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  

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

作　　者：曹家樊 孙跃 丁鑫[1,2,3,4] 李盛文 陈博 兰天 CAO Jiafan;SUN Yue;DING Xin;LI Shengwen;CHEN Bo;LAN Tian(School of Pharmacy,Guangdong Pharmaceutical University,Guangzhou 510006,China;Institute of Chinese Medicine,Guangdong Pharmaceutical University,Guangzhou 510006,China;Key Laboratory of Glucolipid Metabolic Disorder of the Ministry of Education,Guangdong Pharmaceutical University,Guangzhou 510006,China;Guangdong Metabolic Diseases Research Center of Integrated Chinese and Western Medicine,Guangdong Pharmaceutical University,Guangzhou 510006,China)

机构地区：[1]广东药科大学药学院,广东广州510006 [2]广东药科大学中医药研究院,广东广州510006 [3]广东药科大学糖脂代谢病教育部重点实验室,广东广州510006 [4]广东药科大学、广东省代谢性疾病中医药防治重点实验室,广东广州510006

出　　处：《南方医科大学学报》2024年第4期652-659,共8页Journal of Southern Medical University

基　　金：国家自然科学基金(82070590);广东省基础与应用基础研究重大项目(2019B030302005)。

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(25mg/kg)和Arb高剂量给药组(50mg/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;Westernblot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24h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24h,Westernblot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。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,and low-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 ofα-SMA,Pdgfb,Col1α1,Timp-1,Ccl2 and Tnf-a,and Western blotting was performed to detectα-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 andα-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 andα-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 ofα-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.

关 键 词：熊果苷 炎症 肝纤维化 巨噬细胞募集 肝星状细胞 

分 类 号：R285.5[医药卫生—中药学]