南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (7): 1073-1078.doi: 10.12122/j.issn.1673-4254.2021.07.16

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Exendin-4通过促进自噬减少非酒精性脂肪性肝病细胞模型的脂质沉积

林楚曼，方俊博，向倩如，周 蕊，杨 力   

  • 出版日期:2021-07-20 发布日期:2021-07-19

Exendin-4 promotes autophagy to relieve lipid deposition in a NAFLD cell model by activating AKT/mTOR signaling pathway

  • Online:2021-07-20 Published:2021-07-19

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摘要： 目的 探究Exendin-4改善肝细胞脂质沉积的作用机制，讨论Exendin-4治疗非酒精性脂肪肝病的可能机制。方法 对照组（人正常肝细胞LO2和肝癌细胞HepG2，不做任何处理）；棕榈酸钠（PA）组（LO2与HepG2分别进行PA处理，以构建肝细胞脂肪变性模型）；PA+Exendin-4组（LO2与HepG2分别进行PA与Exendin-4共处理）；PA+Exendin-4+3BDO组（LO2与HepG2分别进行Exendin-4与3BDO共处理）。通过油红O染色实验、CCK8实验，检测对照组、PA组、PA+Exendin-4组的脂质沉积程度与细胞增殖能力，并通过Western blot检测信号通路关键分子p-mTOR、m-TOR、p-AKT、AKT，以及自噬相关蛋白LC3-I/II和p62的表达；通过 Western blot 与免疫荧光实验，检测 LO2 与 HepG2 细胞中 GLP-1R 表达情况；通过 Western blot，检测对照组、PA+Exendin-4组、PA+Exendin-4+3BDO组中LC3-I/II和p62的表达。结果 油红O染色实验表明，PA诱导后细胞中脂质明显增加，PA+Exendin-4组脂质沉积程度较PA组降低；细胞增殖实验表明，PA组细胞增殖水平较对照组明显受抑制（P<0.01）；PA+Exendin-4组细胞增殖水平较PA组升高（P<0.05）；免疫荧光实验表明，人正常肝细胞LO2和肝癌细胞HepG2细胞株中均有GLP-1R表达；Western blot表明，PA+Exendin-4组p-mTOR的表达较PA组下调，p-AKT的表达较PA组上调。Exendin-4可下调自噬相关蛋白p62，而上调LC3-II的表达，而这一结果可被3BDO逆转。结论 Exendin-4可能通过直接作用于细胞上GLP-1R，激活AKT-mTOR信号通路，进而促进自噬；同时，Exendin-4有助于缓解PA作用下肝细胞的脂质沉积过程，并促进细胞增殖。提示Exendin-4在PA作用下肝细胞的脂质沉积过程中具有重要作用。

关键词: 胰高血糖素样肽-1；非酒精性脂肪性肝病；自噬；AKT-mTOR信号通路

Abstract: Objective To investigate the effect of exendin-4 on lipid deposition in hepatocytes and explore its possible mechanism for treatment of nonalcoholic fatty liver disease (NAFLD). Methods Human normal hepatocyte line LO2 and hepatoma cell line HepG2 were treated with palmitic acid (PA) to mimic hepatocyte steatosis or with combined treatments with PA+exendin-4 or PA+exendin-4+3BDO. Lipid deposition and proliferation of the two cell lines following treatment with PA or PA+exendin-4 were detected using Oil Red O staining and CCK8 assay, and the expression of p-mTOR, m-TOR, p-AKT, AKT and autophagy-related proteins LC3- I/II and p62 were detected with Western blotting; the expression of GLP-1R was detected with both Western blotting and immunofluorescence assay. The expression of LC3-I/II and p62 in the cells following treatment with PA+exendin-4 and PA+exendin-4+3BDO was detected with Western blotting. Results Lipid deposition in the two cell lines increased significantly after PA treatment, but was alleviated by co-treatment with exendin- 4. PA treatment significantly inhibited the proliferation of the two cell lines (P<0.01), and this inhibitory effect was obviously attenuated by exendin-4 (P<0.05). Immunofluorescence assay showed that both LO2 and HepG2 cells expressed GLP-1R. The expression of p-mTOR was significantly lower and that of p-AKT was higher in cells treated with PA +exendin-4 than in PA- treated cells. Exendin-4 also down-regulated the autophagy-associated protein p62 and up-regulated the expression of LC3-II in PA-treated cells, and this effect was obviously reversed by 3BDO. Conclusion Exendin-4 may activate the AKT-mTOR signal pathway to promote autophagy via its direct action on GLP-1R. Exendin-4 can also alleviate lipid deposition and promote proliferation of PA-treated hepatocytes, suggesting its important role in PA-induced lipid deposition in hepatocytes.

Key words: GLP-1; nonalcoholic fatty liver disease; autophagy; AKT-mTOR signaling pathway