Sirt1通过HNF-1α/FXR-1通路调控脓毒症肝损伤的动物研究

doi:10.16150/j.1671-2870.2020.03.014

摘要/Abstract

摘要：

目的：探讨沉默信息调节因子1（Sirtuin 1,Sirt1）在脓毒症肝损伤中的作用及机制。方法： 采用腹腔注射脂多糖（lipopolysaccharide, LPS）法构建脓毒症小鼠模型,并设置腹腔注射生理盐水小鼠作为对照组,用苏木精-伊红染色（hematoxylin-eosin staining,HE染色）检测脓毒症小鼠模型的肝损伤情况,采用定量实时聚合酶链反应（quantitative real-time polymerase chain reaction, qrt-PCR）检测脓毒症小鼠及对照组小鼠肝组织中的Sirt1/肝细胞核因子-1α（hepatic nuclear factor-1α,HNF-1α）/法尼酯X受体1（farnesoid X receptor-1,FXR-1）通路表达情况。分离鉴定原代小鼠肝实质细胞,采用CCK8法检测不同质量浓度的LPS对小鼠肝实质细胞活力的影响;利用细胞增殖成像分析试剂盒(EdU法)检测不同质量浓度的LPS对小鼠肝实质细胞增殖能力的影响;应用酶联免疫吸附测定（enzyme-linked immunosorbent assay,ELISA）法检测不同质量浓度的LPS对小鼠肝实质细胞炎症因子[白细胞介素-6（interleukin-6, IL-6）/肿瘤坏死因子α（tumor necrosis factor-α,TNF-α）]分泌的影响。构建过表达Sirt1质粒,然后以Lipofectamine 2000为载体,转染进细胞,48 h后采用qrt-PCR检测Sirt1/HNF-1α/FXR-1通路的表达情况,分别采用CCK8、EdU法以及ELISA法检测Sirt1对LPS在肝实质细胞中功能的影响。结果： 与对照组相比,LPS诱导的脓毒症小鼠肝损伤样本中Sirt1/HNF-1α/FXR-1通路表达下调;LPS会降低小鼠肝实质细胞的活力,抑制肝实质细胞的增殖能力,并促进肝实质细胞中炎症因子IL-6及TNF-α的分泌。转染过表达Sirt1的肝实质细胞中,HNF-1α/FXR-1通路的表达上调,且能够抑制LPS引起的细胞活力及增殖降低以及炎症因子的分泌。结论： Sirt1/HNF-1α/FXR-1通路参与调控LPS诱导的脓毒症肝损伤,过表达Sirt1能够抑制LPS在肝实质细胞中的损伤效应。

关键词: 脓毒症, 脂多糖, 沉默信息调节因子1, 肝细胞核因子-1α/法尼酯X受体1通路

Abstract:

Objective: To investigate the role of Silent information regulator 1 (SIRT1) in sepsis-induced liver injury and the related mechanism in a mouse model. Methods: A mouse sepsis model was established by intraperitoneal injection (IP) of LPS, and the mice in control group were only given same volume sterilized 0.9% saline via IP. Sections of the liver from the mice in both groups were stained with hematoxylin-eosin and hepatic injury was examined, and quantitative real-time polymerase chain reaction (qrt-PCR) was performed to detect expression of Sirt1/HNF-1α/FXR-1 in hepatic tissue. The isolated primary mouse hepatic parenchymal cells were treated with different concentrations of LPS. Viabilityand proliferation of hepatic parenchymal cells were evaluated by CCK8 and EdU assay respectively. In addition, enzyme linked immunosorbent assay (ELISA) was conducted to measure interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) production in hepatic parenchymal cells treated with different concentration of LPS. Sirt1 overexpressing plasmids were constructed and transfected into hepatic cells in mediation of Lipofectamine 2000. After 48 hour, the Sirt1/HNF-1α/FXR-1 pathway expression was tested by qrt-PCR and the impact of overexpressing Sirt1 on the function of the cells was evaluated by CCK8, EdU and ELISA. Results: Compared with control, Sirt1/HNF-1α/FXR-1 pathway was down-regulated in LPS-induced sepsis mouse model. LPS lowered cell viability, inhibited proliferation and increased secretion of inflammatory factors IL-6 and TNF-α in hepatocytes. Over-expression of Sirt1 upregulated the expression of HNF-1α/FXR-1 pathway in parenchymal cells, alleviated LPS-induced inhibition of cell viability and proliferation, and decreased LPS-induced inflammatory factor secretion. Conclusions: The Sirt1/HNF-1α/FXR-1 pathway is involved in LPS-induced septic liver injury. Overexpression of Sirt1 is able to attenuate the toxic effects of LPS on parenchymal cells.

Key words: Sepsis, LPS, Silent information regulator 1 (SIRT1), HNF-1α/FXR-1 pathway

中图分类号:

R575.3

王秋云, 陈影, 赵冰, 孙思磊, 杨之涛, 毛恩强, 陈尔真. Sirt1通过HNF-1α/FXR-1通路调控脓毒症肝损伤的动物研究[J]. 诊断学理论与实践, 2020, 19(03): 279-285.

WANG Qiuyun, CHEN Ying, ZHAO Bing, SUN Silei, YANG Zhitao, MAO Enqiang, CHEN Erzhen. Sirt1 involved in sepsis-induced acute liver injury via regulating HNF-1α/FXR-1 pathway: The mechanism study in a rodent model[J]. Journal of Diagnostics Concepts & Practice, 2020, 19(03): 279-285.

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基因	引物序列
Sirtq1上游	5'-GATACGGAGAGGCCCGAATG-3'
Sirtq1下游	5'-AGTTCCCAATGCTGGTGGAG-3'
FXR-1上游	5'-TCCTTGTCCTCCTCGGAACA-3'
FXR-1下游	5'-TGGGGTTTCCTGAAGCCTTG-3'
HNF-1α上游	5'-GTACGGACAGTCTGCAACCA-3'
HNF-1α下游	5'-TACTTGGTGTAAGGCCGCAG-3'
GAPDH上游	5'-AGGTCGGTGTGAACGGATTTG-3'
GAPDH下游	5'-GGGGTCGTTGATGGCAACA-3'