南方医科大学学报

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (4): 652-659.doi: 10.12122/j.issn.1673-4254.2024.04.05

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熊果苷通过抑制巨噬细胞募集并调控Akt/NF-κB和Smad信号通路改善小鼠肝纤维化

曹家樊，孙 跃，丁 鑫，李盛文，陈 博，兰 天   

  1. 广东药科大学药学院，中医药研究院，糖脂代谢病教育部重点实验室，广东省代谢性疾病中医药防治重点实验室，广东 广州 510006
  • 发布日期:2024-04-29

Arbutin ameliorates liver fibrosis in mice by inhibiting macrophage recruitment and regulating the Akt/NF-κB and Smad signaling pathways

CAO Jiafan, SUN Yue, DING Xin, LI Shengwen, CHEN Bo, LAN Tian   

  1. School of Pharmacy, Institute of Chinese Medicine, Key Laboratory of Glucolipid Metabolic Disorder of the Ministry of Education, Guangdong Metabolic Diseases Research Center of Integrated Chinese and Western Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, China
  • Published:2024-04-29

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摘要： 目的 探究熊果苷（Arb）对CCl4诱导的小鼠肝纤维化的保护作用及其机制。方法 将24只C57BL/6小鼠随机分为对照组、模型组、Arb低剂量给药组（25 mg/kg）和Arb高剂量给药组（50 mg/kg），6只/组。通过腹腔注射CCl4建立肝纤维化小鼠模型，灌胃给药持续6周后取材。取血清进行生化指标检测，取肝组织进行HE染色、天狼猩红染色和免疫组化染色；q-PCR检测肝组织纤维化相关指标a-SMA，Pdgfb，Col1α1，Timp-1基因以及炎症相关指标Ccl2和Tnf-a基因的mRNA水平；Western blot法检测肝组织α-SMA蛋白表达水平。Transwell迁移实验使用Arb处理THP-1和RAW264.7细胞24 h后，DAPI染色检测迁移细胞数目。体外实验使用Arb处理LX-2细胞24 h，Western blot法检测Akt、p65、Smad3及其磷酸化p-Akt、p-p65、p-Smad3和α-SMA的蛋白水平。结果 与模型组小鼠相比，Arb给药组小鼠血清谷丙转氨酶、谷草转氨酶水平降低（P<0.05），肝组织损伤和胶原沉积减轻（P<0.001），肝脏巨噬细胞浸润程度和α-SMA蛋白表达水平也降低（P<0.001）。Arb降低CCl4诱导的小鼠肝脏a-SMA，Pdgfb，Col1α1，Timp-1，Ccl2和Tnf-a基因的mRNA水平（P<0.05）。Transwell迁移实验显示，Arb可抑制THP-1和RAW264.7细胞的迁移募集作用。体外实验显示，Arb可抑制LX-2细胞Akt、p65和Smad3的磷酸化，并且降低α-SMA的蛋白表达水平。结论 Arb可通过减少巨噬细胞募集和浸润，同时干预Akt/NF-κB和Smad信号通路抑制肝星状细胞活化，从而改善小鼠肝脏炎症及纤维化。

关键词: 熊果苷；炎症；肝纤维化；巨噬细胞募集；肝星状细胞

Abstract: Objective To investigate the protective effect of arbutin against CCl4-induced hepatic fibrosis in mice and explore the underlying mechanisms. Methods Twenty- four C57BL/6 mice were randomly divided into control group, model group, andlow- and high-dose arbutin treatment (25 and 50 mg/kg, respectively) groups. Mouse models of liver fibrosis were established by intraperitoneal injection of CCl4, and arbutin was administered daily via gavage for 6 weeks. After the treatments, serum biochemical parameters of the mice were tested, and liver tissues were taken for HE staining, Sirius Red staining and immunohistochemical staining. RT-qPCR was used to detect the mRNA levels of α-SMA, Pdgfb, Col1α1, Timp-1, Ccl2 and Tnf-a, and Western blotting was performed to detect α-SMA protein expression in the liver tissues. In the cell experiment, the effect of arbutin treatment for 24 h on THP-1 and RAW264.7 cell migration and recruitment was examined using Transwell migration assay and DAPI staining; The changes in protein levels of Akt, p65, Smad3, p-Akt, p-p65, p-Smad3 and α-SMA in arbutin-treated LX-2 cells were detected with Western blotting. Results Arbutin treatment significantly lowered serum alanine aminotransferase and aspartate aminotransferase levels, alleviated liver tissue damage and collagen deposition, and reduced macrophage infiltration and α-SMA protein expression in the liver of the mouse models (P<0.05 or 0.001). Arbutin treatment also significantly reduced CCl4-induced elevation of a-SMA, Pdgfb, Col1α1, Timp-1, Ccl2 and Tnf- a mRNA levels in mice (P<0.05). In the cell experiment, arbutin treatment obviously inhibited migration and recruitment of THP-1 and RAW264.7 cells and lowered the phosphorylation levels of Akt, p65 and Smad3 and the protein expression level of α-SMA in LX-2 cells. Conclusion Arbutin ameliorates liver inflammation and fibrosis in mice by inhibiting hepatic stellate cell activation via reducing macrophage recruitment and infiltration and suppressing activation of the Akt/NF-κB and Smad signaling pathways.

Key words: arbutin; inflammation; liver fibrosis; macrophage recruitment; hepatic stellate cells