Xiayuxue Decoction(下瘀血汤)Attenuates Hepatic Stellate Cell Activation and Sinusoidal Endothelium Defenestration in CCI_4-Induced Fibrotic Liver of Mice

Objective:To investigate the effects of ancient Chinese medical formula Xiayuxue Decoction(下瘀血汤,XYXD) on activation of hepatic stellate cells(HSCs) and defenestration of sinusoidal endothelial cells(SECs) in CCI_4-induced fibrotic liver of mice.Methods:High performance liquid chromatography was used to identify the main components of XYXD and control the quality of extraction.C57BL/6 mice were induced liver fibrosis by CCl_4 exposure and administered with XYXD for 6 weeks simultaneously.Liver tissue was investigated by hematoxylin-eosin and Sirius-red staining.Sinusoidal fenestrations were observed by scanning electronic microscopy and fluorescent immunohistochemistry of PECAM-1(CD31).Whole liver lysates were detected of α-smooth muscle actin(α-SMA) and type-1 collagen by Western blot.Primary rat HSCs-T6 cells were analyzed by detecting a-SMA,F-actin,DNA fragmentation through confocal microscopy,Western blot,terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL) assay and cellomics arrayscan,respectively.Results:Amygdalin and emodin in XYXD were identified.XYXD(993 mg/kg) inhibited Sinus red positive area up to 70.1%(P0.01),as well as protein levels of α-SMA and type-1 collagen by42.0%and 18.5%(P0.05) respectively.In vitro,XYXD(12.5 μg/mL,50 μg/mL) suppressed the activation of HSCs and reversed the myofibroblastic HSCs into quiescent,demonstrated as inhibition of fluorescent F-actin by 32.3%and 46.6%(P0.05).Besides,XYXD induced the apoptosis of HSC-T6 cells by 20.0%(P0.05)and 49.5%(P0.01),evidenced by enhanced TUNEL positivity.Moreover,ultrastructural observation suggested XYXD inhibited defenestration of SECs,which was confirmed by 31.1%reduction of protein level of CD31(P0.05).Conclusions:XYXD inhibited both HSCs activation and SECs defenestration which accompany chronic liver injuries.These data may help to understand the underlying mechanisms of XYXD for prevetion of chronic liver diseases.     

野菊花提取物舒张血管及抗炎机制研究

目的 研究野菊花舒张血管及抗炎机制,同时探索其活性物质基础.方法 通过离体血管环张力实验观察野菊花提取物及其主要单体对大鼠胸主动脉环经苯肾上腺素引起血管环收缩的影响.以Griess反应检测野菊花提取物及其单体对LPs(1 μg/ml)/IFN-γ(10 U/ml)刺激巨噬细胞RAW264.7释放NO的抑制作用;噻唑兰(MTT)比色法检测二者对细胞活力的影响;western blot检测二者对诱导型一氧化氮合酶(iNOS)及环氧合酶-2(COX-2)的抑制作用.结果 野菊花提取物、木犀草素对血管的舒张效应为部分内皮依赖性、部分非内皮依赖性;二者对均能剂量依赖性地抑制炎症巨噬细胞生成NO、抑制iNOS表达,同时无细胞毒性,对COX-2表达无影响.结论 野菊花提取物的血管舒张效应,抑制炎症细胞内NO及其诱导型合酶iNOS表达可能是其药理作用机制.     

下瘀血汤减少CCl4诱导小鼠肝纤维化肝星状细胞激活及肝窦内皮细胞死亡的相关研究

Objective： To investigate the effects of ancient Chinese medical formula Xiayuxue Decoction （下瘀血汤, XYXD） on activation of hepatic stellate cells （HSCs） and defenestration of sinusoidal endothelial cells （SECs） in CCI4-induced fibrotic liver of mice. Methods： High performance liquid chromatography was used to identify the main components of XYXD and control the quality of extraction. C57BL/6 mice were induced liver fibrosis by CCI4 exposure and administered with XYXD for 6 weeks simultaneously. Liver tissue was investigated by hematoxylin-eosin and Sirius-red staining. Sinusoidal fenestrations were observed by scanning electronic microscopy and fluorescent immunohistochemistry of PECAM-1 （CD31）. Whole liver lysates were detected of α-smooth muscle actin （α-SMA） and type-I collagen by Western blot. Primary rat HSCs-T6 cells were analyzed by detecting α-SMA, F-actin, DNA fragmentation through confocal microscopy, Western blot, terminal-deoxynucleoitidyl transferase mediated nick end labeling （TUNEL） assay and cellomics arrayscan, respectively. Results： Amygdalin and emodin in XYXD were identified. XYXD （993 mg/kg） inhibited Sirius red positive area up to 70.1% （P〈0.01）, as well as protein levels of α-SMA and type- I collagen by 42.0% and 18.5% （P〈0.05） respectively. In vitro, XYXD （12.5 μg/mL, 50 μg/mL） suppressed the activation of HSCs and reversed the myofibroblastic HSCs into quiescent, demonstrated as inhibition of fluorescent F-actin by 32.3% and 46.6% （P〈0.05）. Besides, XYXD induced the apoptosis of HSC-T6 cells by 20.0% （P〈0.05） and 49.5% （P〈0.01）, evidenced by enhanced TUNEL positivity. Moreover, ultrastructural observation suggested XYXD inhibited defenestration of SECs, which was confirmed by 31.1% reduction of protein level of CD31 （P〈0.05）. Conclusions： XYXD inhibited both HSCs activation and SECs defenestration which accompany chronic liver injuries. These data may help to understand the underlying mechanisms of XYXD for prevetion of chronic liver diseases.