siRNA抑制饥饿素-O-乙酰基转移酶对肝细胞脂肪变性的影响及作用机制

张绍仁; 胡静娴; 樊晓明

doi:10.3969/j.issn.1001-5256.2018.05.027

siRNA抑制饥饿素-O-乙酰基转移酶对肝细胞脂肪变性的影响及作用机制

DOI: 10.3969/j.issn.1001-5256.2018.05.027

复旦大学附属金山医院消化内科

基金项目:

上海市卫计委青年研究项目(20154Y0096)；上海市金山区卫计委优秀青年人才培养计划项目(JSYQ201606);上海市金山区卫计委面上项目(JSKJ-KTMS-2015-02)；上海市金山区重点临床学科建设项目(JSZK2015A06)；

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中图分类号: R575.5
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出版历程
- 收稿日期: 2017-11-17
- 出版日期: 2018-05-20

Effect of ghrelin O-acyltransferase inhibition by small interfering RNA on hepatocyte fatty degeneration and related mechanism of action

Department of Gastroenterology, Jinshan Hospital Affiliated to Fudan University

Research funding:

摘要

摘要: 目的探讨siRNA抑制饥饿素-O-乙酰基转移酶（GOAT）对肝脂肪变性影响及作用机制。方法游离脂肪酸（FFA）作用于人肝细胞系LO2细胞以诱导肝细胞脂肪变性。FFA与siRNA-GOAT单独或联合处理LO2细胞,分为4组,即空白对照（NC）组:仅用PBS处理;siRNA-GOAT组:仅以终浓度为10 nmol/L的siRNA-GOAT处理;FFA组:仅以终浓度为1 mmol/L的FFA处理;FFA+siRNA-GOAT组:采用上述浓度FFA和siRNA-GOAT混合处理。肝细胞油红O染色检测脂肪滴形成情况;TG检测试剂盒检测LO2细胞脂质水平;免疫印迹法（Western Blot）、实时定量逆转录聚合酶链反应（RT-PCR）、免疫荧光染色、电子显微镜检测自噬活性;酶联免疫吸附测定法、RT-PCR检测TNFα、IL-6水平。Western Blot检测雷帕霉素（m TOR）、磷酸化m TOR（pm TOR）、AMP-活化蛋白激酶（AMPK）以及磷酸化AMPK（p-AMPK）的蛋白水平变化。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果 FFA+siRNA-G...
- 脂肪肝 /
- 酰基转移酶类 /
- 脂肪酸类,非酯化 /
- 自噬
Abstract: Objective To investigate the effect of inhibition of ghrelin O-acyltransferase ( GOAT) by small interfering RNA ( siRNA) on hepatocyte fatty degeneration and related mechanism of action. Methods Human LO2 hepatocytes were treated with free fatty acid ( FFA) to induce hepatocyte fatty degeneration. LO2 hepatocytes were treated with FFA and siRNA-GOAT alone or in combination and then divided into normal control ( NC) group ( treated with phosphate buffered saline alone) , siRNA-GOAT group ( treated with siRNA-GOAT at a final concentration of 10 nm) , FFA group ( treated with FFA at a final concentration of 1 mm) , and FFA + siRNA-GOAT group ( treated with FFA at a final concentration of 1 mm and siRNA-GOAT at a final concentration of 10 nm) . Oil red O staining was performed for hepatocytes to identify lipid droplets; the triglyceride ( TG) test kit was used to measure the lipid level in LO2 hepatocytes; Western blot, qRT-PCR, immunofluorescent staining, and electron microscopy were used to measure autophagy; ELISA and RT-PCR were used to measure the levels of tumor necrosis factor-α ( TNFα) and interleukin-6 ( IL-6) ; ELISA was used to measure the changes in the levels of mammalian target of rapamycin ( m TOR) , phosphorylated m TOR ( p-m TOR) , AMP-activated protein kinase ( AMPK) , and phosphorylated AMPK. A one-way analysis of variance was used for comparison between multiple groups, and the least significant difference t-test was used for further comparison between any two groups. Results Compared with the FFA group, the FFA + siRNA-GOAT group had a significant reduction in the formation of lipid droplets and a significantly lower TG level ( P < 0. 001) . Compared with the FFA group, the FFA +siRNA-GOAT group had significant reductions in the protein and mRNA expression of TNFα and IL-6 ( all P < 0. 005) . The siRNA +GOAT group had significantly higher mRNA expression of LC3-II and Beclin-1 than the NC group ( all P < 0. 001) . The FFA + siRNA-GOAT group had significantly higher mRNA expression of LC3-II and Beclin-1 than the FFA group ( all P < 0. 001) . The siRNA + GOAT group had significantly higher protein expression of LC3-II and Beclin-1 than the NC group ( all P < 0. 05) . The FFA + siRNA-GOAT group had significantly higher protein expression of LC3-II and Beclin-1 than the FFA group ( all P < 0. 05) . Immunofluorescent staining showed that compared with the FFA group and the siRNA-GOAT group, the FFA + siRNA-GOAT group had a significant increase in the expression of endogenous LC3-II in LO2 hepatocytes. Electron microscopy showed that compared with the FFA group, the FFA + siRNA-GOAT group had a significant increase in the expression of autophagosome. After the LO2 hepatocytes were treated by autophagy inhibitors siRNA-ATG5 and 3-MA or an autophagy stimulant, rapamycin, there was a significant difference in TG level between the FFA + siRNA-ATG5 group and the FFA + siRNA-GOAT group ( P < 0. 001) , as well as between the FFA + 3-MA group and the FFA + rapamycin group ( P < 0. 001) . The FFA + siRNA-GOAT group had a significantly higher level of LC3-I/II than the FFA + siRNA-ATG5 group ( P <0. 05) , and the FFA + rapamycin group had a significantly higher level of LC3-I/II than the FFA + 3-MA group ( P < 0. 05) . Compared with the FFA group, the FFA + siRNA-GOAT group had significantly higher protein expression of p-AMPK ( P < 0. 05) and significantly lower protein expression of p-m TOR ( P < 0. 05) . Conclusion GOAT inhibition by siRNA can upregulate autophagy and alleviate hepatocyte fatty degeneration, possibly by regulating the AMPK/m TOR pathway.
- fatty liver /
- acyltransferases /
- fatty acids, nonesterified /
- autophagy

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参考文献(37)

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siRNA抑制饥饿素-O-乙酰基转移酶对肝细胞脂肪变性的影响及作用机制

DOI: 10.3969/j.issn.1001-5256.2018.05.027

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Effect of ghrelin O-acyltransferase inhibition by small interfering RNA on hepatocyte fatty degeneration and related mechanism of action

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siRNA抑制饥饿素-O-乙酰基转移酶对肝细胞脂肪变性的影响及作用机制

DOI: 10.3969/j.issn.1001-5256.2018.05.027

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Effect of ghrelin O-acyltransferase inhibition by small interfering RNA on hepatocyte fatty degeneration and related mechanism of action

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