目的:探讨熊果苷对CCl4诱导的小鼠肝纤维化的保护作用及其机制。
方法:24只C57BL/6小鼠随机分为对照组,模型组,以及低剂量和高剂量熊果苷治疗(25和50mg/kg,分别)组。腹腔注射CCl4建立小鼠肝纤维化模型,熊果苷每日灌胃给药6周。治疗后,对小鼠的血清生化指标进行了测试,取肝脏组织进行HE染色,天狼星红染色和免疫组织化学染色。RT-qPCR检测α-SMAmRNA水平,pdgfb,Col1α1,Timp-1,Ccl2和Tnf-a,免疫印迹法检测肝组织中α-SMA蛋白的表达。在细胞实验中,使用Transwell迁移试验和DAPI染色检查熊果苷处理24h对THP-1和RAW264.7细胞迁移和募集的影响;Akt蛋白水平的变化,P65,Smad3,p-Akt,用蛋白质印迹法检测经阿布丁素处理的LX-2细胞中的p-p65、p-Smad3和α-SMA。
结果:熊果苷治疗显著降低血清谷丙转氨酶和谷草转氨酶水平,减轻肝组织损伤和胶原沉积,小鼠模型肝脏中巨噬细胞浸润和α-SMA蛋白表达降低(P<0.05或0.001)。熊果苷治疗也显著降低CCl4诱导的a-SMA升高,pdgfb,小鼠Col1α1、Timp-1、Ccl2和Tnf-amRNA水平(P<0.05)。在细胞实验中,熊果苷处理明显抑制THP-1和RAW264.7细胞的迁移和募集,降低Akt的磷酸化水平,p65和Smad3及α-SMA蛋白在LX-2细胞中的表达水平。
结论:熊果苷通过减少巨噬细胞募集和浸润抑制肝星状细胞活化,抑制Akt/NF-κB和Smad信号通路的活化,从而改善小鼠肝脏炎症和纤维化。
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 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 arbutintreated 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.
CONCLUSIONS: 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.