Stages based molecular mechanisms for generating cholangiocytes from liver stem/progenitor cells

Except for the most organized mature hepatocytes,liver stem/progenitor cells(LSPCs)can differentiate into many other types of cells in the liver including cholangiocytes.In addition,LSPCs are demonstrated to be able to give birth to other kinds of extra-hepatic cell types such as insulin-producing cells.Even more,under some bad conditions,these LSPCs could generate liver cancer stem like cells(LCSCs)through malignant transformation.In this review,we mainly concentrate on the molecular mechanisms for controlling cell fates of LSPCs,especially differentiation of cholangiocytes,insulin-producing cells and LCSCs.First of all,to certificate the cell fates of LSPCs,the following three features need to be taken into account to perform accurate phenotyping:(1)morphological properties;(2)specific markers;and(3)functional assessment including in vivo transplantation.Secondly,to promote LSPCs differentiation,systematical attention should be paid to inductive materials(such as growth factors and chemical stimulators),progressive materials including intracellular and extracellular signaling pathways,and implementary materials(such as liver enriched transcriptive factors).Accordingly,some recommendations were proposed to standardize,optimize,and enrich the effective production of cholangiocyte-like cells out of LSPCs.At the end,the potential regulating mechanisms for generation of cholangiocytes by LSPCs were carefully analyzed.The differentiation of LSPCs is a gradually progressing process,which consists of three main steps:initiation,progression and accomplishment.It’s the unbalanced distribution of affecting materials in each step decides the cell fates of LSPCs.

Histamine regulation of hyperplastic and neoplastic cell growth in cholangiocytes

Histamine has long been known to be involved in inflammatory events.The discovery of antihistamines dates back to the first half of the 20th century when a Swiss-Italian pharmacologist,Daniel Bovet began his work.In 1957 he was awarded a Nobel Prize for his production of antihistamines for allergy relief.Since that time,histamine has been found to play a role in other events besides allergic reaction.Possiblyun believable to Bovet and his peers,histamine has now been marked as playing a role in liver pathologies including hepatobiliary diseases.

Danhongqing formula alleviates cholestatic liver fibrosis by downregulating long non-coding RNA H19 derived from cholangiocytes and inhibiting hepatic stellate cell activation

Objective:This study explores the mechanism of action of Danhongqing formula(DHQ),a compoundbased Chinese medicine formula,in the treatment of cholestatic liver fibrosis.Methods:In vivo experiments were conducted using 8-week-old multidrug resistance protein 2 knockout(Mdr2-/-)mice as an animal model of cholestatic liver fibrosis.DHQ was administered orally for 8 weeks,and its impact on cholestatic liver fibrosis was evaluated by assessing liver function,liver histopathology,and the expression of liver fibrosis-related proteins.Real-time polymerase chain reaction,Western blot,immunohistochemistry and other methods were used to observe the effects of DHQ on long non-coding RNA H19(H19)and signal transducer and activator of transcription 3(STAT3)phosphorylation in the liver tissue of Mdr2-/-mice.In addition,cholangiocytes and hepatic stellate cells(HSCs)were cultured in vitro to measure the effects of bile acids on cholangiocyte injury and H19 expression.Cholangiocytes overexpressing H19 were constructed,and a conditioned medium containing H19 was collected to measure its effects on STAT3 protein expression and cell activation.The intervention effect of DHQ on these processes was also investigated.HSCs overexpressing H19 were constructed to measure the impact of H19 on cell activation and assess the intervention effect of DHQ.Results:DHQ alleviated liver injury,ductular reaction,and fibrosis in Mdr2-/-mice,and inhibited H19expression,STAT3 expression and STAT3 phosphorylation.This formula also reduced hydrophobic bile acid-induced cholangiocyte injury and the upregulation of H19,inhibited the activation of HSCs induced by cholangiocyte-derived conditioned medium,and decreased the expression of activation markers in HSCs.The overexpression of H19 in a human HSC line confirmed that H19 promoted STAT3 phosphorylation and HSC activation,and DHQ was able to successfully inhibit these effects.Conclusion:DHQ effectively alleviated spontaneous cholestatic liver fibrosis in Mdr2-/-mice by inhibiting H19 upregulation in cholangiocytes and preventing the inhibition of STAT3 phosphorylation in HSC,thereby suppressing cell activation.

Fibroblast Growth Factor 19 Induced Changes in Non-malignant Cholangiocytes

Background and Aims:Fibroblast growth factor(FGF)19 has been implicated in the pathogenesis of murine hepatocellular carcinoma.Whether it plays a role in the development or course of human cholangiocarcinoma remains to be determined.The aim of this study was to determine whether prolonged exposure to FGF19 results in the transformation of non-malignant human cholangiocytes into cells with malignant features.Methods:Human SV-40 transfected non-malignant H69 cholangiocytes were cultured with FGF19(0-50 ng/mL)for 6 weeks,followed by 6 weeks with medium alone.Cell proliferation,invasion,stem cell surface markers,oncofetoprotein expression,state of differentiation,epithelial-mesenchymal transition(EMT)and interleukin(IL)-6 expression were documented at various time intervals throughout the 12-week period.Results:FGF19 exposure was associated with significant increases in cell proliferation,de-differentiation,EMT and IL-6 expression.However,each of these effects returned to baseline or control values during the 6-week FGF19 free follow-up period.The remaining cell properties remained unaltered.Conclusions:Six weeks of FGF19 exposure did not result in the acquisition of permanent malignant features in non-malignant,human cholangiocytes.

Secondary bile acids and the biliary epithelia: The good and the bad

The biliary tract has been considered for several decades a passive system just leading the hepatic bile to the intestine.Nowadays several researches demonstrated an important role of biliary epithelia(i.e.cholangiocytes)in bile formation.The study of biliary processes therefore maintains a continuous interest since the possible important implications regarding chronic cholestatic human diseases,such as primary biliary cholangitis or primary sclerosing cholangitis.Bile acids(BAs),produced by the liver,are the most represented organic molecules in bile.The physiologic importance of BAs was initially attributed to their behavior as natural detergents but several studies now demonstrate they are also important signaling molecules.In this minireview the effect of BAs on the biliary epithelia are reported focusing in particular on secondary(deriving by bacterial manipulation of primary molecules)ones.This class of BAs is demonstrated to have relevant biological effects,ranging from toxic to therapeutic ones.In this family ursodeoxycholic and lithocholic acid present the most interesting features.The molecular mechanisms linking ursodeoxycholic acid to its beneficial effects on the biliary tract are discussed in details as well as data on the processes leading to lithocholic damage.These findings suggest that expansion of research in the field of BAs/cholangiocytes interaction may increase our understanding of cholestatic diseases and should be helpful in designing more effective therapies for biliary disorders.

Morphological aspects of small-duct cholangiopathies:A minireview

The biliary system consists of intrahepatic and extrahepatic bile ducts lined by biliary epithelial cells(cholangiocytes).Bile ducts and cholangiocytes are affected by a variety of disorders called cholangiopathies,which differ in aetiology,pathogenesis,and morphology.Classification of cholangiopathies is complex and reflects pathogenic mechanisms(immune-mediated,genetic,drug-and toxininduced,ischaemic,infectious,neoplastic),predominant morphological patterns of biliary injury(suppurative and non-suppurative cholangitis,cholangiopathy),and specific segments of the biliary tree affected by the disease process.While the involvement of large extrahepatic and intrahepatic bile ducts is typically visualised using radiology imaging,histopathological examination of liver tissue obtained by percutaneous liver biopsy still plays an important role in the diagnosis of cholangiopathies affecting the small intrahepatic bile ducts.To increase the diagnostic yield of a liver biopsy and determine the optimal therapeutic approach,the referring clinician is tasked with interpreting the results of histopathological examination.This requires knowledge and understanding of basic morphological patterns of hepatobiliary injury and an ability to correlate microscopic findings with results obtained by imaging and laboratory methods.This minireview describes the morphological aspects of small-duct cholangiopathies pertaining to the diagnostic process.

Sequence of events leading to primary biliary cholangitis

Primary biliary cholangitis(PBC)is a chronic cholestatic liver disease that is observed more frequently in middle-aged women.This disorder is considered an autoimmune disease,since liver injury is sustained by the presence of selfdirected antimitochondrial antibodies targeting the bile duct cells.The prognosis may vary depending on an early diagnosis and response to therapy.However,nearly a third of patients can progress to liver cirrhosis,thus requiring a liver transplant.Traditional immunosuppressive therapies,commonly employed for other autoimmune diseases,have limited effects on PBC.In fact,dramatic functional changes that occur in the biliary epithelium in the course of inflammation play a major role in perpetuating the injury.In this minireview,after a background on the disease and possible predisposing factors,the sequential cooperation of cellular/molecular events leading to end-stage PBC is discussed in detail.The rise and maintenance of the autoimmune process,as well as the response of the biliary epithelia during inflammatory injury,are key factors in the progression of the disease.The so-called“ductular reaction(DR)”,intended as a reactive expansion of cells with biliary phenotype,is a process frequently observed in PBC and partially understood.However,recent findings suggest a strict relationship between this pathological picture and the progression to liver fibrosis,cell senescence,and loss of biliary ducts.All these issues(onset of chronic inflammation,changes in secretive and proliferative biliary functions,DR,and its relationship with other pathological events)are discussed in this manuscript in an attempt to provide a snapshot,for clinicians and researchers,of the most relevant and sequential contributors to the progression of this human cholestatic disease.We believe that interpreting this disorder as a multistep process may help identify possible therapeutic targets to prevent evolution to severe disease.

To explore the mechanism of Dahuang Lingxian Formula in relieving inflammatory response of bile duct cells based on IL-6/JAK/STAT3 signaling pathway

Objective:To explore the mechanism of action of Dahuang Lingxian Formula in alleviating the inflammatory response of bile duct cells in LPS-induced intrahepatic bile duct inflammation model rats based on IL-6/JAK/STAT3 signaling pathway.Methods:Fifty SD rats were randomly divided into five groups,blank group,model group,choling tablets(0.5 g/kg),and low and high concentration groups(2.4 g/kg and 4.8 g/kg)of Dahuang Lingxian Formula,ten rats in each group.Except for the blank group,the rats in each group were injected with 1.25 mg/kg LPS at the common bile duct at one time to construct an animal model of intrahepatic bile duct infection.After gavage on day 8,liver tissues were taken from rats at the hepatic hilum,and the histopathological changes of the hepatic hilum and biliary tree were observed by HE staining.The expression levels of serum glutamic alanine transaminase(ALT),glutamic oxalacetic transaminase(AST),malondialdehyde(MDA)and superoxide dismutase(SOD)were measured by biochemical method.The expression levels of interleukin 6(IL-6),Janus protein tyrosine kinase 2(JAK2),signal transducer and activator of transcription 3(STAT3)in rat serum were measured by enzyme-linked immunosorbent assay(ELISA).Protein immunoblotting(WB)and real-time fluorescence quantitative PCR(RT-qPCR)were used to detect the expression levels of IL-6,JAK2,STAT3 protein and mRNA in biliary tree tissues.Results:①Compared with the blank group,the structures such as interlobular bile ducts in the hepatic sinusoids and portal duct area of the model rats were destroyed,and inflammatory cells infiltrated around them.The expression of ALT,AST,MDA,IL-6,JAK2 and STAT3 in the serum increased significantly,the expression level of SOD decreased,and the expression levels of IL-6,JAK2 and STAT3 proteins and mRNA increased.②Compared with the model group,the degree of liver pathological damage in rats in the Chiling Ning tablet group and the low and high concentration groups of Dahuang Lingxian Formula were improved,which could significantly reduce the expression levels of ALT,AST,MDA,IL-6,JAK2,STAT3 and up-regulate SOD in serum,and down-regulate the expression of IL-6,JAK2,STAT3 protein and mRNA,with the best effect in the high concentration group of Dahuang Lingxian Formula.③Compared with the choling tablet group,the rats in the low and high concentration groups of Dahuang Lingxian Formula tended to normalize the degree of liver pathological damage,without obvious inflammatory cell infiltration,and the expression levels of ALT,AST,MDA,IL-6,JAK2,STAT3 and the expression levels of IL-6,JAK2,STAT3 protein and mRNA in serum were reduced,and the expression levels of SOD were increased,with the best effect of Dahuang Lingxian Formula The treatment effect was best in the high concentration group.Conclusion:The mechanism may be related to the down-regulation of IL-6/JAK/STAT3 signaling pathway activation,and the best therapeutic effect was achieved by the high concentration group of Dahuang Lingxian Formula.

New insights on cholangiocarcinoma

Cholangiocarcinoma(CCA) is a devastating cancer arising from the neoplastic transformation of the biliary epithelium.It is characterized by a progressive increase in incidence and prevalence.The only curative therapy is radical surgery or liver transplantation but,unfortunately,the majority of patients present with advanced stage disease,which is not amenable to surgical therapies.Recently,proposed serum and bile biomarkers could help in the screening and surveillance of categories at risk and in diagnosing CCA at an early stage.The molecular mechanisms triggering neoplastic transformation and growth of biliary epithelium are still undef ined,but signif icant progress has been achieved in the last few years.This review deals with the most recent advances on epidemiology,biology,and clinical management of CCA.

Role of autophagy in cholangiocarcinoma:Pathophysiology and implications for therapy

Cholangiocarcinoma(CCA)is a malignant tumour of the biliary system that originates from the neoplastic transformation of cholangiocytes.CCA is characterized by late diagnosis and poor outcome,with surgery considered as the last option for management.Autophagy is a physiological lysosomal degradation process,essential for cellular homeostasis and ubiquitous in all eukaryotic cells.Several studies have reported a potential involvement of autophagy in cancer,but it remains unclear whether activation of this process represents a survival mechanism of cancer cells.In the present review,we examine the autophagic process and summarize the current knowledge about the involvement of autophagy in the progression of cancer.The link between autophagy and chemoresistance and the use of autophagic markers in diagnosis are also considered in detail.Preliminary evidence shows that the combination of autophagy modulators(activators or inhibitors)with conventional chemotherapeutic agents offers a possible treatment option against signalling pathways that are hyperactivated or altered in CCA.In vitro evidence suggests that combination of chemotherapy agents,such as cisplatin,under activation or inhibition of autophagic processes,in two different CCA cell lines,may improve chemosensitivity and reduce cell survival,respectively.A deeper understanding of these pathways,in both cancer and non-cancer cells,could unveil possible therapeutic targets to treat CCA patients.

Presence of disease specific autoantibodies against liver sinusoidal cells in primary biliary cirrhosis

AIM: To investigate the presence of autoantibodies directed against liver sinusoidal cells in primary biliary cirrhosis(PBC).METHODS: Liver biopsies from 21 PBC patients were studied and compared with 12 liver biopsies from disease controls [3 patients with hepatitis B(HBV) virus,3 patients with hepatitis C virus(HCV), 3 patients with non-alcoholic steatohepatitis and 3 patients with acute alcoholic hepatitis(AAH)]. As healthy controls, we used tissue specimens adjacent to metastatic liver adenocarcinoma. Normal serum was taken from staff members of the unit. The determination of the cell type targetedby autoantibodies present in the patients sera was performed by indirect immunofluorescence(IIF) analysis using paraffin-embedded liver sections as a substrate.Sera from homologous or heterologous PBC patients or sera from the disease control group were used as primary antibodies. The presence of autoantibodies was identified using confocal microscopy.RESULTS: In total, 18/21(85.7%) PBC patients exhibited positive staining in the sinusoidal cells, 10/21(47.6%) in lymphocytes, 8/21(38%) in cholangiocytes and 7/21(33.3%) in hepatocytes, when homologous serum and fluorescein isothiocyanate-conjugated immunoglobulin type G(IgG) secondary antibody were used. PBC sections incubated with heterologous PBC serum showed reduced staining(20% for sinusoidal cells, 20% for lymphocytes, 20% for cholangiocytes and 13.3% for hepatocytes). When IgM immunoglobulin, instead of IgG, was used as secondary antibody,positive staining was observed in 75% of lymphocytes,62.5% of cholangiocytes, 37.5% of hepatocytes and50% of the sinusoidal cells with a much stronger staining intensity. No staining was observed when either normal or PBC sera were used as a primary antibody on liver sections from the disease control group. When PBC sera were incubated with healthy control sections,weak positive staining of cholangiocytes was observed in 3/21(14.3%) PBC serum samples. Steatohepatitis serum on PBC sections gave a positive staining of some hepatocytes and lymphocytes but no staining on viral hepatitis sections. Incubation with HBV sera stained some hepatocytes, cholangiocytes and intra-sinusoidal or portal lymphocytes of PBC, HBV and AAH patients but not HCV patients.CONCLUSION: In this study, for the first time in diseased liver tissue, we have demonstrated that a large proportion of PBC patients have disease specific autoantibodies against liver sinusoidal cells.

Notch signalling pathway in development of cholangiocarcinoma

Cholangiocarcinoma(CCA)comprises of extra-hepatic cholangiocarcinoma and intrahepatic cholangiocarcinoma cancers as a result of inflammation of epithelium cell lining of the bile duct.The incidence rate is increasing dramatically worldwide with highest rates in Eastern and South Asian regions.Major risk factors involve chronic damage and inflammation of bile duct epithelium from primary sclerosing cholangitis,chronic hepatitis virus infection,gallstones and liver fluke infection.Various genetic variants have also been identified and as CCA develops on the background of biliary inflammation,diverse range of molecular mechanisms are involved in its progression.Among these,the Notch signalling pathway acts as a major driver of cholangiocarcinogenesis and its components(receptors,ligands and downstream signalling molecules)represent a promising therapeutic targets.Gamma-Secretase Inhibitors have been recognized in inhibiting the Notch pathway efficiently.A comprehensive knowledge of the molecular pathways activated by the Notch signalling cascade as well as its functional crosstalk with other signalling pathways provide better approach in developing innovative therapies against CCA.

COVID-19 and liver disease: Are we missing something?

Since the coronavirus disease 2019(COVID-19)has hit the world as a pandemic,researchers all over the world have worked on its diagnostics,prognosticating factors,etc.The present study showed liver enzymes,especially aspartate aminotransferase(AST)levels,to be high in non-survivors with raised AST/alanine aminotransferase ratio.Considering the non-specific nature of AST with its presence in organs other than liver such as muscle,heart,kidney and brain makes it difficult to interpret.Even pre-existing metabolic syndrome and non-alcoholic fatty liver disease are confounding factors for deranged liver functions detected during COVID-19 disease.Therefore,the results of the study should be taken with caution.

Inositol 1,4,5-trisphosphate receptor in the liver:Expression and function

The liver is a complex organ that performs several functions to maintain homeostasis.These functions are modulated by calcium,a second messenger that regulates several intracellular events.In hepatocytes and cholangiocytes,which are the epithelial cell types in the liver,inositol 1,4,5-trisphosphate(InsP3)receptors(ITPR)are the only intracellular calcium release channels.Three isoforms of the ITPR have been described,named type 1,type 2 and type 3.These ITPR isoforms are differentially expressed in liver cells where they regulate distinct physiological functions.Changes in the expression level of these receptors correlate with several liver diseases and hepatic dysfunctions.In this review,we highlight how the expression level,modulation,and localization of ITPR isoforms in hepatocytes and cholangiocytes play a role in hepatic homeostasis and liver pathology.

Hepatic senescence, the good and the bad

Gradual alterations of cell’s physiology and functions due to age or exposure to various stresses lead to the conversion of normal cells to senescent cells.Once becoming senescent,the cell stops dividing permanently but remains metabolically active.Cellular senescence does not have a single marker but is characterized mainly by a combination of multiple markers,such as,morphological changes,expression of cell cycle inhibitors,senescence associatedβ-galactosidase activity,and changes in nuclear membrane.When cells in an organ become senescent,the entire organism can be affected.This may occur through the senescence-associated secretory phenotype(SASP).SASP may exert beneficial or harmful effects on the microenvironment of tissues.Research on senescence has become a very exciting field in cell biology since the link between age-related diseases,including cancer,and senescence has been established.The loss of regenerative and homeostatic capacity of the liver over the age is somehow connected to cellular senescence.The major contributors of senescence properties in the liver are hepatocytes and cholangiocytes.Senescent cells in the liver have been implicated in the etiology of chronic liver diseases including cirrhosis and hepatocellular carcinoma and in the interference of liver regeneration.This review summarizes recently reported findings in the understanding of the molecular mechanisms of senescence and its relationship with liver diseases.

Somatic second-hit mutations leads to polycystic liver diseases

Polycystic liver diseases(PCLDs) are a heterogeneous group of genetic disorders characterized by the development of multiple fluid-filled cysts in the liver,which derive from cholangiocytes,the epithelial cells lining the bile ducts.When these cysts grow,symptoms such as abdominal distension,nausea,and abdominal pain may occur.PCLDs may exist isolated(i.e.,autosomal dominant polycystic liver disease,ADPLD) or in combination with renal cystogenesis(i.e.,autosomal dominant polycystic kidney disease and autosomal recessive polycystic liver disease).The exact prevalence of PCLDs is unknown,but is estimated to occur in approximately 1:1000 persons.Although the pathogenesis of each form of PCLD appears to be different,increasing evidences indicate that hepatic cystogenesis is a phenomenon that may involve somatic loss of heterozygosity(LOH) in those pathological conditions inherited in a dominant form.A recent report,using highly sophisticated methodology,demonstrated that ADPLD patients with a germline mutation in the protein kinase C substrate 80K-H(PRKCSH) gene mostly develop hepatic cystogenesis through a second somatic mutation.While hepatocystin,the PRKCSH-encoding protein,was absent in the hepatic cysts with LOH,it was still expressed in the heterozygous cysts.On the other hand,no additional trans-heterozygous mutations on the SEC63 homolog(S.cerevisiae /SEC63) gene(also involved in the development of PCLDs) were observed.These data indicate that PCLD is recessive at the cellular level,and point out the important role of hepatocystin loss in cystogenesis.In this commentary,we discuss the knowledge regarding the role of somatic second-hit mutations in the development of PCLDs,and the most relevant findings have been highlighted.

Chuanxiong Rhizoma extracts prevent liver fibrosis via targeting CTCF-c-MYC-H19 pathway

Objective:Hepatic fibrosis has been widely considered as a conjoint consequence of almost all chronic liver diseases.Chuanxiong Rhizoma(Chuanxiong in Chinese,CX)is a traditional Chinese herbal product to prevent cerebrovascular,gynecologic and hepatic diseases.Our previous study found that CX extracts significantly reduced collagen contraction force of hepatic stellate cells(HSCs).Here,this study aimed to compare the protection of different CX extracts on bile duct ligation(BDL)-induced liver fibrosis and investigate plausible underlying mechanisms.Methods:The active compounds of CX extracts were identified by high performance liquid chromatography(HPLC).Network pharmacology was used to determine potential targets of CX against hepatic fibrosis.Bile duct hyperplasia and liver fibrosis were evaluated by serologic testing and histopathological evaluation.The expression of targets of interest was determined by quantitative real-time PCR(qPCR)and Western blot.Results:Different CX extracts were identified by tetramethylpyrazine,ferulic acid and senkyunolide A.Based on the network pharmacological analysis,42 overlap targets were obtained via merging the candidates targets of CX and liver fibrosis.Different aqueous,alkaloid and phthalide extracts of CX(CX_(AE),CX_(AL) and CXP_(HL))significantly inhibited diffuse severe bile duct hyperplasia and thus suppressed hepatic fibrosis by decreasing CCCTC binding factor(CTCF)-c-MYC-long non-coding RNA H19(H19)pathway in the BDL-induced mouse model.Meanwhile,CX extracts,especially CX_(AL) and CX_(PHL) also suppressed CTCF-c-MYC-H19 pathway and inhibited ductular reaction in cholangiocytes stimulated with taurocholate acid(TCA),lithocholic acid(LCA)and transforming growth factor beta(TGF-β),as illustrated by decreased bile duct proliferation markers.Conclusion:Our data supported that different CX extracts,especially CX_(AL) and CX_(PHL) significantly alleviated hepatic fibrosis and bile duct hyperplasia via inhibiting CTCF-c-MYC-H19 pathway,providing novel insights into the anti-fibrotic mechanism of CX.

Liver cell therapies:cellular sources and grafting strategies

The liver has a complex cellular composition and a remarkable regenerative capacity.The primary cell types in the liver are two parenchymal cell populations,hepatocytes and cholangiocytes,that perform most of the functions of the liver and that are helped through interactions with non-parenchymal cell types comprising stellate cells,endothelia and various hemopoietic cell populations.The regulation of the cells in the liver is mediated by an insoluble complex of proteins and carbohydrates,the extracellular matrix,working synergistically with soluble paracrine and systemic signals.In recent years,with the rapid development of genetic sequencing technologies,research on the liver’s cellular composition and its regulatory mechanisms during various conditions has been extensively explored.Meanwhile breakthroughs in strategies for cell transplantation are enabling a future in which there can be a rescue of patients with end-stage liver diseases,offering potential solutions to the chronic shortage of livers and alternatives to liver transplantation.This review will focus on the cellular mechanisms of liver homeostasis and how to select ideal sources of cells to be transplanted to achieve liver regeneration and repair.Recent advances are summarized for promoting the treatment of end-stage liver diseases by forms of cell transplantation that now include grafting strategies.

Chuanxiong Rhizoma extracts prevent cholestatic liver injury by targeting H3K9ac-mediated and cholangiocyte-derived secretory protein PAI-1 and FN

Chuanxiong Rhizoma(CX,the dried rhizome of Ligusticum wallichii Franch.),a well-known traditional Chinese medicine,is clinically used for treating cardiovascular,cerebrovascular and hepatobiliary diseases.Cholestatic liver damage is one of the chronic liver diseases with limited effective therapeutic strategies.Currently,little is known about the mechanism links between CX-induced anti-cholestatic action and intercellular communication between cholangiocytes and hepatic stellate cells(HSCs).The study aimed to evaluate the hepatoprotective activity of different CX extracts including the aqueous,alkaloid,phenolic acid and phthalide extracts of CX(CX_(AE),CX_(AL),CX_(PA)and CX_(PHL))and investigate the intercellular communication-related mechanisms by which the most effective extracts work on cholestatic liver injury.The active compounds of different CX extracts were identified by UPLC-MS/MS.A cholestatic liver injury mouse model induced by bile duct ligation(BDL),and transforming growth factor-β(TGF-β)-treated human intrahepatic biliary epithelial cholangiocytes(HIBECs)and HSC cell line(LX-2 cells)were used for in vivo and in vitro studies.Histological and other biological techniques were also applied.The results indicated that CX_(AE),CX_(AL)and CX_(PHL)significantly reduced ductular reaction(DR)and improved liver fibrosis in the BDL mice.Meanwhile,both CX_(AE)and CX_(PHL)suppressed DR in injured HIBECs and reduced collagen contraction force and the expression of fibrosis biomarkers in LX-2 cells treated with TGF-β.CX_(PHL)suppressed the transcription and transfer of plasminogen activator inhibitor-1(PAI-1)and fibronectin(FN)from the‘DR-like’cholangiocytes to activated HSCs.Mechanistically,the inhibition of PAI-1 and FN by CX_(PHL)was attributed to the untight combination of the acetyltransferase KAT2A and SMAD3,followdd by the suppression of histone 3 lysine 9 acetylation(H3K9ac)-mediated transcription in cholangiocytes.In conclusion,CX_(PHL)exerts stronger anti-cholestatic activity in vivo and in vitro than other CX extracts,and its protective effect on the intracellular communication between cholangiocytes and HSCs is achieved by reducing KAT2A/H3K9ac-mediated transcription and release of PAI-1 and FN.

Generation and metabolomic characterization of functional ductal organoids with biliary tree networks in decellularized liver scaffolds

Developing functional ductal organoids(FDOs)is essential for liver regenerative medicine.We aimed to construct FDOs with biliary tree networks in rat decellularized liver scaffolds(DLSs)with primary cholangiocytes isolated from mouse bile ducts.The developed FDOs were dynamically characterized by functional assays and metabolomics for bioprocess clarification.FDOs were reconstructed in DLSs retaining native structure and bioactive factors with mouse primary cholangiocytes expressing enriched biomarkers.Morphological assessment showed that biliary tree-like structures gradually formed from day 3 to day 14.The cholangiocytes in FDOs maintained high viability and expressed 11 specific biomarkers.Basal-apical polarity was observed at day 14 with immunostaining for E-cadherin and acetylatedα-tubulin.The rhodamine 123 transport assay and active collection of cholyl-lysyl-fluorescein exhibited the specific functions of bile secretion and transportation at day 14 compared to those in monolayer and hydrogel culture systems.The metabolomics analysis with 1075 peak pairs showed that serotonin,as a key molecule of the tryptophan metabolism pathway linked to biliary tree reconstruction,was specifically expressed in FDOs during the whole period of culture.Such FDOs with biliary tree networks and serotonin expression may be applied for disease modeling and drug screening,which paves the way for future clinical therapeutic applications.