Zhao Zhi-Min, Liu Hong-Liang, Sun Xin, Guo Tao, Shen Li, Tao Yan-Yan, Liu Cheng-Hai
1 Institute of Liver Diseases, Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
2 Shanghai Key Lab of Liver Diseases of TCM, Shanghai 201203, China.
Exp Biol Med (Maywood). 2017 May;242(9):974-985. doi: 10.1177/1535370217701005. Epub 2017 Mar 22.
Levistilide A (CHO, molecular weight = 380.48) derived from Angelica sinensis (Danggui) has been reported to inhibit hepatic stellate cell proliferation. This study investigated the effects of levistilide A on liver fibrosis relating to angiogenesis, particularly on the characteristic change in liver sinusoidal endothelial cells. LX-2 cells were activated by TGF-β1, and the human hepatic sinusoidal endothelial cells (HHSECs) were induced by endothelial cell growth supplement. Cell viability was detected using a methylthiazoldiphenyl-tetrazolium bromide assay; F-actin was visualized through the fluorescence probe method; cell proliferation was examined using the EdU kit; antiangiogenesis activity was assessed using the tube formation assay and transgenic zebrafish model. To verify the results in vivo, rats were subcutaneously injected with CCl twice a week for six weeks to duplicate the liver fibrosis model and then treated with 10 mL/kg of normal saline, 4 mg/kg of sorafenib, and 3 and 6 mg/kg of levistilide A for three weeks from the fourth week. Collagen deposition was detected through Sirius Red staining; liver microvasculature was examined through vWF labeling and X-ray 2D imaging; sinusoidal fenestrations were observed through scanning electron microscopy; collagen I, α-SMA, CD31, vascular endothelial growth factor (VEGF), and VEGF-R2 were detected through Western blotting. Our results indicated that levistilide A attenuated LX-2 cell activation and HHSEC proliferation. The ability of HHSECs to form tubelike structures in Matrigel was inhibited, and the number of functional vessels in transgenic zebrafish decreased. In in vivo experiments, levistilide A reduced collagen deposition and the number of new microvessels; ameliorated sinusoid capillarization; and downregulated the expression of CD31, VEGF, and VEGF-R2. These findings suggest that levistilide A can inhibit liver fibrosis through antiangiogenesis by alleviating sinusoid capillarization via the VEGF signaling pathway. Impact statement Levistilide A has been reported to inhibit hepatic stellate cell (HSC) proliferation. In this study, we further investigated the mechanisms of levistilide A on liver fibrosis relating to angiogenesis, particularly on the characteristic change in liver sinusoidal endothelial cells. The cell models of HSC and liver sinusoidal endothelial cell and CCl induced liver fibrosis model were used. These results suggest that levistilide A can inhibit liver fibrosis through antiangiogenesis by alleviating sinusoid capillarization via the vascular endothelial growth factor signaling pathway. The effect of levistilide A on liver fibrosis was confirmed, and its detailed mechanism was also discussed. These findings suggest that levistilide A may be a great potential drug for treating liver fibrosis through antiangiogenesis, and this effect will be verified in other fibrotic animal model studies or by clinical trials.
据报道，源自当归的阿魏酸A（化学式C₁₉H₂₈O₄，分子量 = 380.48）可抑制肝星状细胞增殖。本研究调查了阿魏酸A对与血管生成相关的肝纤维化的影响，特别是对肝窦内皮细胞特征性变化的影响。用转化生长因子-β1激活LX-2细胞，并用内皮细胞生长补充剂诱导人肝窦内皮细胞（HHSEC）。使用甲基噻唑二苯基溴化四氮唑法检测细胞活力；通过荧光探针法观察F-肌动蛋白；使用EdU试剂盒检测细胞增殖；使用管形成试验和转基因斑马鱼模型评估抗血管生成活性。为了在体内验证结果，每周两次给大鼠皮下注射四氯化碳，持续六周以复制肝纤维化模型，然后从第四周开始用10 mL/kg生理盐水、4 mg/kg索拉非尼以及3和6 mg/kg阿魏酸A治疗三周。通过天狼星红染色检测胶原沉积；通过血管性血友病因子标记和X射线二维成像检查肝脏微血管；通过扫描电子显微镜观察肝血窦窗孔；通过蛋白质免疫印迹法检测I型胶原、α-平滑肌肌动蛋白、CD31、血管内皮生长因子（VEGF）和VEGF-R2。我们的结果表明，阿魏酸A可减弱LX-2细胞的活化和HHSEC的增殖。HHSEC在基质胶中形成管状结构的能力受到抑制，转基因斑马鱼中的功能性血管数量减少。在体内实验中，阿魏酸A减少了胶原沉积和新生微血管的数量；改善了肝血窦毛细血管化；并下调了CD31、VEGF和VEGF-R2的表达。这些发现表明，阿魏酸A可通过VEGF信号通路减轻肝血窦毛细血管化，从而通过抗血管生成作用抑制肝纤维化。影响声明 据报道，阿魏酸A可抑制肝星状细胞（HSC）增殖。在本研究中，我们进一步研究了阿魏酸A对与血管生成相关的肝纤维化的作用机制，特别是对肝窦内皮细胞特征性变化的影响。使用了HSC和肝窦内皮细胞的细胞模型以及四氯化碳诱导的肝纤维化模型。这些结果表明，阿魏酸A可通过VEGF信号通路减轻肝血窦毛细血管化，从而通过抗血管生成作用抑制肝纤维化。证实了阿魏酸A对肝纤维化的作用，并讨论了其详细机制。这些发现表明，阿魏酸A可能是一种通过抗血管生成治疗肝纤维化的极具潜力的药物，这种作用将在其他纤维化动物模型研究或临床试验中得到验证。
