Background In clinical liver transplantation, whether the delay of hepatic arterial ischaemia increases biliary fibrosis or not is controversial. We designed a liver transplantation model to test this controversy and explore its mechanism. Methods Twelve dogs were divided into two groups randomly： hepatic arterial ischaemia （HAI） and control groups. In HAI group, hepatic artery was perfused 60 minutes after portal perfusion, but in control group, hepatic arterial perfusion was simultaneous with portal perfusion. The pathological changes of intrahepatic bile ducts were observed. Transforming growth factor beta 1 （TGF-β1）, expressed in epithelial cells of intrahepatic bile duct, was detected by immunohistochemical streptoadividin-biotin complex method. Expressions of Smad3, P-Smad3 and the transcriptional levels of alpha smooth muscle actin （α-SMA） mRNA in intrahepatic bile ducts were detected by Western blotting and RT-PCR respectively.Results Compared with the control group, more collagen deposition and leucocytic infiltration could be seen in biliary vessel walls. Significantly more buffy particles, which are the proteins of TGF-β1, could be seen in biliary epithelial cells. P-Smad3 and α-SMA mRNA （as ratio to corresponding β-actin） in intrahepatic bile ducts were 1.82±0.18 and 1.86±0.73 respectively in HAI group, significantly higher than those in control group （0.59±0.09 and 0.46±0.18, respectively）. Conclusions Hepatic arterial ischaemia could increase the deposition of collagen fibres, trigger the transdifferentiation of myofibroblasts in intrahepatic bile duct and might result in biliary fibrosis by activating the TGF-β1 signalling pathway.
LU Hong-weiCHEN Yong-bingLI Yi-mingDONG Jia-hongYANG Hui-ning
Objective：To investigate the effect of hepatic ischemia-reperfusion（I/R） on bile canalicular F-actin microfilaments in rats. Methods： A rat model of hepatic ischemia-reperfusion was employed and the ischemia time was 35 min. The activity of serum alanine aminotransferase （ALT）, aspartate aminotransferase（AST）, γ-glutamyl transferase（GGT） and the level of total bilirubin（TBIL） were measured. Changes in the bile canaliculi were observed by transmission electron microscope. The modification of F-actin microfilaments was quantified by using FITC-Phalloidin and analyzed by confocal laser scanning microscopy imaging. Results：Modifications of F-actin staining were consistent with the observations made by transmission electron microscopy. The staining of F-actin was normal in hepatocytes before reperfusion but decreased significantly after reperfusion, and there was a marked loss of canalicular microvilli after reperfusion, which coincided with abnormal serum GGT and TBIL levels. Conclusion：Reperfusion, not short-term ischemia, induced a disruption of F-actin microfilaments and a loss of microvilli. These modifications could lead to the impaired bile secretion by damaging canalicular contraction, and could be the main mechanisms of cholestasis after hepatic ischemia-reperfusion in rats.
Objective： To investigate whether the method of bridgy duct established between the recipient＇s spleen artery and the donor＇s gastroduodenal artery could inhibit the apoptosis of liver and bile duct cells caused by hepatic artery ischemia. Methods： Twenty-four mongrel dogs from Xi＇an area were used to establish simplified models of dog orthotopic liver transplantation and divided into three groups randomly： HAI group （hepatic artery ischemia group）,BBB group（bypassing the blood by a bridgy duct） and control group. After cold perfusion, The samples were collected from liver and bile duct in each group at different time and fixed in glutaraldehyde and 4% polyformaldehyde respectively. At last, the apoptosis of liver and bile duct cells were observed and the apoptotic indexes were calculated. Results： Two hours after cold perfusion, apoptotic phenomenon was common in HAI group, rare in BBB group, while no apoptotic phenomenon was observed in control group. TUNEL staining showed that there was no significant difference in apoptotic index among the three groups immediately after cold perfusion. However, with time going, the apoptotic cells were increased in three groups, and the difference in apoptotic index was significant among three groups （P 〈 0.01 ）. Conclusions： Bridgy duct of hepatic artery can inhibit the apoptosis of liver and bile duct cells caused by HAI significantly.
