干扰对高脂诱导的大鼠非酒精性脂肪性肝病合并动脉粥样硬化的影响  

Effects of Pcsk9 gene interference on high fat-induced nonalcoholic fatty liver disease with atherosclerosis in rats

作　　者：张晓文 王祎 张婵 张迪 贠航 黄笛 ZHANG Xiaowen;WANG Yi;ZHANG Chan;ZHANG Di;YUN Hang;HUANG Di(Department of Nursing,School of Health Management,Shangluo University,Shangluo 726000,China;Shaanxi"Four Subjects and One Union"Qinling Health Food Ingredients and Walnut Industry Technology School-Enterprise Joint Research Center,Shangluo 726000,China)

机构地区：[1]商洛学院健康管理学院护理系,商洛726000 [2]陕西“四主体一联合”秦岭健康食品配料及核桃产业技术校企联合研究中心,商洛726000

出　　处：《上海交通大学学报（医学版）》2022年第2期150-157,共8页Journal of Shanghai Jiao tong University:Medical Science

基　　金：陕西省科技厅2021年科技计划项目(2021SF-326)。

摘　　要：目的·探讨前蛋白转化酶枯草溶菌素9(proprotein convertase subtilisin kexin 9,Pcsk9)基因干扰对高脂诱导的大鼠非酒精性脂肪性肝病及动脉粥样硬化损伤的影响。方法·构建非酒精性脂肪性肝病SD大鼠模型。40只大鼠随机分为4组:对照组、模型(high fat)组、shRNA-阴性对照(negative control,NC)干扰模型(high fat+shRNA-NC)组及Pcsk9-shRNA干扰模型(high fat+Pcsk9-shRNA)组。实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)检测Pcsk9基因干扰效率。放射免疫法测定空腹血清胰岛素。全自动生化分析仪检测大鼠血脂指标。苏木精-伊红染色(hematoxylin-eosin staining,H-E染色)观察肝脏组织及主动脉组织损伤。TUNEL染色检测肝脏组织细胞凋亡情况。酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测外周血中白细胞介素-1β(interleukin-1β,IL-1β)、IL-6及诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)的表达水平。蛋白印迹法检测PCSK9、Toll样受体4(Toll-like receptor 4,TLR4)、核因子κB(nuclear factorκB,NF-κB)P65及肿瘤坏死因子α(tumor necrosis factorα,TNF-α)的表达。结果·与对照组比较,模型组的肥胖指数和胰岛素水平显著升高(均P=0.000);肝脏细胞凋亡率显著升高(P=0.000);高密度脂蛋白胆固醇(high density lipoprotein cholesterol,HDL-C)水平显著降低,而低密度脂蛋白胆固醇(low density lipoprotein cholesterol,LDL-C)、总胆固醇(total cholesterol,TC)和三酰甘油(triacylglycerol,TAG)水平显著升高(均P=0.000);炎症因子IL-1β、IL-6、iNOS水平均显著升高(均P=0.000);TLR4、NF-κB P65蛋白活化及TNF-α表达均显著升高(均P=0.000);肝脏组织和主动脉组织均呈现明显损伤。干扰Pcsk9基因表达后,与模型组比较,high fat+Pcsk9-shRNA组肥胖指数和胰岛素水平显著降低(P=0.007,P=0.000);肝脏细胞凋亡率显著降低(P=0.000);HDL-C水平显著升高,而LDL-C、TC和TAG水平显著降Objective·To investigate the effects of proprotein convertase subtilisin kexin 9(Pcsk9)gene knockdown on nonalcoholic fatty liver disease(NAFLD)and atherosclerotic lesions in rats induced by high fat.Methods·The SD rat model of NAFLD was established.Rats were randomly divided into 4 groups:control group,model group(high fat),shRNA-negative control(NC)interference model group(high fat+shRNA-NC)and Pcsk9-shRNA interference model group(high fat+Pcsk9-shRNA).Realtime quantitative polymerase chain reaction(RT-qPCR)was used to detect Pcsk9 gene interference efficiency.Fasting serum insulin was determined by radioimmunoassay.Automatic biochemical analyzer was used to detect levels of blood lipid in rats.Hematoxylin-eosin staining(H-E staining)was used to observe the injury of liver tissue and aorta tissue.Apoptosis of liver tissue was detected by TUNEL staining.The levels of interleukin-1β(IL-1β),IL-6 and inducible nitric oxide synthase(iNOS)in peripheral blood were detected by enzyme linked immunosorbent assay(ELISA).The expression of PCSK9,Toll-like receptor 4(TLR4),nuclear factorκB(NF-κB)P65 and tumor necrosis factorα(TNF-α)were detected by Western blotting.Results·Compared with the control group,obesity index and insulin level in the model group were significantly increased(all P=0.000);the apoptosis rate of liver cells was significantly increased(P=0.000);the level of high density lipoprotein cholesterol(HDL-C)was significantly decreased,while the levels of low density lipoprotein cholesterol(LDL-C),total cholesterol(TC)and triacylglycerol(TAG)were significantly increased(all P=0.000);the levels of IL-1β,IL-6 and iNOS were significantly increased(all P=0.000);TLR4,NF-κB P65 protein activation and TNF-αexpression were significantly increased(all P=0.000);the liver tissue and aorta tissue were significantly damaged.After interference of Pcsk9 gene expression,compared with the model group,obesity index and insulin level in the high fat+Pcsk9-shRNA group were significantly reduced(P=0.007,P=0.000);the apopt

关 键 词：前蛋白转化酶枯草溶菌素9 基因干扰 非酒精性脂肪性肝病 动脉粥样硬化 炎症因子 

分 类 号：R54[医药卫生—心血管疾病] R575.5[医药卫生—内科学]