目的:乌灵胶囊(WL)在慢性乙型肝炎肝损伤的临床治疗中具有较好的疗效。肝纤维化是慢性肝病的常见病理特征,可发展为不可逆的肝硬化和肝癌。越来越多的证据表明,调节巨噬细胞极化有助于肝纤维化的治疗。然而,WL对调节巨噬细胞极化以减轻肝纤维化的作用尚不清楚。本研究探讨了WL在四氯化碳(CCl4)诱导的大鼠肝纤维化中的抗肝纤维化作用。以及对巨噬细胞极化的调节作用和潜在的分子机制。
方法:通过腹腔注射40%CCl4橄榄油混合物建立大鼠肝纤维化模型。在第2、4、6和8周,采用苏木精-伊红(HE)和Masson染色评估肝脏的组织病理学状况;在大鼠肝脏组织中测量肝脏生化指标。ELISA法检测肝组织中炎性细胞因子的表达水平。不同表型巨噬细胞标记物的mRNA水平和蛋白表达,ELISA法独立检测TLR4-NF-κB信号通路指标,免疫荧光,RT-PCR和蛋白质印迹。
结果:体内,WL治疗减轻了体重的异常变化,器官指数和生化指标,减轻病理变化,肝组织中胶原纤维沉积和α-SMA表达减少。进一步的研究表明,WL降低了巨噬细胞M1极化标记物诱导型一氧化氮合酶(iNOS)的表达,TNF-α,IL-6和CD86促进M2巨噬细胞极化标志物IL-10,CD206和精氨酸酶-1(Arg-1)的表达,并通过几个关键信号蛋白抑制TLR4-NF-κB信号通路的激活。体外,WL显著抑制巨噬细胞M1极化,并促进了LPS激活的RAW264.7细胞的M2极化,同时增强了M1极化向M2极化的转变。
结论:本研究表明,WL主要通过抑制TLR4-NF-κB信号通路的激活来调控巨噬细胞极化抗肝纤维化。
OBJECTIVE: Wuling capsule (WL) has good efficacy in the clinical treatment of chronic hepatitis B and liver injury. Liver fibrosis is a common pathological feature of chronic liver disease and may progress to irreversible cirrhosis and liver cancer. Accumulating evidence reveals that modulating macrophage polarization contribute to the therapy of liver fibrosis. However, the effects of WL on modulating macrophage polarization to relive liver fibrosis remain unclear. This study investigated the anti-liver fibrosis effects of WL in carbon tetrachloride (CCl4)-induced liver fibrosis in rats, and the modulation effects and underlying molecular mechanism on macrophage polarization.
METHODS: A rat liver fibrosis model was constructed by intraperitoneal injection of 40 % CCl4 olive oil mixture. At 2, 4, 6, and 8 weeks, the histopathological status of the liver was assessed by hematoxylin-eosin (HE) and Masson staining; the liver biochemical indexes were measured in rat liver tissue. The expression levels of inflammatory cytokines in liver tissue were detected by ELISA. The mRNA levels and proteins expression of macrophage markers of different phenotypes, TLR4-NF-κB signaling pathway indicators were detected independently by ELISA, immunofluorescence, RT-PCR and western blotting.
RESULTS: In vivo, WL treatment attenuated abnormal changes in weight, organ indices and biochemical indices, alleviated pathological changes, and reduced collagen fiber deposition as well as the expression of α-SMA in liver tissues. Further studies revealed that WL decreased the expression of the macrophage M1 polarization markers inducible nitric oxide synthase (iNOS), TNF-α, IL-6, and CD86, promoted the expression of the M2 macrophage polarization markers IL-10, CD206, and arginase-1 (Arg-1), and inhibited the activation of the TLR4-NF-κB signaling pathway via several key signaling proteins. In vitro, WL significantly suppressed macrophage M1 polarization, and promoted M2 polarization while boosted M1 polarization transform to M2 polarization in LPS-activated RAW264.7 cells.
CONCLUSIONS: This study demonstrated that WL modulated macrophage polarization against liver fibrosis mainly by inhibiting the activation of the TLR4-NF-κB signaling pathway.