Xiayuxue Decoction（下瘀血汤）Attenuates Hepatic Stellate Cell Activation and Sinusoidal Endothelium Defenestration in CCI-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 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 CCl4 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%（P<0.01）,as well as protein levels of α-SMA and type-1 collagen by42.0%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.