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 （SEC...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.展开更多
基金Supported by China Postdoctoral Science Foundation fundedproject(No.20110490750)Shanghai Leading AcademicDiscipline Project(No.Y0302)+2 种基金E-institute of ShanghaiMunicipal Education Commission(No.E03008)Innovative Research Team in Universities,Shanghai Municipal Education Commission,Key Disciplines of Liver and Gallbladder Diseases and Key Laboratory of Chronic Liver Disease of State Administration of Traditional Chinese Medicine of China,National Basic Research Program of China(No.2006CB504801)National S&T Major Project(No.2009ZX09311-003)
文摘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.
