南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (7): 1164-1171.doi: 10.12122/j.issn.1673-4254.2023.07.13

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LRG1抑制小鼠肝巨噬细胞活化从而改善代谢相关脂肪性肝病：基于增强TGF-β1信号通路

徐龙飞，韩 晶，杨 喆，杨燕平，陈金慧，吴西军，王 琪，洪 艳   

  1. 贵州医科大学基础医学院组织学与胚胎学教研室，金阳医院检验科，贵州 贵阳 550025；中国医学科学院成体干细胞转化研究重点实验室，贵州医科大学干细胞与组织工程研究中心，贵州 贵阳 550025
  • 出版日期:2023-07-20 发布日期:2023-07-20

LRG1 inhibits hepatic macrophage activation by enhancing TGF-β1 signaling to alleviate MAFLD in mice

XU Longfei, HAN Jing, YANG Zhe, YANG Yanping, CHEN Jinhui, WU Xijun, WANG Qi, HONG Yan   

  1. Department of Histology and Embryology, School of Basic Medical Sciences, Guizhou Medical University, Guiyang 550025, China; Clinical Laboratory, Jinyang Hospital, Guizhou Medical University, Guiyang 550025, China; Key Laboratory of Adult Stem Cell Transformation Research, Chinese Academy of Medical Sciences, Stem Cell and Tissue Engineering Research Center, Guizhou Medical University, Guiyang 550025, China
  • Online:2023-07-20 Published:2023-07-20

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摘要： 目的 探讨肝细胞来源的富含亮氨酸α-2糖蛋白1（LRG1）对M1型肝巨噬细胞活化的影响。方法 BALB/c小鼠通过高脂饮食（HFD）喂养16周来建立代谢相关脂肪性肝病（MAFLD）模型，油酸诱导小鼠原代肝细胞发生脂肪变性，用RT-PCR和Western blot检测肝组织和肝细胞中LRG1的mRNA和蛋白水平的表达。将原代肝巨噬细胞分为5组，对照组、脂肪变性肝细胞来源的上清（CM）刺激组、CM+LRG1组、CM+转化生长因子-β1（TGF-β1）组、CM+TGF-β1+LRG1组，各组培养24 h后，Westernbot法检测一氧化氮合酶（iNOS）蛋白水平的表达，RT-PCR法检测iNOS、趋化因子1（CXCL-1）和白细胞介素-1β（IL-1β）的mRNA水平的表达。MAFLD小鼠分为4组，1组作为对照（6只），其余3组经尾静脉分别注射LRG1（6只）、TGF-β1（6只）或者TGF-β1+LRG1（6只），收集肝组织，经HE染色观察肝脏病理学变化，免疫组化法观察肝组织中F4/80+细胞的分布，RT-PCR法检测肝组织iNOS、CXCL-1和IL-1β的mRNA水平的表达。结果 HFD小鼠肝组织和脂肪变性的肝细胞中LRG1的mRNA和蛋白水平的表达显著降低（P<0.05）。脂肪变性肝细胞来源的CM刺激明显促进肝巨噬细胞中iNOS、CXCL-1和IL-1β的mRNA水平的表达，而TGF-β1和LRG1联合刺激时明显抑制这些分子mRNA水平的表达（P<0.05）。小鼠HFD16周后，单独注射LRG1或TGF-β1均可减少肝内脂质沉积以及肝内巨噬细胞的浸润，二者联合上述改变更加明显。同时，TGF-β1和LRG1联合刺激时也明显抑制iNOS、CXCL-1和IL-1β的mRNA水平的表达（P<0.05）。结论 LRG1通过增强TGF-β1信号通路抑制肝巨噬细胞浸润，从而缓解非酒精性脂肪肝的炎性反应。

关键词: 富亮氨酸α2-糖蛋1；肝巨噬细胞；代谢相关脂肪性肝病；转化生长因子-β1；脂肪变性

Abstract: Objective To explore the effect of leucine- rich α-2-glycoprotein (LRG1) derived from hepatocytes on activation of hepatic M1 Kupffer cells. Methods A metabolic dysfunction-associated fatty liver disease (MAFLD) model was established in BALB/c mice by high-fat diet (HFD) feeding for 16 weeks. Oleic acid was used to induce steatosis in primary cultures of mouse hepatocytes. The mRNA and protein expressions of LRG1 in mouse liver tissues and hepatocytes were detected by real-time PCR and Western blotting. Primary hepatic macrophages were stimulated with the conditioned medium (CM) from steatotic hepatocyte along with LRG1 or transforming growth factor-β1 (TGF-β1), or both for 24 h, and the expression levels of inducible nitric oxide synthase (iNOS) was detected with Western botting, and the mRNA expressions of iNOS, chemokine ligand 1 (CXCL-1) and interleukin-1β (IL-1β) were measured by RT-PCR. The MAFLD mice were injected with LRG1 (n=6), TGF-β1 (n=6), or both (n=6) through the caudal vein, and the live tissues were collected for HE staining and immumohistochemical detection of F4/80 expression; the mRNA expressions of iNOS, CXCL-1 and IL-1β in liver tissues were detected using RT-PCR. Results The mRNA and protein expression levels of LRG1 were significantly downregulated in the liver tissues of MAFLD mice and steatotic hepatocytes (P<0.05). Treatment of the hepatic macrophages with CM from steatosis hepatocytes significantly enhanced the mRNA expression levels of iNOS, CXCL-1 and IL-1β, and these changes were significantly inhibited by the combined treatment with TGF-β1 and LRG1 (P<0.05). In MAFLD mice, injections with either LRG1 or TGF-β1 alone reduced hepatic lipid deposition and intrahepatic macrophage infiltration, and these effects were significantly enhanced by their combined treatment, which also more strongly inhibited the mRNA expression levels of iNOS, CXCL-1 and IL-1β (P<0.05). Conclusion LRG1 inhibits hepatic macrophage infiltration by enhancing TGF-β1 signaling to alleviate fatty liver inflammation in MAFLD mice.

Key words: leucine-rich α-2-glycoprotein 1; hepatic macrophages; metabolic dysfunction-associated fatty liver disease; transforming growth factor-β1; steatosis