Extrahepatic Bile Duct Ligation Research Articles

Analysis of gut microbiota in rats with bile duct obstruction after biliary drainage

The abundance of specific gut microorganisms is strongly associated with the concentrations of microbially modified bile acids. This study aimed to investigate the composition of intestinal microbiota in rats subjected to bile duct ligation or biliary drainage. Extrahepatic bile duct ligation was conducted to induce bile duct obstruction in rats. The bile was drained via a percutaneous biliary drainage catheter to cause bile deficiency. The total DNA extracted from fecal samples was sequenced with 16S DNA sequencing. Taxonomic classifications were conducted using the Mothur algorithm and SILVA138 database and were presented along with the abundance presented using a heatmap. The inter- and intra-group differences in the intestinal microbiome composition were analyzed by ANOSIM test. The biomarker microorganisms were screened using the Linear discriminant analysis Effect size method. The possible functional pathways were predicted using the Tax4Fun package. A total of 3277 operational taxonomic units (OTUs) were examined, with 2410 in the Kongbai group, 2236 in the Gengzu group, and 1763 in the Yinliu group. The composition of microorganisms at the levels of phylum, class, order, family, and genus was altered in rats with bile duct obstruction. This composition was then restored by biliary drainage. The top 10 predominant microorganisms were identified that led to the inter-group differences. Functional annotation revealed that the potential functions of the microorganisms with significant differences were enriched in metabolism, cellular processes, and genetic and environmental information processing. The intestinal microbial community was significantly changed in rats with bile duct obstruction. The changes in the abundance of intestinal microbiota Prevotellaceae and Enterobacteriaceae were statistically significant after biliary drainage treatment.

Progressive Collecting Liver Lymphatic Vessels Dysfunction over the Course of Extrahepatic Cholestasis

Liver produces a large fraction of total lymph flow (25–50%). Transport function of lymphatics is altered during inflammation, which impacts the pathogenesis of numerous diseases. Despite their importance in liver function, the functional characteristics of liver lymphatic vessels (LLVs) have been never evaluated in health and disease. We evaluated liver lymphatic vessel (LLV) function in normal and cholestatic rats following extrahepatic bile duct ligation (BDL). LLV contractility/pumping characteristics were determined in LLVs isolated from Sprague‐Dawley rats in control, 1, 3 and 7 days after BDL (BDL1, BDL3, and BDL7) and sham groups. LLV pumping/contractility was analyzed in all groups. Control LLVs had outer diameters of ~500 um, typical lymphatic tone (~7%), moderate contraction amplitude (15–35%), high contraction frequency (12–21 contractions/min) and strong active pumping (5–7.5 vol/min). Over the course of its duration, the BDL significantly altered LLV contractile/pump function, inducing an enlargement of LLVs; their decreased tone; lowering of phasic contractions frequency responses to increases in stretch. Cumulatively, BDL induced progressive decline in minute pumping of LLVs with complete cessation of phasic lymphatic contractions after seven days of BDL. In addition, BDL even after one day completely ablated the light imposed flow‐dependent relaxatory/inhibitory reactions of LLVs observed in control. Progressive dysfunction of LLVs correlates with cholestasis‐induced alterations in liver and bile ducts. We believe that observed BDL‐induced changes in LLVs are likely the result of the liver inflammation and will further promote the inflammatory edema known to occur in the liver during BDL. Thus, LLV dysfunction may play important roles in the ensuing pathogenesis of cholestasis and merit further study.Support or Funding InformationNIH/NIDDK R01 DK110035; NIH/NHLBI 1U01HL123420; funds from TAMU HSC COM and Dept. of Medical Physiology.

Inhibition of thrombospondin‐1 reduces transforming growth factor beta 1 signaling, hepatic fibrosis and neurological deficits during cholestasis in rats

IntroductionPrimary sclerosing cholangitis (PSC) is a liver disease associated with biliary inflammation, fibrosis and ultimately liver cirrhosis. During decompensated liver cirrhosis, hepatic encephalopathy (HE) can develop leading to increased morbidity and mortality. One rodent model that mimics clinical aspects of PSC is extrahepatic bile duct ligation (BDL). Transforming growth factor beta 1 (TGFβ1) has been shown to induce activation of hepatic stellate cells and promote liver fibrosis. For TGFβ1 to bind and activate its receptor, it must be released from a protein complex by interactions with other proteins. One of these is a matricellular protein called thrombospondin‐1 (TSP‐1). Therefore, we hypothesize that the expression of TSP‐1 is upregulated during BDL and promotes TGFβ1 signaling, liver injury, fibrosis and HE.MethodsMale Sprague Dawley rats (150–175 g) underwent BDL surgery and were implanted with osmotic minipumps that infused a TSP‐1 inhibitory peptide (LSKL, 100 g/day for 5 weeks) or inactive peptide (SLLK, 100 g/day for 5 weeks) into the peritoneum. Rotarod performance and open‐field tests were performed bi‐weekly starting at 2 weeks post‐surgery. After 5 weeks, rats were euthanized and tissue was collected. Serum ammonia, alanine and aspartate transaminases, total bilirubin and alkaline phosphatase were measured. TGFβ1, SMAD2/3, phosphorylated SMAD2/3 and TSP‐1 expression were assessed in liver, serum and cortex. Fibrosis was determined in liver sections by Sirius Red staining, hydroxyproline assays and qPCR analyses for alpha smooth muscle actin, fibronectin 1, and matrix metalloproteinase 9. In order to assess HE, cerebral edema, microglia activation and proinflammatory cytokine expression were assessed in the cortex.ResultsTGFβ1 signaling and TSP‐1 expression were increased in the liver and serum, but not the cortex, of BDL rats. Infusion of LSKL into BDL rats reduced liver injury, as determined by improved serum chemistry, in comparison to SLLK‐infused BDL rats. Sirius red staining, hydroxyproline content and fibrosis‐associated gene expression were reduced in livers from LSKL‐infused BDL rats compared to SLLK‐infused BDL rats. Time and distance traversed on a rotarod and overall activity were greater in BDL rats infused with LSKL compared to BDL rats infused with SLLK. Ammonia concentrations in serum, cerebral edema, microglia activation and cortex cytokine expression were significantly decreased in BDL rats infused with LSKL compared to SLLK‐infused BDL rats.ConclusionsInhibition of TSP‐1 via LSKL reduced TGFβ1 signaling, liver injury, fibrosis and neurological deficits in BDL rats giving support that antagonism of TSP‐1 could be a therapeutic option for the management of cholestatic liver disease.Support or Funding InformationThis study was funded by a VA Career Development award (BX003486) from the United States Department of Veterans Affairs to Dr. Matthew McMillin. This study was also funded by NIH R01 awards (DK082435 and DK112803) and a VA Merit award (BX002638) from the United States Department of Veterans Affairs to Dr. Sharon DeMorrow.

Progressive dysfunction of collecting liver lymphatics during the development of extrahepatic cholestasis

Lymphatics are necessary anatomical structures of body systems to maintain tissue fluid and molecular homeostasis and are tightly involved in immune reactions of the body. Transport function of lymphatics is altered during inflammation, which impacts the pathogenesis of many diseases. Despite their importance in liver function, the functional characteristics of liver lymphatic vessels (LLVs) have not been evaluated. In this study we, for the first time, measured the function of LLVs in normal and cholestatic rats following extrahepatic bile duct ligation (BDL). In this study parameters of lymphatic contractility/pumping of isolated, cannulated and pressurized segments of LLVs were determined in male 5 moth old Sprague‐Dawley rats. The following animal groups were used: control, 1, 3 and 7 days, after BDL (BDL1, BDL3 and BDL7) and sham control 3 days after surgery. Segments of LLVs located in close proximity to the liver were dissected for the study. Lymphatic contractility parameters were measured and analyzed in isolated, cannulated and pressurized LLV segments under standard transmural pressures and imposed flow ranges in all groups. Healthy LLVs with outer diameter of ~500±50 μm have significant lymphatic tone (7–9%), moderate contraction amplitude (10–25%), high contraction frequency (14–25 contractions/min) demonstrating therefore strong pumping (4–7 volumes/min). Contractility of LLVs from sham and control group were not significantly different. BDL induced specific changes in tone and phasic contractions of LLVs. BDL caused an increase in vessel size (up to 900±100 μm of outer diameter in BDL7 group), progressive lowering of lymphatic tone over period of time after BDL (up to ~2–3‐fold lower lymphatic tone in BDL7 group). Effects of BDL on lymphatic contraction amplitude were as follows: BDL1‐LLVs contraction amplitude was ~2–2.5 times higher than control, BDL3– back to control values, BDL7– amplitude was 0 (a complete cessation of phasic contractions). At the same time contraction frequency in BDL1 and BDL3 LLVs was ~2–2.2 fold lower than control with no contractions in BDL7 group. Cumulatively, we observed a progressive decline in LLVs' pumping in BDL1 and BDL3 groups with complete lymphatic pump failure in BDL7 group. Additionally, LLVs possess comparatively light flow‐dependent responses, which were ablated by BDL. The data indicate that over the time course of cholestasis, the transport capabilities of LLVs are progressively diminished introducing severe, harmful disturbances in fluid/macromolecular homeostasis of the cholestatic liver, which could alter proper trafficking of immune cells and promote liver inflammation. Further research on underlying mechanisms of liver lymphatic dysfunction will shed light on the pathogenesis of cholestatic liver disease and may reveal novel therapeutic approaches.Support or Funding Information1U01HL123420 (DZ, AG); DK110035 (SG, GA)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A20 Attenuates Liver Fibrosis in NAFLD and Inhibits Inflammation Responses.

We previously reported A20 was able to inhibit lipid accumulation in nonalcoholic steatohepatitis. We want to investigate whether A20 influences liver fibrosis in this study. Liver tissues from patients with hepatic fibrosis (n = 9) and healthy individuals (n = 7) were studied for A20 protein level by immunohistochemistry. A20 messenger RNA (mRNA) and protein level were also analyzed in two murine hepatic fibrosis models: methionine- and choline-deficient (MCD) diet and extrahepatic bile duct ligation (BDL) operation by real-time PCR and western blot. In vitro, the LX-2 human hepatic stellate cell line was treated by LPS at 0, 0.001, 0.01, 0.1, and 1μg/mL for 6h or at the concentration of 0.1μg/mL for 0, 6, 12, and 24h, then A20 expression levels were detected by western blot and PCR. The mRNA level of α-SMA, collagen I, collagen III, TGF-β, IL-6, MCP-1, and TLR4 was also examined by PCR. We then overexpressed A20 in LX-2 cells using adenovirus technique. Levels of α-SMA, collagen I, collagen III, TGF-β, IL-6, MCP-1, and TLR4 were examined in A20-overexpression LX-2 cells. Patients with hepatic fibrosis showed significantly higher A20 protein level compared with healthy controls. A20 mRNA and protein levels were also increased in livers from MCD feeding or BDL operation mice in comparison to normal controls. In LX-2 cells, LPS induced A20 protein in a concentration-dependent manner. The mRNA levels of α-SMA, collagen I, collagen III, TGF-β, IL-6, MCP-1, and TLR4 were increased after LPS treatment. Overexpression of A20 in LX-2 cells inhibited α-SMA deposition and collagen I, collagen III secretion. TGF-β, IL-6, MCP-1, and TLR4 mRNA levels were also reduced in A20-overexpression LX-2 cells in response to LPS stimulation. A20 overexpression inhibits hepatic stellate cell activation, which could be the mechanism for high A20 expression protected livers from fibrosis. Enhancement of A20 expression seems to be rational therapeutic strategies for liver fibrosis.

Regulators of Cholangiocyte Proliferation.

Cholangiocytes, a small population of cells within the normal liver, have been the focus of a significant amount of research over the past two decades because of their involvement in cholangiopathies such as primary sclerosing cholangitis and primary biliary cholangitis. This article summarizes landmark studies in the field of cholangiocyte physiology and aims to provide an updated review of biliary pathogenesis. The historical approach of rodent extrahepatic bile duct ligation and the relatively recent utilization of transgenic mice have led to significant discoveries in cholangiocyte pathophysiology. Cholangiocyte physiology is a complex system based on heterogeneity within the biliary tree and a number of signaling pathways that serve to regulate bile composition. Studies have expanded the list of neuropeptides, neurotransmitters, and hormones that have been shown to be key regulators of proliferation and biliary damage. The peptide histamine and hormones, such as melatonin and angiotensin, angiotensin, as well as numerous sex hormones, have been implicated in cholangiocyte proliferation during cholestasis. Numerous pathways promote cholangiocyte proliferation during cholestasis, and there is growing evidence to suggest that cholangiocyte proliferation may promote hepatic fibrosis. These pathways may represent significant therapeutic potential for a subset of cholestatic liver diseases that currently lack effective therapies.

Activation of the renin-angiotensin system stimulates biliary hyperplasia during cholestasis induced by extrahepatic bile duct ligation.

Cholangiocyte proliferation is regulated in a coordinated fashion by many neuroendocrine factors through autocrine and paracrine mechanisms. The renin-angiotensin system (RAS) is known to play a role in the activation of hepatic stellate cells and blocking the RAS attenuates hepatic fibrosis. We investigated the role of the RAS during extrahepatic cholestasis induced by bile duct ligation (BDL). In this study, we used normal and BDL rats that were treated with control, angiotensin II (ANG II), or losartan for 2 wk. In vitro studies were performed in a primary rat cholangiocyte cell line (NRIC). The expression of renin, angiotensin-converting enzyme, angiotensinogen, and angiotensin receptor type 1 was evaluated by immunohistochemistry (IHC), real-time PCR, and FACs and found to be increased in BDL compared with normal rat. The levels of ANG II were evaluated by ELISA and found to be increased in serum and conditioned media of cholangiocytes from BDL compared with normal rats. Treatment with ANG II increased biliary mass and proliferation in both normal and BDL rats. Losartan attenuated BDL-induced biliary proliferation. In vitro, ANG II stimulated NRIC proliferation via increased intracellular cAMP levels and activation of the PKA/ERK/CREB intracellular signaling pathway. ANG II stimulated a significant increase in Sirius red staining and IHC for fibronectin that was blocked by angiotensin receptor blockade. In vitro, ANG II stimulated the gene expression of collagen 1A1, fibronectin 1, and IL-6. These results indicate that cholangiocytes express a local RAS and that ANG II plays an important role in regulating biliary proliferation and fibrosis during extraheptic cholestasis.

Extrahepatic bile duct ligation in broiler chickens: ultrastructural study of Ito cell

The Ito cell (fat-storing cell) is a cell lying in perisinusoidal space of liver. The function of Ito cell is expanding from a site of fat-storing site to a center of extracellular matrix metabolism and mediator production in the liver. This study was performed in order to evaluate the Ito cells in cholestatic condition. The artificial cholestatic was conducted by ligation of extrahepatic bile ducts (bile duct ligation = BDL) in broilers. The results showed that BDL induced bile congestion, fibrosis, proliferation of Ito cells and intrahepatic bile ductules. Immunohistochemistry demonstrated that Ito cells were scattered throughout the fibrotic areas, and larger in size with more extensive immunoreactivity than those in normal livers. Ultrastructural study demonstrated that Ito cells were closely associated with the production of extracellular collagen fibers. Ito cells actively react against hepatocytic injuries, especially in fibrogenesis of cholestatic livers. Key words: Bile duct, ito cell, broiler

Hepcidin protects against lipopolysaccharide-induced liver injury in a mouse model of obstructive jaundice

Obstructive jaundice (OJ) increases the risk of liver injury and sepsis, leading to increased mortality. Cholestatic liver injury is associated with a downregulation of hepcidin expression levels. In fact, hepcidin has an important antimicrobial effect, especially against Escherichia coli. It is unknown whether supplementing recombinant hepcidin is effective in alleviating cholestasis-induced liver injury and mortality in mice with superimposed sepsis. A mouse model of cholestasis was developed using extrahepatic bile duct ligation for 3 days. In addition, sepsis due to E. coli 0111:B4 lipopolysaccharide (LPS) was induced in the model. The serum levels of total bilirubin, AST, ALT, and LDH and the mRNA levels of IL-1β, TNF-α, and MCP-1 in the liver were significantly higher in the OJ mice receiving LPS than in the sham-operated mice receiving LPS. Compared to the OJ mice receiving LPS, the hepcidin-pretreated OJ mice receiving LPS showed a significant decrease in the above mentioned parameters, as well as a reversal in the downregulation of LC3B-II and upregulation of cleaved caspase-3; this, in turn, led to significantly decreased lethality in 24h. In conclusion, these results indicate that hepcidin pretreatment significantly reduced hepatic proinflammatory cytokine expression and liver injury, leading to reduced early lethality in OJ mice receiving LPS. Enhanced autophagy and reduced apoptosis may account for the protective effects of hepcidin.

Recent advances in the regulation of cholangiocyte proliferation and function during extrahepatic cholestasis

Bile duct epithelial cells (i.e., cholangiocytes), which line the intrahepatic biliary epithelium, are the target cells in a number of human cholestatic liver diseases (termed cholangiopathies). Cholangiocyte proliferation and death is present in virtually all human cholangiopathies. A number of recent studies have provided insights into the key mechanisms that regulate the proliferation and function of cholangiocytes during the pathogenesis of cholestatic liver diseases. In our review, we have summarised the most important of these recent studies over the past 3 years with a focus on those performed in the animal model of extrahepatic bile duct ligation. In the first part of the review, we provide relevant background on the biliary ductal system. We then proceed with a general discussion of the factors regulating biliary proliferation performed in the cholestatic animal model of bile duct ligation. Further characterisation of the factors that regulate cholangiocyte proliferation and function will help in elucidating the mechanisms regulating the pathogenesis of biliary tract diseases in humans and in devising new treatment approaches for these devastating diseases.

KUPFFER CELL BLOCKADE IMPROVES THE ENDOTOXIN-INDUCED MICROCIRCULATORY INFLAMMATORY RESPONSE IN OBSTRUCTIVE JAUNDICE

Cholestasis predisposes to hypersensitivity to LPS, leading to potential septic complications. We set out to characterize the involvement of Kupffer cell (KC) activation in the hepatic microcirculatory and structural consequences of obstructive jaundice in the presence and absence of acute endotoxemia. The hepatic microcirculatory consequences of 3-day extrahepatic bile duct ligation (BDL) were assessed in rats. The contributions of changes in hepatic perfusion, leukocyte influx, and proinflammatory cytokine release to the development of hepatic structural damage were also determined. Furthermore, the corresponding consequences of BDL in combination with acute (2-h) endotoxemia (1 mg kg(-1) LPS, i.v.) were compared with those observed after LPS alone. In a second series, the same protocols were applied in identical groups of rats where the KC function was inhibited with 24-h gadolinium chloride pretreatment (10 mg kg(-1), i.v.). Bile duct ligation induced minor inflammatory reactions but caused a marked reduction in hepatic sinusoidal perfusion and severe histological damage. LPS treatment, however, elicited an approximately 5-fold increase in leukocyte adherence in the central venules and pronounced IL-6 and TNF-alpha release, but without significant structural damage. The combination of BDL with LPS enhanced the perfusion failure, leukocyte sticking/deposition, and proinflammatory cytokine release; most of these changes can be effectively ameliorated by gadolinium chloride. In conclusion, when obstructive jaundice is followed by a second hit of LPS, perfusion failure, liver inflammation, and structural damage are enhanced, the KCs playing a decisive role in this scenario. Therapeutic strategies aimed at KC blockade can potentially reduce the risk of inflammatory complications in cholestasis.

Pathomorphological studies of cholangiohepatitis in broiler chickens

Enlarged, discolorated and firm livers, often associated with cholecystitis and extra­ hepatic bile duct distended with bile, have been reported in broiler chickens. Histopa­ thologically' these livers showed a diffuse fi­ brosis with marked proliferation of biliary ductules, cholestasis, accompanied by hetero­ philic and lymphocytic infiltration. Although Clostridium perfringens (c. perfringens) has been suggested as the causative agent, the precise pathogenesis of the hepatic lesions re­ mains unclear. In the present study, the rela­ tionship between cholangiohepatitis and mal­ formation of extrahepatic bile ducts, patho­ morphologic features of Ito cells in livers treated with extrahepatic bile duct ligation (BDL) and in livers of field cases were inves­ tigated in broiler chickens. The role of Ito cells in the hepatic fibrogenesis in these hepatic le­ sions was also discussed. Six of the 88 abnormal livers from a

Immunohistochemical and ultrastructural study of ito cells (fat-storing cells) in response to extrahepatic bile duct ligation in broiler chickens.

The Ito cell (fat-storing cell) lies in perisinusoidal space of liver and has a variety of functions. We investigated the immunohistochemistry and ultrastructure of Ito cells in normal and cholestatic livers of broiler chickens. Immunohistochemistry demonstrated that Ito cells expressed HHF35 muscle actin, vimentin, desmin, glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), chromogranin A and cytokeratins in normal livers. These cells were diffusely scattered throughout the lobules. Livers treated with extrahepatic bile duct ligation (BDL) showed cholestasis, fibrosis, proliferation of biliary ductules and Ito cells. The Ito cells were frequently found in fibrotic areas and were larger in size with more extensive immunoreactivity than those of normal livers. Ultrastructural study demonstrated that Ito cells were closely associated with the production of collagen fibers in BDL livers. These findings suggest that Ito cells actively react against hepatocytic injuries and play a major role in the hepatic fibrogenesis of cholestatic livers of chickens.