利拉鲁肽通过SIRT1/AMPK通路改善小鼠肝脏脂质变性的机制研究

作者：路瑶1  焦谊2  杨晶晶1  马雪莲1

单位：1. 新疆医科大学第二附属医院 内分泌科 新疆 乌鲁木齐 830000 2. 新疆医科大学 基础医学院 生物化学与分子生物学教研室 新疆 乌鲁木齐 830000

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出版年,卷(期):页码：2024,16(3):36-44

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摘要：目的 探讨利拉鲁肽通过沉默信息调节因子1（silent information regulator 1，SIRT1） /腺苷酸活化蛋白激酶（adenosine monophosphate-activated protein kinase，AMPK）通路 改善小鼠肝脏脂质变性的机制。方法 将24只健康雄性6周龄C57BL/6J小鼠随机分为对照 组、模型组、模型+利拉鲁肽组、模型+ SIRT1组、模型+ SIRT1 + 利拉鲁肽组、模型+ SIRT1- NC组，每组4只。对照组普通饲料饲养，予以等容积生理盐水皮下注射，模型组高脂饲养 12周建立代谢相关脂肪性肝病（metabolic dysfunction-associated fatty liver disease，MAFLD） 模型，之后3周尾静脉注射SIRT1干扰慢病毒及利拉鲁肽。检测各组小鼠血清甘油三酯和 丙氨酸氨基转移酶（alanine aminotransferase，ALT）水平，采用HE染色和油红O染色观察 肝组织病理，采用实时荧光定量RT-PCR检测AMPK、SIRT1、肝激酶B1（liver kinase B1， LKB1）和去乙酰化固醇调节元件结合蛋白（sterol regulatory element binding protein-1c， SREBP-1c）基因表达水平，采用Western blot检测蛋白表达水平。结果 利拉鲁肽可降低高 脂喂养的C57BL/6J小鼠体质量、肝湿重、血清甘油三酯及ALT水平，油红O染色可见肝细 胞脂滴减少。与模型组相比，模型+利拉鲁肽组SIRT1（0.212 ± 0.110比0.076 ± 0.010）、 AMPK（0.518 ± 0.051比0.248 ± 0.023）、LKB1（1.023 ± 0.039比0.576 ± 0.029）基因表 达量和AMPK（0.212 ± 0.026比0.100 ± 0.006）、LKB1（0.413 ± 0.016比0.221 ± 0.015）蛋 白表达水平均上调，而SREBP-1c基因（0.727 ± 0.249比9.007 ± 1.530）和蛋白（0.187 ± 0.008比0.824 ± 0.114）表达水平均下调（P均＜ 0.05）。与模型组相比，模型+ SIRT1组 SIRT1（0.029 ± 0.003比0.076 ± 0.010）、AMPK（0.105 ± 0.013比0.248 ± 0.023）、LKB1（0.333 ± 0.106比0.576 ± 0.029）基因表达量均下调（P均＜ 0.05）。与模型+ SIRT1组相比，模型+ SIRT1 +利拉鲁肽组LKB1（0.945 ± 0.110比0.333 ± 0.106；0.380 ± 0.004比0.145 ± 0.014）、 AMPK（0.319 ± 0.051比0.105 ± 0.013；0.181 ± 0.039比0.051 ± 0.012）基因表达量和蛋白 表达量均上调，而SREBP-1c基因表达量（4.239 ± 0.554比12.740 ± 0.976）下调（P均＜ 0.05）。结论 利拉鲁肽改善C57BL/6J小鼠肝脏脂毒性可能通过直接上调SIRT1/AMPK通 路信号分子基因和蛋白表达水平，或间接激活LKB1并增强AMPK基因和蛋白表达、拮抗 SREBP-1c基因和蛋白表达，从而降低脂质合成相关分子水平，而干扰SIRT1表达削弱了 利拉鲁肽上调SIRT1/AMPK通路改善肝脏脂肪变性的作用。

Abstract: Objective To investigate the mechanism of liraglutide ameliorates hepatic steatosis by regulation silent information regulator 1 (SIRT1) / adenosine monophosphate-activated protein kinase (AMPK) pathway in mice. Methods Twenty-four healthy 6-week-old C57BL/6J male mice were randomly divided into control group, model group, model + liraglutide group, model + SIRT1 group, model + SIRT1 + liraglutide group and model + SIRT1-NC group, 4 mice in each group. Mice in control group were fed with regular feed and received subcutaneous injection of equal volume physiological saline. Mice in model group were fed with high-fat diet for 12 weeks to establish a metabolic dysfunction-associated fatty liver disease (MAFLD) model, followed by tail vein injection of SIRT1 interference lentivirus and liraglutide for 3 weeks. The levels of serum triglycerides and alanine aminotransferase (ALT) of mice in each group were detected. HE staining and oil red O staining were used to observe liver tissue pathology. Realtime fluorescence quantitative RT-PCR was used to detect the gene expression levels of AMPK, SIRT1, liver kinase B1 (LKB1) and sterol regulatory element binding protein-1c (SREBP-1c). Western blot was used to detect the protein expression levels. Results Liraglutide could reduce body mass, liver wet weight, serum triglycerides and ALT level of C57BL/6J mice with high-fat diet, and oil red O staining showed a decrease in hepatic lipid droplets. Compared with model group, SIRT1 (0.212 ± 0.110 vs. 0.076 ± 0.010), AMPK (0.518 ± 0.051 vs. 0.248 ± 0.023), LKB1 (1.023 ± 0.039 vs. 0.576 ± 0.029) gene expression levels and AMPK (0.212 ± 0.026 vs. 0.100 ± 0.006), LKB1 (0.413 ± 0.016 vs. 0.221 ± 0.015) protein expression levels were upregulated of mice in the model + liraglutide group, while SREBP-1c gene (0.727 ± 0.249 vs. 9.007 ± 1.530) and protein (0.187 ± 0.008 vs. 0.824 ± 0.114) expression levels were downregulated (all P ＜ 0.05). Compared with model group, gene expression levels of SIRT1 (0.029 ± 0.003 vs 0.076 ± 0.010), AMPK (0.105 ± 0.013 vs 0.248 ± 0.023) and LKB1 (0.333 ± 0.106 vs 0.576 ± 0.029) were all downregulated in the model + SIRT1 group (all P ＜ 0.05). Compared with model + SIRT1 group, LKB1 (0.945 ± 0.110 vs. 0.333 ± 0.106; 0.380 ± 0.004 vs. 0.145 ± 0.014) and AMPK (0.319 ± 0.051 vs. 0.105 ± 0.013; 0.181 ± 0.039 vs. 0.051 ± 0.012) gene expression and protein expression in model + SIRT1 + liraglutide group were upregulated, while SREBP-1c gene expression was downregulated (4.239 ± 0.554 vs. 12.740 ± 0.976) (all P ＜ 0.05). Conclusions Liraglutide may improve liver lipid toxicity in C57BL/6J mice by directly upregulating the gene and protein expression levels of SIRT1/AMPK signaling molecules, or indirectly activating LKB1 and enhancing AMPK gene and protein expression, antagonizing SREBP-1c gene and protein expression, thereby reducing lipid synthesis related molecular levels. Interference with SIRT1 expression weakens the effect of liraglutide upregulating the SIRT1/AMPK pathway on improving liver steatosis.

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