自噬参与原发性胆汁性胆管炎患者Th17/ Treg免疫失衡的研究

目的:Th17/Treg免疫失衡在原发性胆汁性胆管炎(PBC)的发病机制中起到关键作用。自噬是调节T细胞稳态的重要环节。本研究旨在探索自噬对PBC患者Th17/Treg免疫失衡的影响。方法:选取20例初次诊断的PBC患者,20例健康对照者,收集其外周血单个核细胞(PBMCs)。利用流式细胞术检测PBC患者与健康对照者PBMCs中Th17和Treg细胞分布及其胞内自噬的水平,进一步体外利用氯喹抑制自噬,证实自噬对PBC患者Th17/Treg平衡的影响。结果:相较于健康对照者,PBC患者Th17细胞占CD4~+T比例上升(P0.05),且血清中IL-17含量较高(P0.01);Treg细胞比例下降(P0.05),且血清中TGF-β含量较低(P0.01)。而PBC患者Th17细胞和Treg细胞内自噬水平均升高。另外,体外实验结果证实,自噬抑制剂氯喹能够有效地恢复PBC患者PBMCs中Th17/Treg平衡。结论:异常活化的自噬可导致PBC患者Th17/Treg免疫失衡,抑制自噬能够在体外恢复此平衡。因此,对异常自噬的干预可能将成为PBC疾病治疗的新方法。     

Mechanisms of cholangiocyte responses to injury

Cholangiocytes, epithelial cells that line the biliary epithelium, are the primary target cells for cholangiopathies including primary sclerosing cholangitis and primary biliary cholangitis. Quiescent cholangiocytes respond to biliary damage and acquire an activated neuroendocrine phenotype to maintain the homeostasis of the liver. The typical response of cholangiocytes is proliferation leading to bile duct hyperplasia, which is a characteristic of cholestatic liver diseases. Current studies have identified various signaling pathways that are associated with cholangiocyte proliferation/loss and liver fibrosis in cholangiopathies using human samples and rodent models. Although recent studies have demonstrated that extracellular vesicles and microRNAs could be mediators that regulate these messenger/receptor axes, further studies are required to confirm their roles. This review summarizes current studies of biliary response and cholangiocyte proliferation during cholestatic liver injury with particular emphasis on the secretin/secretin receptor axis. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.     

Genetic determinants of cholangiopathies: Molecular and systems genetics

Familial cholangiopathies are rare but potentially severe diseases. Their spectrum ranges from fairly benign conditions as, for example, benign recurrent intrahepatic cholestasis to low-phospholipid associated cholelithiasis and progressive familial intrahepatic cholestasis (PFIC). Many cholangiopathies such as primary biliary cholangitis (PBC) or primary sclerosing cholangitis (PSC) affect first the bile ducts (“ascending pathophysiology”) but others, such as PFIC, start upstream in hepatocytes and cause progressive damage “descending” down the biliary tree and leading to end-stage liver disease. In recent years our understanding of cholestatic diseases has improved, since we have been able to pinpoint numerous disease-causing mutations that cause familial cholangiopathies. Accordingly, six PFIC subtypes (PFIC type 1–6) have now been defined. Given the availability of genotyping resources, these findings can be introduced in the diagnostic work-up of patients with peculiar cholestasis. In addition, functional studies have defined the pathophysiological consequences of some of the detected variants. Furthermore, ABCB4 variants do not only cause PFIC type 3 but confer an increased risk for chronic liver disease in general. In the near future these findings will serve to develop new therapeutic strategies for patients with liver diseases. Here we present the latest data on the genetic background of familial cholangiopathies and discuss their application in clinical practice for the differential diagnosis of cholestasis of unknown aetiology. As look in the future we present “system genetics” as a novel experimental tool for the study of cholangiopathies and disease-modifying genes. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.     

Development of liver microtissues with functional biliary ductular network

Liver tissue engineering aims to create transplantable liver grafts that can serve as substitutes for donor's livers. One major challenge in creating a fully functional liver tissue has been to recreate the biliary drainage in an engineered liver construct through integration of bile canaliculi (BC) with the biliary ductular network that would enable the clearance of bile from the hepatocytes to the host duodenum. In this study, we show the formation of such a hepatic microtissue by coculturing rat primary hepatocytes with cholangiocytes and stromal cells. Our results indicate that within the spheroids, hepatocytes maintained viability and function for up to 7 days. Viable hepatocytes became polarized by forming BC with the presence of tight junctions. Morphologically, hepatocytes formed the core of the spheroids, while cholangiocytes resided at the periphery forming a monolayer microcysts and tubular structures extending outward. The spheroids were subsequently cultured in clusters to create a higher order ductular network resembling hepatic lobule. The cholangiocytes formed functional biliary ductular channels in between hepatic spheroids that were able to collect, transport, and secrete bile. Our results constitute the first step to recreate hepatic building blocks with biliary drainage for repopulating the whole liver scaffolds to be used as transplantable liver grafts.     

Stereoselectivity of biliary excretion of 2-arylpropionates in rats

To examine the stereoselectivity of biliary excretion, the optically pure enantiomers of ketoprofen (KT), ibuprofen (IBU), and flurbiprofen (FLU) were intravenously administered to normal and bile duct-cannulated rats at 10 mg/kg. The recovery of total KT in bile was significantly higher after administration of (S)-KT than after (R)-KT [90.1 ± 3.5% vs 68.8 ± 8.2%, n =3, P < 0.05]. In normal rats the terminal half-life of (R)-KT was significantly shorter than that of (S)-KT after administration of (R)-KT (2.2 ± 0.6 h vs 14.3 ± 4.9 h, n = 3, P < 0.05). The terminal half-life of both enantiomers was significantly shorter in rats with continuous bile drainage as compared to normal rats. No significant differences in pharmacokinetic parameters could be found between both enantiomers in bile duct-cannulated animals. The total amount of IBU in bile was slightly higher after administration of (S)-IBU than after (R)-IBU administration. The percentage of (R)-IBU after (R)-IBU administration, however, was very low [(R)-IBU: 1.5 ± 0.9%, (S)-IBU: 23.4 ± 5.8%]. In normal rats the clearance of (R)-IBU was significantly higher as compared to (S)-IBU. Differences in pharmacokinetic parameters between normal and bile duct-cannulated rats were not statistically significant due to high interindividual variability. The total recovery of FLU, which was excreted in bile to a lower extent than either KT or IBU, also tended to be greater after S-enantiomer administration. Only small amounts of (S)-FLU could be recovered in bile after (R)-FLU administration. The pharmacokinetic parameters did not differ significantly between (R)- and (S)-FLU or between normal and bile duct-cannulated rats due to its low inversion rate and low excretion via bile. © 1993 Wiley-Liss, Inc.     

原发性胆汁性肝硬化患者血清IL-6 水平与UDCA疗效的相关性研究

目的:观察原发性胆汁性肝硬化(primary biliary cirrhosis,PBC)患者治疗前后血清IL-6表达水平,探索其与熊去氧胆酸(Ursodeoxycholic acid,UDCA)疗效的临床相关性。方法:本研究回顾性纳入自2013年-2015年就诊于第四军医大学西京消化病医院的40例新诊断PBC患者,及40例健康对照者。收集PBC患者治疗前后的相关临床资料和血清样本,采用ELISA方法检测患者血清IL-6表达水平,并进一步分析其临床意义。结果:1)治疗前PBC患者血清IL-6表达水平明显高于健康对照者(P0.001);2)PBC患者在接受UDCA治疗后的第3,6和12个月血清IL-6水平与治疗前相比明显降低(P0.05),且在第3个月时下降最明显。3)无论是依据Paris I标准还是Barcelona标准,结果显示,UDCA应答者与应答不佳者相比其治疗前血清IL-6水平无统计学差异(P=0.373;P=0.409)。但UDCA应答者在治疗3个月时其血清IL-6表达水平比治疗前明显下降(P0.05),而应答不佳者治疗3个月时血清IL-6表达水平与治疗前相比无明显差异(P=0.667;P=0.186)。结论:IL-6可能在PBC发病的免疫机制中发挥着重要的作用。目前尚不能认为PBC患者治疗前血清IL-6表达水平能独立评价UDCA疗效,但是治疗三个月后患者血清IL-6水平下降趋势能够提示PBC患者对UDCA的应答情况。     

The role of ERK-RSK signaling in the proliferation of intrahepatic biliary epithelial cells exposed to microcystin-leucine arginine

Microcystin-leucine arginine (MC-LR) is a potent specific hepatotoxin produced by cyanobacteria in diverse water systems, and it has been documented to induce liver injury and hepatocarcinogenesis. However, its toxic effects on intrahepatic biliary epithelial cells have not been invested in detail. In this study, we aimed to investigate the effects of MC-LR exposure on the intrahepatic biliary epithelial cells in the liver. MC-LR was orally administered to mice at 1 μg/L, 7.5 μg/L, 15 μg/L, or 30 μg/L for 180 consecutive days for histopathological and immunoblot analysis. We observed that MC-LR can enter intrahepatic bile duct tissue and induce hyperplasia of mice. Human primary intrahepatic biliary epithelial cells (HiBECs) were cultured with various concentrations of MC-LR for 24 h, meanwhile the cell viability and proteins level were detected. Western blotting analysis revealed that MC-LR increased RSK phosphorylation via ERK signaling. RSK participated in cell proliferation and cell cycle progression. Taken together, after chronic exposure, MC-LR-treated mice exhibited abnormal bile duct hyperplasia and thickened bile duct morphology through activating the ERK-RSK signaling. These data support the potential toxic effects of MC-LR on bile duct tissue of the liver.     

How the biliary tree maintains immune tolerance?

The liver is a vital organ with distinctive anatomy, histology and heterogeneous cell populations. These characteristics are of particular importance in maintaining immune homeostasis within the liver microenvironments, notably the biliary tree. Cholangiocytes are the first line of defense of the biliary tree against foreign substances, and are equipped to participate through various immunological pathways. Indeed, cholangiocytes protect against pathogens by TLRs-related signaling; maintain tolerance by expression of IRAK-M and PPARγ; limit immune response by inducing apoptosis of leukocytes; present antigen by expressing human leukocyte antigen molecules and costimulatory molecules; recruit leukocytes to the target site by expressing cytokines and chemokines. However, breach of tolerance in the biliary tree results in various cholangiopathies, exemplified by primary biliary cholangitis, primary sclerosing cholangitis and biliary atresia. Lessons learned from immune tolerance of the biliary tree will provide the basis for the development of effective therapeutic approaches against autoimmune biliary tract diseases. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.     

Estimation of ursodeoxycholic acid in human and bear biles using Clostridium absonum 7 beta-hydroxysteroid dehydrogenase

Ursodeoxycholic acid was estimated in bile samples from humans and wild North American black bears using 7 beta-hydroxysteroid dehydrogenase purified from Clostridium absonum by Procion Red affinity chromatography. The percentage ursodeoxycholic acid was calculated by two methods: (a) 7 beta-hydroxyl groups were quantified using 7 beta-hydroxysteroid dehydrogenase and 3 alpha-hydroxyl groups (total bile acids) were quantified using 3 alpha-hydroxysteroid dehydrogenase. The percentage ursodeoxycholic acid was calculated on the basis of [7 beta-hydroxyl groups]/[3 alpha-hydroxyl groups] X 100. (b) Bile was hydrolyzed with sodium hydroxide and subjected to thin-layer chromatography. Bands corresponding to cholic acid, chenodeoxycholic acid plus deoxycholic acid, and ursodeoxycholic acid were identified by the use of standards and Komarowsky's spray reagent. Total bile acids and total ursodeoxycholic acid were measured by elution of silica gel in unsprayed areas corresponding to the bile acid standards and quantification of the total bile acid in each eluate. Direct comparison of these methods validated the use of 7 beta-hydroxysteroid dehydrogenase in the estimation of ursodeoxycholic acid in the biles of black bears and of patients fed ursodeoxycholic acid for cholesterol gallstone dissolution. Relative percentages of ursodeoxycholic acid were 8-24% in four bears and 22 and 27% in the patients ingesting 500 and 750 mg ursodeoxycholic acid per day for 3 months, respectively. Predictably lower values were obtained in two control subjects and one patient ingesting 750 mg chenodeoxycholic acid per day for 3 months.