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

doi:10.3969/j.issn.1674-8115.2022.02.003

摘要/Abstract

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 factor κB，NF-κB）P65及肿瘤坏死因子α（tumor necrosis factor α，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水平显著降低（均P=0.000）；炎症因子IL-1β、IL-6及iNOS水平均显著降低（均P=0.000）；TLR4、NF-κB P65蛋白活化及TNF-α表达均显著降低（均P=0.000）；肝脏组织和主动脉组织病理损伤均得到改善。结论·干扰Pcsk9基因可减轻非酒精性脂肪性肝病合并动脉粥样硬化大鼠的肥胖指数、胰岛素水平、血脂指标及炎症反应，并抑制TLR4、NF-κB P65蛋白激活，进而改善大鼠肝脏及主动脉组织的损伤。

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

Abstract:

Objective·To investigate the effects of proprotein convertase subtilisin kexin 9 (Pcsk9) gene knockdown on non-alcoholic 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). Real-time 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 factor κB (NF-κB) P65 and tumor necrosis factor α (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-α expression were significantly increased (all P=0.000); the liver tissue and aorta tissue were significantly damaged. After interferenceof 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 apoptosis rate of liver cells was significantly reduced (P=0.000); the level of HDL-C was significantly increased while the levels of LDL-C, TC and TAG were significantly decreased (all P=0.000); the levels of IL-1β, IL-6 and iNOS were significantly decreased (all P=0.000); TLR4, NF-κB P65 protein activation and TNF-α expression were significantly decreased (all P=0.000); the histopathological lesions of liver tissue and aorta tissue were improved.

Conclusion·Knockdown of Pcsk9 gene can reduce obesity index, insulin level, blood lipid index and inflammatory response in the rats with NAFLD and atherosclerosis, and inhibit the activation of TLR4 and NF-κB P65 protein, thereby improving the injury of liver and aortic tissue in rats.

Key words: proprotein convertase subtilisin kexin 9 (Pcsk9), gene interference, non-alcoholic fatty liver disease (NAFLD), atherosclerosis, inflammatory cytokine

中图分类号:

张晓文, 王祎, 张婵, 张迪, 贠航, 黄笛. 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[J]. 上海交通大学学报(医学版), 2022, 42(2): 150-157.

Xiaowen ZHANG, Yi WANG, Chan ZHANG, Di ZHANG, Hang YUN, Di HUANG. Effects of Pcsk9 gene interference on high fat-induced nonalcoholic fatty liver disease with atherosclerosis in rats[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2022, 42(2): 150-157.

图/表 8

图1 4组 Pcsk9 基因干扰的效率检测Note：A. Relative mRNA level of Pcsk9. B. Relative protein level of PCSK9. ①P=0.000, compared with the control group; ②P=0.000, compared with the high fat group.

Fig 1 Efficiency detection of Pcsk9 gene interference in 4 groups

表1 4组肥胖指数和胰岛素水平比较

Tab 1 Obesity index and insulin levels in 4 groups

Item	Control group	High fat group	High fat+shRNA-NC group	High fat+Pcsk9-shRNA group
Obesity index	322.79±19.61	387.53±23.57^①	384.74±18.76	343.22±21.82^②
Insulin/（mU·L^-1）	12.32±3.64	34.23±6.12^①	33.78±5.21	17.32±4.16^③

图2 大鼠肝脏H-E染色(×200)

Fig 2 H-E staining of rat livers (×200)

图3 TUNEL检测大鼠肝脏细胞凋亡Note：A. Apoptosis of liver tissues in rats by TUNEL staining (×200). The arrows in the figures indicate apoptotic cells (brown). B. The statistical histogram of TUNEL assay. ①P=0.000, compared with the control group; ②P=0.000, compared with the high fat group.

Fig 3 Apoptosis of rat liver cells detected by TUNEL

表2 4组大鼠血脂指标比较

Tab 2 Blood lipid indicators of rats in 4 groups

Item	Control group	High fat group	High fat +shRNA-NC group	High fat +Pcsk9-shRNA group
HDL-C/（mmol·L^-1）	1.43±0.29	0.45±0.12^①	0.47±0.14	1.18±0.31^②
LDL-C/（mmol·L^-1）	0.13±0.04	0.82±0.13^①	0.81±0.10	0.24±0.06^②
TC/（mmol·L^-1）	1.14±0.56	4.65±0.41^①	4.58±0.43	1.72±0.29^②
TAG/（mmol·L^-1）	0.43±0.08	1.47±0.27^①	1.39±0.21	0.66±0.09^②

图4 大鼠主动脉血管壁H-E染色(×200)Note：→ represents atherosclerotic or fibrous plaque formed by foam cells and cholesterol crystals; ? indicates lipid cell infiltration.

Fig 4 H-E staining of the aortic wall in rats (×200)

表3 4组大鼠外周血中炎性因子的表达水平比较

Tab 3 Expression levels of inflammatory factors in peripheral blood of rats in 4 groups

Item	Control group	High fat group	High fat＋shRNA-NC group	High fat＋Pcsk9-shRNA group
IL-1β/（pg·mL^-1）	21.24±7.38	151.69±24.03^①	149.81±30.67	58.93±12.47^②
IL-6/（pg·mL^-1）	13.38±4.31	98.92±13.05^①	95.46±10.54	35.92±9.72^②
iNOS/（pg·mL^-1）	23.03±8.32	136.35±32.16^①	137.93±34.74	65.56±13.28^②

图5 TLR4及NF-κB P65的磷酸化和TNF-α的表达水平Note：A. Protein bands in Western blotting. B. Quantitative histogram. ①P=0.000, compared with the control group; ②P=0.000, compared with the high fat group.

Fig 5 Phosphorylation levels of TLR4 and NF-κB P65 and the expression of TNF-α

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