Classic Lesions of the Biliary Tree.

Abnormal findings in the biliary tree are frequently encountered in response to acute and chronic exposures to various compounds. The more common findings are described here in an overview of previous publications such as the INHAND Proliferative and Nonproliferative Lesions of the Rodent Liver and the Liver-Nonneoplastic Lesion Atlas NTP with comments regarding current considerations. This was presented at the 2023 Annual Meeting of the Society of Toxicologic Pathology. Histologic descriptions and some discussions regarding the pathogenesis of the various categories of non-neoplastic lesions in the biliary tree are presented. Discussions regarding the use of the term oval cell versus ductular reaction and the potentially neoplastic nature of cholangiofibrosis are presented in some detail.

Total astragalus saponins attenuate primary sclerosing cholangitis in mice by upregulation of TGR5.

Total astragalus saponins (TAS) are the main active components of astragali radix, and have potent anti-hepatic fibrosis effect. However, the therapeutic efficacy of TAS and their potential mechanisms in the treatment of primary sclerosing cholangitis (PSC) remain unclear. In this study, two mouse models of PSC, including 3,5-Diethoxycarbonyl-1,4-Dihydro-2,4,6-Collidine (DDC)-induced PSC and Mdr2-/- spontaneous PSC, and the Tgr5-/- mice were used to investigate the therapeutic effect and mechanisms of TAS. Treatment with TAS, particularly with a dose of 56 mg/kg, significantly ameliorated the PSC-related liver injury, cholestasis, collagen deposition, ductular reaction (DR), and fibrosis in the DDC-induced and Mdr2-/-spontaneous PSC mice. Furthermore, treatment with TAS significantly mitigated the PSC-related inflammatory responses in vivo and HIBEpiC cells by inhibiting the expression of TNF-α, IL-6, and IL-1ß. Mechanistically, treatment with TAS rescued the PSC-decreased hepatic TGR5 expression to attenuate the NF-κB p65 phosphorylation. Notably, the therapeutic efficacy of TAS on PSC in DDC-induced mice was abrogated in Tgr5-/- mice, suggesting the anti-PSC effect of TAS may depend on enhancing TGR5 expression. In conclusion, TAS ameliorated DR, inflammation and liver fibrosis in both models of PSC mice by rescuing TGR5 expression. Our findings may aid in the design of new therapeutic strategies for the treatment of PSC.

Aberrant differentiation and proliferation of hepatocytes in chronic liver injury and liver tumors.

Chronic liver injury induces liver cirrhosis and facilitates hepatocarcinogenesis. However, the effects of this condition on hepatocyte proliferation and differentiation are unclear. We showed that rodent hepatocytes display a ductular phenotype when they are cultured within a collagenous matrix. This process involves transdifferentiation without the emergence of hepatoblastic features and is at least partially reversible. During the ductular reaction in chronic liver diseases with progressive fibrosis, some hepatocytes, especially those adjacent to ectopic ductules, demonstrate ductular transdifferentiation, but the majority of increased ductules originate from the existing bile ductular system that undergoes extensive remodeling. In chronic injury, hepatocyte proliferation is weak but sustained, and most regenerative nodules in liver cirrhosis are composed of clonally proliferating hepatocytes, suggesting that a small fraction of hepatocytes maintain their proliferative capacity in chronic injury. In mouse hepatocarcinogenesis models, hepatocytes activate the expression of various fetal/neonatal genes, indicating that these cells undergo dedifferentiation. Hepatocyte-specific somatic integration of various oncogenes in mice demonstrated that hepatocytes may be the cells of origin for a broad spectrum of liver tumors through transdifferentiation and dedifferentiation. In conclusion, the phenotypic plasticity and heterogeneity of mature hepatocytes are important for understanding the pathogenesis of chronic liver diseases and liver tumors.

Navigating the liver landscape: upcoming pharmacotherapies for primary sclerosing cholangitis.

INTRODUCTION: Primary sclerosing cholangitis (PSC) is a bile duct disorder characterized by ductular reaction, hepatic inflammation, and liver fibrosis. The pathogenesis of PSC is still undefined, and treatment options for patients are limited. Previous clinical trials evaluated drug candidates targeting various cellular functions and pathways, such as bile acid signaling and absorption, gut bacteria and permeability, and lipid metabolisms. However, most of phase III clinical trials for PSC were disappointing, except vancomycin therapy, and there are still no established medications for PSC with efficacy and safety confirmed by phase IV clinical trials. AREAS COVERED: This review summarizes the currently ongoing or completed clinical studies for PSC, which are phase II or further, and discusses therapeutic targets and strategies, limitations, and future directions and possibilities of PSC treatments. A literature search was conducted in PubMed and ClinicalTrials.gov utilizing the combination of the searched term 'primary sclerosing cholangitis' with other keywords, such as 'clinical trials,' 'antibiotics,' or drug names. Clinical trials at phase II or further were included for consideration. EXPERT OPINION: Only vancomycin demonstrated promising therapeutic effects in the phase III clinical trial. Other drug candidates showed futility or inconsistent results, and the search for novel PSC treatments is still ongoing.

Mucosal-associated invariant T cells promote ductular reaction through amphiregulin in biliary atresia.

BACKGROUND: Biliary atresia (BA) is a neonatal fibro-inflammatory cholangiopathy with ductular reaction as a key pathogenic feature predicting poor survival. Mucosal-associated invariant T (MAIT) cells are enriched in human liver and display multiple roles in liver diseases. We aimed to investigate the function of MAIT cells in BA. METHODS: First, we analyzed correlations between liver MAIT cell and clinical parameters (survival, alanine transaminase, bilirubin, histological inflammation and fibrosis) in two public cohorts of patients with BA (US and China). Kaplan-Meier survival analysis and spearman correlation analysis were employed for survival data and other clinical parameters, respectively. Next, we obtained liver samples or peripheral blood from BA and control patients for bulk RNA sequencing, flow cytometry analysis, immunostaning and functional experiments of MAIT cells. Finally, we established two in vitro co-culture systems, one is the rhesus rotavirus (RRV) infected co-culture system to model immune dysfunction of human BA which was validated by single cell RNA sequencing and the other is a multicellular system composed of biliary organoids, LX-2 and MAIT cells to evaluate the role of MAIT cells on ductular reaction. FINDINGS: Liver MAIT cells in BA were positively associated with low survival and ductular reaction. Moreover, liver MAIT cells were activated, exhibited a wound healing signature and highly expressed growth factor Amphiregulin (AREG) in a T cell receptor (TCR)-dependent manner. Antagonism of AREG abrogated the proliferative effect of BA MAIT cells on both cholangiocytes and biliary organoids. A RRV infected co-culture system, recapitulated immune dysfunction of human BA, disclosed that RRV-primed MAIT cells promoted cholangiocyte proliferation via AREG, and further induced inflammation and fibrosis in the multicellular system. INTERPRETATION: MAIT cells exhibit a wound healing signature depending on TCR signaling and promote ductular reaction via AREG, which is associated with advanced fibrosis and predictive of low survival in BA. FUNDING: This work was funded by National Natural Science Foundation of China grant (82001589 and 92168108), National Key R&D Program of China (2023YFA1801600) and by Basic and Applied Basic Research Foundation of Guangdong (2020A1515110921).

Hepatic progenitor cell-originated ductular reaction facilitates liver fibrosis through activation of hedgehog signaling.

Background: It is poorly understood what cellular types participate in ductular reaction (DR) and whether DR facilitates recovery from injury or accelerates hepatic fibrosis. The aim of this study is to gain insights into the role of hepatic progenitor cell (HPC)-originated DR during fibrotic progression. Methods: DR in liver specimens of PBC, chronic HBV infection (CHB) or NAFLD, and four rodent fibrotic models by different pathogenic processes was evaluated. Gli1 expression was inhibited in rodent models or cell culture and organoid models by AAV-shGli1 or treating with GANT61. Results: Severity of liver fibrosis was positively correlated with DR extent in patients with PBC, CHB or NAFLD. HPCs were activated, expanded, differentiated into reactive cholangiocytes and constituted "HPC-originated DR", accompanying with exacerbated fibrosis in rodent models of HPC activation & proliferation (CCl4/2-AAF-treated), Μdr2-/- spontaneous PSC, BDL-cholestatic fibrosis or WD-fed/CCl4-treated NASH-fibrosis. Gli1 expression was significantly increased in enriched pathways in vivo and in vitro. Enhanced Gli1 expression was identified in KRT19+-reactive cholangiocytes. Suppressing Gli1 expression by administration of AAV-shGli1 or GANT61 ameliorated HPC-originated DR and fibrotic extent. KRT19 expression was reduced after GANT61 treatment in sodium butyrate-stimulated WB-F344 cells or organoids or in cells transduced with Gli1 knockdown lentiviral vectors. In contrast, KRT19 expression was elevated after transducing Gli1 overexpression lentiviral vectors in these cells. Conclusions: During various modes of chronic injury, Gli1 acted as an important mediator of HPC activation, expansion, differentiation into reactive cholangiocytes that formed DR, and subsequently provoked hepatic fibrogenesis.

Ductular Reactions in Liver Injury, Regeneration, and Disease Progression-An Overview.

Ductular reaction (DR) is a complex cellular response that occurs in the liver during chronic injuries. DR mainly consists of hyper-proliferative or reactive cholangiocytes and, to a lesser extent, de-differentiated hepatocytes and liver progenitors presenting a close spatial interaction with periportal mesenchyme and immune cells. The underlying pathology of DRs leads to extensive tissue remodeling in chronic liver diseases. DR initiates as a tissue-regeneration mechanism in the liver; however, its close association with progressive fibrosis and inflammation in many chronic liver diseases makes it a more complicated pathological response than a simple regenerative process. An in-depth understanding of the cellular physiology of DRs and their contribution to tissue repair, inflammation, and progressive fibrosis can help scientists develop cell-type specific targeted therapies to manage liver fibrosis and chronic liver diseases effectively.

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 (CXAE, CXAL and CXPHL) 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 CXAL and CXPHL 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-ß), as illustrated by decreased bile duct proliferation markers. Conclusion: Our data supported that different CX extracts, especially CXAL and CXPHL 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.

Cellular Interactions and Crosstalk Facilitating Biliary Fibrosis in Cholestasis.

Biliary fibrosis is seen in cholangiopathies, including primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). In PBC and PSC, biliary fibrosis is associated with worse outcomes and histologic scores. Within the liver, both hepatic stellate cells (HSCs) and portal fibroblasts (PFs) contribute to biliary fibrosis, but their roles can differ. PFs reside near the bile ducts and may be the first responders to biliary damage, whereas HSCs may be recruited later and initiate bridging fibrosis. Indeed, different models of biliary fibrosis can activate PFs and HSCs to varying degrees. The portal niche can be composed of cholangiocytes, HSCs, PFs, endothelial cells, and various immune cells, and interactions between these cell types drive biliary fibrosis. In this review, we discuss the mechanisms of biliary fibrosis and the roles of PFs and HSCs in this process. We will also evaluate cellular interactions and mechanisms that contribute to biliary fibrosis in different models and highlight future perspectives and potential therapeutics.

Chuanxiong Rhizoma extracts prevent liver fibrosis via targeting CTCF-c-MYC-H19 pathway / 中草药·英文版

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 (CXAE, CXAL and CXPHL) 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 CXAL and CXPHL 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 CXAL and CXPHL 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.

Unraveling the complexities of fibrosis and ductular reaction in liver disease: pathogenesis, mechanisms, and therapeutic insights.

Ductular reaction and fibrosis are hallmarks of many liver diseases including primary sclerosing cholangitis, primary biliary cholangitis, biliary atresia, alcoholic liver disease, and metabolic dysfunction-associated steatotic liver disease/metabolic dysfunction-associated steatohepatitis. Liver fibrosis is the accumulation of extracellular matrix often caused by excess collagen deposition by myofibroblasts. Ductular reaction is the proliferation of bile ducts (which are composed of cholangiocytes) during liver injury. Many other cells including hepatic stellate cells, hepatocytes, hepatic progenitor cells, mesenchymal stem cells, and immune cells contribute to ductular reaction and fibrosis by either directly or indirectly interacting with myofibroblasts and cholangiocytes. This review summarizes the recent findings in cellular links between ductular reaction and fibrosis in numerous liver diseases.

VEGFA mRNA-LNP promotes biliary epithelial cell-to-hepatocyte conversion in acute and chronic liver diseases and reverses steatosis and fibrosis.

The liver is known for its remarkable regenerative ability through proliferation of hepatocytes. Yet, during chronic injury or severe hepatocyte death, proliferation of hepatocytes is exhausted. To overcome this hurdle, we propose vascular-endothelial-growth-factor A (VEGFA) as a therapeutic means to accelerate biliary epithelial-cell (BEC)-to-hepatocyte conversion. Investigation in zebrafish establishes that blocking VEGF receptors abrogates BEC-driven liver repair, while VEGFA overexpression promotes it. Delivery of VEGFA via nonintegrative and safe nucleoside-modified mRNA encapsulated into lipid nanoparticles (mRNA-LNPs) in acutely or chronically injured mouse livers induces robust BEC-to-hepatocyte conversion and elimination of steatosis and fibrosis. In human and murine diseased livers, we further identified VEGFA-receptor KDR-expressing BECs associated with KDR-expressing cell-derived hepatocytes. This work defines KDR-expressing cells, most likely being BECs, as facultative progenitors. This study reveals unexpected therapeutic benefits of VEGFA delivered via nucleoside-modified mRNA-LNP, whose safety is widely validated with COVID-19 vaccines, for harnessing BEC-driven repair to potentially treat liver diseases.

CD24+LCN2+ liver progenitor cells in ductular reaction contributed to macrophage inflammatory responses in chronic liver injury.

BACKGROUND: CD24+CK19+/CD24+SOX9+ resident liver cells are activated and expanded after chronic liver injury in a ductular reaction. However, the sources and functions of these cells in liver damage remain disputed. RESULTS: The current study combined genetic lineage tracing with in vitro small-molecule-based reprogramming to define liver progenitor cells (LPCs) derived from hepatic parenchymal and non-parenchymal tissues. tdTom+ hepatocytes were isolated from ROSA26tdTomato mice following AAV8-Tbg-Cre-mediated recombination, EpCAM+ biliary epithelial cells (BECs) from wild-type intrahepatic bile ducts and ALB/GFP-EpCAM- cells were isolated from AlbCreERT/R26GFP mice. A cocktail of small molecules was used to convert the isolated cells into LPCs. These in vitro cultured LPCs with CD24 and SOX9 expression regained the ability to proliferate. Transcriptional profiling showed that the in-vitro cultured LPCs derived from the resident LPCs in non-parenchymal tissues expressed Lipocalin-2 (Lcn2) at high levels. Accordingly, endogenous Cd24a+Lcn2+ LPCs were identified by integration of sc-RNA-sequencing and pathological datasets of liver dysfunction which indicates that LPCs produced by ductular reactions might also originate from the resident LPCs. Transplantation of in-vitro cultured Cd24a+Lcn2+ LPCs into CCl4-induced fibrotic livers exacerbated liver damage and dysfunction, possibly due to LCN2-dependent macrophage inflammatory response. CONCLUSIONS: CD24+LCN2+ LPCs constituted the expanding ductular reaction and contributed to macrophage-mediated inflammation in chronic liver damage. The current findings highlight the roles of LPCs from distinct origins and expose the possibility of targeting LPCs in the treatment of chronic hepatic diseases.

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 self-directed 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.

PGC1α deficiency reverses cholestasis-induced liver injury via attenuating hepatic inflammation and promoting bile duct remodeling.

OBJECTIVES: Cholestatic liver diseases are characterized by hepatocellular damage, cholangiocyte proliferation, and progressive fibrosis. Bile duct ligation (BDL) is widely used to resemble liver injuries induced by cholestasis. Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC1α) was reported to play a critical role in multiple biological responses. Nevertheless, whether PGC1α is involved in bile acid metabolism and biliary disorders remains unclear. This study aimed to investigate the effect of PGC1α on hepatic responses after cholestatic injury. MATERIALS AND METHODS: Wild-type mice were subjected to BDL or sham surgery for 14 days and human liver specimens from patients with primary biliary cholangitis (PBC) were collected to detect the expression of PGC1α. Hepatic-specific PGC1α knockout mice (HKO) were constructed and subjected to BDL, in which the effects of PGC1α on cholestatic liver injury were demonstrated by biochemical and histopathological assessments, immunoblotting, and metabolomics. RESULTS: The expression of PGC1α was upregulated in the liver of PBC patients and murine models. Both in vivo and in vitro experiments supported the protective effects of PGC1α on cholestasis-induced hepatocyte injury. Infiltrated inflammatory cells after BDL were decreased in HKO mice. Inhibited Wnt/ß-Catenin pathway and enhanced Notch signaling promoted transdifferentiation of hepatic progenitor cells (HPC)/ hepatocytes into cholangiocytes, leading to the greater ductular reaction observed in the HKO mice. But bile acids metabolism and mitochondrial function were not affected due to hepatic PGC1α deficiency in cholestasis. CONCLUSIONS: Hepatic-specific deletion of PGC1α regulated liver regeneration by promoting ductular reactions, thereby exerting protective effects against BDL-induced liver injury, which could be a new potential therapeutic target.

Gentiopicroside Ameliorated Ductular Reaction and Inflammatory Response in DDC-induced Murine Cholangiopathies Model.

BACKGROUND: Cholangiopathies comprise a spectrum of diseases without curative treatments. Pharmacological treatments based on bile acid (BA) metabolism regulation represent promising therapeutic strategies for the treatment of cholangiopathies. Gentiopicroside (GPS), derived from the Chinese medicinal herb Gentianae Radix, exerts pharmacological effects on bile acid metabolism regulation and oxidative stress. OBJECTIVE: The present study aims to investigate the effect of GPS on 3,5-diethoxycarbonyl-1,4dihydrocollidine (DDC)-induced cholangiopathy. METHODS: Two independent animal experiments were designed to evaluate the comprehensive effect of GPS on chronic DDC diet-induced cholangiopathy, including bile duct obliteration, ductular reaction, BA metabolism reprogramming, liver fibrosis, oxidative stress and inflammatory responses. RESULTS: In the first pharmacological experiment, three doses of GPS (5, 25 and 125 mg/kg) were injected intraperitoneally into mice fed a DDC diet for 14 days. DDC induced a typical ductular reaction, increased periductal fibrosis and mixed inflammatory cell infiltration in the portal areas. GPS treatment showed dose-dependent improvements in the ductular reaction, BA metabolism, fibrosis, oxidative stress and inflammatory response. In the second experiment, a high dose of GPS was injected intraperitoneally into control mice for 28 days, resulting in no obvious histologic changes and significant serologic abnormalities in liver function. However, GPS inhibited DDC-induced oxidative stress, serum and hepatic BA accumulation, proinflammatory cytokine production, and immunocyte infiltration. Specifically, the GPS-treated groups showed decreased infiltration of monocyte-derived macrophages and CD4+ and CD8+ T lymphocytes, as well as preserved Kupffer cells. CONCLUSION: GPS alleviated chronic DDC diet-induced cholangiopathy disorder by improving the ductular reaction, periductal fibrosis, oxidative stress and inflammatory response. Its dosage-dependent pharmacological effects indicated that GPS warrants its further evaluation in clinical trials for cholangiopathy.

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 (CXAE, CXAL, CXPA and CXPHL) 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-ß (TGF-ß)-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 CXAE, CXAL and CXPHL significantly reduced ductular reaction (DR) and improved liver fibrosis in the BDL mice. Meanwhile, both CXAE and CXPHL suppressed DR in injured HIBECs and reduced collagen contraction force and the expression of fibrosis biomarkers in LX-2 cells treated with TGF-ß. CXPHL 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 CXPHL 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, CXPHL 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.

Three-dimensional analysis of ductular reactions and their correlation with liver regeneration and fibrosis.

The liver has multiple regeneration modes, including hepatocellular hypertrophy and self-renewal of hepatocytes. When hepatocyte proliferation is impaired, hepatic progenitor cells may proliferate through ductular reaction (DR), differentiate into hepatocytes, and contribute to fibrosis. However, the three-dimensional spatial relationship between DR and regenerating hepatocytes and dynamic changes in DR associated with fibrosis remain poorly understood. Here, we performed three-dimensional (3D) imaging of cleared 42 liver explants with chronic and acute liver diseases and 4 normal livers to visualize DR. In chronic hepatic liver diseases, such as viral hepatitis, steatohepatitis, autoimmune hepatitis, and cryptogenic cirrhosis, the total length and number of branches of DR showed a significant positive correlation. We studied the spatial relationship between DR and GS-expressing cells using glutamine synthetase (GS) and cytokeratin 19 (CK19) as markers of liver regeneration and DR, respectively. The percentage of CK19-positive cells that co-expressed GS was less than 10% in chronic liver diseases. In contrast, nearly one-third of CK19-positive cells co-expressed GS in acute liver diseases, and chronic cholestatic liver diseases, including primary biliary cholangitis and primary sclerosing cholangitis, showed no co-expression. We also found that DR was longer and had more branching in livers with progressive fibrosis compared to those with regressive fibrosis. Our results suggest that DR displays varying degrees of spatial complexity and contribution to liver regeneration. DR may serve as hepatobiliary junctions that maintain continuity between hepatocytes and bile ducts rather than hepatocyte regeneration in chronic liver diseases.

Costunolide alleviated DDC induced ductular reaction and inflammatory response in murine model of cholestatic liver disease.

Purpose: Cholestatic liver diseases are groups of hepatobiliary diseases without curative drug-based therapy options. Regulation of bile acid (BA) metabolism, hepatoperiductal fibrosis, and inflammatory response indicated present novel methods for the treatment of cholestatic liver disease. Costunolide (COS) from herb Saussurea lappa exerts a pharmacological effect of regulation of BA metabolism, liver fbrosis and inflammatory response. The present study aimed to clarify the pharmacodynamic effects of COS against the murine model of cholestatic liver disease. Methods: We established a murine model of cholestatic liver disease through chronic feeding of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet for 28 days. Two independent in vivo experiments were designed to reveal the pharmacological effect of COS against cholestatic liver disease. In the first experiment, two dosages of COS (10 and 30 mg/kg) were intraperitoneally injected into model mice daily for 14 days. In the second experiment, high dosage of COS (30 mg/kg) was intraperitoneally injected into control and model mice daily for 28 days. Results: In the evaluation of the hepatoprotective effect of COS, COS showed dosage-dependent improvement of cholestatic liver disease, including ductular reaction, hepatoperiductal fibrosis, and inflammatory response. The mechanism of COS-mediated hepatoprotective effects mainly relies on the regulation of BA metabolism, and the inflammatory response. DDC diet feed induced hepatic BA metabolism, transport and circulation dysfunction. COS treatment not only regulated the BA metabolism and transport gene, but also reprogrammed hepatic primary and secondary BA concentrations. DDC induced hepatic infiltrated monocytes derived macrophages and lymphocytes were inhibited, while Kupffer cells were preserved by COS treatment. The liver elevating inflammatory cytokines of DDC diet feed were alleviated by COS. Moreover, high dosage of 30 mg/kg COS treatment for 28 days resulted in no significant serological changes and no obvious hepatic histopathological changes when compared with control mice. Conclusion: COS protected against DDC diet feeding-induced cholestatic liver disease since COS regulated BA metabolism, ductular reaction, hepatoperiductal fibrosis and inflammatory response. COS is suggested as a potential natural product for the treatment of cholestatic liver disease.

Ductular reaction in non-alcoholic fatty liver disease: When Macbeth is perverted.

Non-alcoholic fatty liver disease (NAFLD) or metabolic (dysfunction)-associated fatty liver disease is the leading cause of chronic liver diseases defined as a disease spectrum comprising hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis, and hepatic carcinoma. NASH, characterized by hepatocyte injury, steatosis, inflammation, and fibrosis, is associated with NAFLD prognosis. Ductular reaction (DR) is a common compensatory reaction associated with liver injury, which involves the hepatic progenitor cells (HPCs), hepatic stellate cells, myofibroblasts, inflammatory cells (such as macrophages), and their secreted substances. Recently, several studies have shown that the extent of DR parallels the stage of NASH and fibrosis. This review summarizes previous research on the correlation between DR and NASH, the potential interplay mechanism driving HPC differentiation, and NASH progression.