候正萍, 李燕平, 赵蕾, 陈压西, 阮雄中*
本研究旨在探讨脂多糖(lipopolysaccharide, LPS)对肝细胞脂质自噬的影响及其机制。体外培养人肝癌细胞株HepG2，用0.1 mmol/L软脂酸(palmitic acid, PA)负荷，分为对照(0 μg/mL LPS)组、LPS (10 μg/mL)组、LPS+DMSO组、LPS+雷帕霉素(rapamycin, RAPA, 10 μmol/L)组。油红O染色观察HepG2细胞内脂质积聚情况；自噬双标腺病毒mRFP-GFP-LC3转染细胞后激光共聚焦显微镜观察细胞自噬流；通过氟硼二吡咯BODIPY 493/503荧光染料和溶酶体标记物溶酶体关联膜蛋白1 (lysosomal associated membrane protein 1, LAMP1)进行脂滴和溶酶体的共定位，反映细胞内脂质自噬水平；Western blot检测哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)、p-mTOR、核糖体S6激酶1 (ribosome protein subunit 6 kinase 1, S6K1)、p-S6K1、LC3II/I、P62蛋白表达。结果显示，与对照组相比，LPS组细胞油红O染色红染脂滴增加，自噬体增加，自噬溶酶体明显降低，LAMP1/BODIPY共定位率降低(P < 0.05)，p-mTOR/mTOR、p-S6K1/S6K1和LC3II/LC3I比值升高，P62蛋白表达增加(P < 0.05)。加入RAPA干预后，与LPS+DMSO组相比，自噬体减少，自噬溶酶体明显增加，LAMP1/BODIPY共定位率升高(P < 0.05)，肝细胞油红O染色红染脂滴减少(P < 0.001)。综上，LPS通过激活mTOR通路抑制HepG2细胞脂质自噬，从而加重细胞内脂质积聚。
Lipopolysaccharide inhibits lipophagy in HepG2 cells via activating mTOR pathway
HOU Zheng-Ping, LI Yan-Ping, ZHAO Lei, CHEN Ya-Xi, RUAN Xiong-Zhong*
Key Laboratory of Metabolism on Lipid and Glucose, Center for Lipid Research, Chongqing Medical University, Chongqing 400016, China
This study aimed to investigate the effect of lipopolysaccharide (LPS) on lipophagy in hepatocytes and the underlying mechanism. Human hepatoma cell line HepG2 was cultured in vitro, treated with 0.1 mmol/L palmitic acid (PA), and then divided into control group (0 μg/mL LPS), LPS group (10 μg/mL LPS), LPS+DMSO group and LPS+RAPA (rapamycin, 10 μmol/L) group. Lipid accumulation in hepatocytes was observed by oil red O staining. The autophagic flux of the cells was assessed using confocal laser scanning microscope after being transfected with autophagy double-labeled adenovirus (mRFP-GFP-LC3). The level of intracellular lipophagy was visualized by the colocalization of lipid droplets (BODIPY 493/503 staining) and lysosomes (lysosome marker, lysosomal associated membrane protein 1, LAMP1). The expression levels of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), ribosome protein subunit 6 kinase 1 (S6K1), p-S6K1, LC3II/I and P62 protein were examined by Western blot. The results showed that the number of red lipid droplets stained with oil red O was significantly increased in LPS group compared with that in control group (P < 0.001). Moreover, in LPS group, the number of autophagosomes was increased, while the number of autophagolysosomes and the colocalization rate of LAMP1 and BODIPY were significantly decreased (P < 0.05). Meanwhile, the ratios of p-mTOR/mTOR and p-S6K1/S6K1, the ratio of LC3II/LC3I and the protein expression of P62 were significantly increased (P < 0.05) in LPS group. Furthermore, compared with LPS+DMSO group, RAPA treatment obviously reduced the number of lipid droplets and autophagosomes, and raised the number of autophagolysosomes and the colocalization rate of LAMP1 and BODIPY (P < 0.05). In conclusion, the results demonstrate that LPS inhibits lipophagy in HepG2 cells via activating mTOR signaling pathway, thereby aggravating intracellular lipid accumulation.
通讯作者：阮雄中 E-mail: firstname.lastname@example.org
候正萍, 李燕平, 赵蕾, 陈压西, 阮雄中. 脂多糖通过mTOR途径抑制肝细胞脂质自噬[J]. 生理学报 2021; 73 (5): 813-820.
HOU Zheng-Ping, LI Yan-Ping, ZHAO Lei, CHEN Ya-Xi, RUAN Xiong-Zhong. Lipopolysaccharide inhibits lipophagy in HepG2 cells via activating mTOR pathway. Acta Physiol Sin 2021; 73 (5): 813-820 (in Chinese with English abstract).