Open Hepatic Artery Flow During Portal Triad Blood Inflow Occlusion Protects Bile Duct And Its Mechanism

Objective(s):The purpose of this study is: 1.Implement the Pringle maneuver and other hepatic inflow occlusion(hepatic artery flow open or short-term intermittently hepatic artery occlusion)to establish a model of hepatic ischemia,and to study the effect of different hepatic inflow occlusion methods on the bile duct.2.Explore the potential mechanism of bile duct ischemic damage,and provide directions for preventing bile duct damage.Methods:1.The model of bile duct ischemia with hepatic inflow occlusion in rats was established by occlusion of hepatic artery and portal vein for different occlusion time.After the completion of ischemia,the clamping was released,after 24 hours of reperfusion,the mortality rate was calculated,biochemical analysis,coagulation function detection,and HE staining were performed to observe liver pathological changes.2.The rat was implemented the classic Pringle maneuver and discriminate hepatic inflow occlusion,and released the occlusion after the occlusion was completed.After 24 hours of reperfusion,the samples were collected for biochemical analysis,histological observation,cytokine detection,blood-biliary barrier integrity detection,and claudin-related molecular analysis.3.Transcriptomic analysis of bile duct tissue was performed,and the sham group was used as a control to analyze the differential gene and functional enrichment of each group;and then according to the results of the second part,CPM group and CHAFO group were analyzed,and differential gene enrichment analysis was performed.4.Based on the results of transcriptomic analysis,kynureninase,a key molecule involved in bile duct injury,was identified as a target for further exploration.Hypoxia reoxygenation with primary bile duct epithelial cells simulates the process of ischemia-reperfusion,detects changes of kynureninase,and detects changes in cytokines in cell culture media.In the persistent hepatic blood flow occlusion group,the expression of kynureninase was interfered,and changes in plasma amino acids,biliary enzyme,biliary histology,blood-biliary barrier,tight-junction related molecules were detected.Results:1.During the groups of continuous clamping the hepatic artery and portal vein indiscriminately,no rats died in the 30 min group,1 rat died in the 40 min group,3 rats died in the 50 min group,and 5 rats died in the 60 min group during the observation time.Biochemical results were significantly increased in the 40 minutes groups and longer than 40 min groups,and the difference was statistically significant compared with the sham group.There were obvious inflammatory cells infiltration in the potal area in the group of 40 minutes or longer groups,and piece-meal necrosis appeared in hepatocytes in the 60 min group.There was no significant difference between the four occlusion groups of coagulation and the sham group.2.Compared with the Pringle maneuver(PM)groups(CPM and IPM group)and the discriminate hepatic flow occlusion(HAFO)groups(CHAFO and IHAFO).The infiltration of inflammatory cells in the portal area was more significant in the PM groups.The apoptosis of cholangiocytes in PM groups were more than that in HAFO groups,and there was no statistical difference between CHAFO group and IHAFO groups.The concentration of LDH in PM groups were higher than that in HAFO groups,and there was no significant difference between CHAFO group and IHAFO group.In blood-bile barrier detection:(1)HRP activity were higher in PM groups than HAFO groups,and there was no significant difference between CHAFO group and IHAFO group;(2)ZO-1 expression were irregular in PM groups,and expressed regularly in HAFO groups;(3)The down-regulation of tight junction-related molecules Claudin-1 and Claudin-3 at the transcriptional and translational levels were more obvious in the PM groups than in the HAFO groups.3.In the transcriptome analysis,compared with the Sham group,the CPM group and the CHAFO group showed that the differentially expressed genes were enriched in cytochrome P450-related signaling pathways and substance metabolism-related signaling pathways;compared with the CPM and CHAFO groups,more differential genes were enriched in metabolism-related signaling pathway.The difference of kynureninase was significant,and it was the key molecule.4.In vitro experiments,the expression of kynureninase was upregulated after hypoxia reoxygenation in primary bile duct epithelial cells.After cell hypoxia and reoxygenation,interfering with expression of KYNU could not antagonize the upregulation of kynureninase expression caused by hypoxia.After cell hypoxia and reoxygenation,the expression of kynureninase intervention could reduce the upregulation of IL-6 and MCP-1 cytokine expression,but the effect on IL-10 expression was not statistically significant.5.In vivo experiments,the levels of serum kynrenine(kynrenine)and3-hydroxykynurenine(3-HK)increased after interfering or inhibiting the expression of kynureninase;and levels of anthranilic acid(AA),3-hydroxyanthranilic acid(3HAA),quinolinic acid(QA)were decreased.The bile associated enzymes were decreased;histological and ultrastructural damage of bile ducts were ameliorated,levels of IL-6,MCP-1,and TNF-α increased,levels of IL-10 were decreased,leakage of Indian ink and HRP activity in bile were lighted,Claudin3 and Claudin5 expression were upregulated,and Occludin expression was downregulated.Conclusion(s):1.Whlie the hepatic artery and portal vein were continuously clamped simultaneouly,ischemic damage to the bile duct occured when the occlusion time is longer than 40 minutes.Hepatocytes are necrosis when the occlusion time up to 60 minutes.The bile duct system is more sensitive to ischemia than hepatocytes;2.Open hepatic artery flow during portal triad blood inflow occlusion can reduce ischemic damage of the bile duct;3.Multiple signaling pathways are involved in ischemic damage of the bile ducts,among which tryptophan-kynuretinase metabolic pathways are significantly different,and kynureninase is a key molecule;4.The tryptophan-kynurate metabolic pathway is involved in ischemic damage of the bile ducts,and downregulation or inhibition of kynureninase expression can reduce the ischemic damage of the bile ducts.