Introduction

Biliary cirrhosis is inevitably accompanied by liver fibrosis, which is characterized by an increase in extracellular matrix (ECM) in the liver along with continuous destruction of liver cells. Until now, many studies have been conducted on the theory of secretion of ECM components by activation of hepatic stellate cells. Ursodeoxycholic acid (UDCA) is a representative hydrophilic endogenous bile acid and has been approved by the FDA. Epithelial-Mesenchymal Transition (EMT) means that epithelial cells lose their cell adhesion and polarity maintenance and are converted into mesenchymal cells. The EMT phenomenon is recognized as a very important molecular/cell physiological factor that causes fibrosis of various organs.

Aims & Methods

We investigated whether UDCA regulates fibrosis-related gene expression and EMT-related protein expression in hepatic stellate cells and conducted an experiment to confirm this in an animal model of biliary cirrhosis.
First, to analyze the regulation of fibrosis-related gene expression in hepatic stellate cells by UDCA, quantitative RT-PCR was performed for α-SMA, Collagen-I, vimentin, and TGF-β1 gene expression in LCX2cells. Second, the level of phosphorylation of Smad3 in the same cells was quantitatively analyzed by WesternBlot method to measure the regulation of EMT-related protein expression. Thirdly, the regulation of EMT-related gene and protein expression by UDCA was tested in the same way in the rat common bile duct ligationmodel.

Results

As a result, UDCA induces a decrease in target protein expression by reducing the expression of genes related to fibrosis in hepatic stellate cells, which is accompanied by inactivation of the TGF-β 1/Smad3 signaling system, which is an upstream regulator of fibrosis and an EMT-related protein. identified. This effect of UDCA was confirmed to be reproduced in an animal model of biliary cirrhosis.

Conclusion

Through this, the researcher identified that UDCA functions to down-regulate EMT and eventually delay liver fibrosis.

Introduction

The treatments for liver fibrosis are still in need so far. As the crucial initiators, activated hepatic stellate cells (aHSCs) undergo a series of epigenetic changes. The study intends to explore transcription factors (TFs) that facilitate HSCs activation and provide novel therapeutic targets for liver fibrosis.

Aims & Methods

Three distinct HSCs activation models were established in present study. Firstly, in vitro activation involved isolating primary HSCs from normal Sprague-Dawley rats and culturing them for two days (n=5) and seven days (n=5), namely natural activation model. Secondly, in vivo activation entailed isolating primary HSCs from bile duct ligated rats (n=4) and normal controls (n=4), followed by culturing until the next day. Lastly, transforming growth factor-β (TGF-β, 2.5ng/ml) (n=10) or control buffer (n=10) were added to the human HSCs cell line LX2 and cultured for 24 hours. Subsequently, assay for transposase-accessible chromatin using sequencing (ATAC-seq) was conducted on the abovementioned cells, along with motif enrichment and footprint analysis. Pathway enrichment analysis was performed on differentially accessible genes (DAGs). RNA sequencing (RNA-seq) was employed to analyze gene expression profiles of primary HSCs in the natural activation model (n=4). The integration of ATAC-seq and RNA-seq further identified potential key TFs. RT-qPCR and differential expression analysis of public datasets was utilized to confirm the dysregulation of TFs in primary HSCs, fibrotic liver and fibrotic model of induced pluripotent stem cells. Finally, enrichment at enhancers and promoters of these genes was validated by H3K27Ac and H3K4me3 chromatin immunoprecipitation followed by sequencing (ChIP-seq) of human HSCs.

Results

For ATAC-seq, primary HSCs exhibited higher chromatin accessibility after activation compared with the controls. After peak annotation, the up-regulated peaks were mainly located in the promoter region in all comparison groups, uncovering the dynamic transcriptional regulation during HSC activation. In the motif enrichment analysis, 150 aHSCs related TFs and 54 quiescent HSCs (qHSCs) related TFs were identified in the in vitro activation model. The three models shared 12 aHSCs related TFs, namely Runx-aml, Runx1, Runx2, Runx, Ets:Runx, Atf2, Atf4, Atf7, c-Jun-Cre, Creb5, Smad3 and CArG; and 19 qHSCs related TFs, including Foxf1, Foxo3, Foxk1, Foxp1, Hoxa1, Hoxa2, Stat3, Hnf4a, Hnf6, Elk1, Elk4, Cux1, Crx, Duxbl, Vdr, Tr4, Ebf2, Gata2, Gata6. Footprint analysis validated the alteration of accessibility in Atf2, Atf4, Atf7, Creb5, Hnf6 and Gata2. Actin filament organization, Ras protein signal transduction and intrinsic apoptotic signal pathways were enriched in DAGs. Integration of ATAC-seq and RNA-seq showed that gene expression was significantly correlated with chromatin accessibility, among which Runx2, Creb5, and Smad3 were significantly upregulated in aHSCs at transcription level. The dysregulation of these three genes was verified in primary HSCs, fibrotic liver and fibrotic model of human induced pluripotent stem cells. Finally, enrichment at active enhancers and promoters of these TFs was validated by H3K27Ac and H3K4me3 ChIP-seq of human HSCs, revealing the crucial role of genes in the transcription.

Conclusion

We generated chromatin accessibility profiles of different HSCs activation models, highlighting the significance of Runx2, Creb5, and Smad3 as key TFs mediating HSCs activation. These findings proposed these TFs as promising targets for future liver fibrosis treatment.

Disclosure

The author declare no competing interests and financial relationships with commercial interests.

Introduction

Patients with primary biliary cholangitis (PBC) are at an increased risk of developing cirrhosis and eventually experiencing hepatic decompensation. While there is increasing evidence for more prevalent aetiologies of cirrhosis that a successful removal/suppression of the underlying aetiological factor can induce significant improvements in liver function and even enable hepatic recompensation, similar insights in PBC are scarce.

Aims & Methods

In this study, we aimed to explore clinical implications of hepatic recompensation according to the novel Baveno VII criteria in PBC, while considering a biochemical response to ursodeoxycholic acid (UDCA) according to Paris-II criteria as surrogate marker of a successful aetiological treatment. Specifically, we included cirrhotic PBC patients at the time of first hepatic decompensation (ascites, hepatic encephalopathy [HE], variceal bleeding) and analysed their subsequent clinical disease course. In accordance with Baveno VII criteria, we defined hepatic recompensation as (i) resolution of ascites and HE despite discontinuation of diuretics and HE treatment, (ii) absence of variceal bleeding and (iii) sustained improvements in biochemical liver function.

Results

This study enrolled 42 PBC patients with decompensated cirrhosis (age: 63.5 [IQR:51.9-69.2] years; 88.1% female) who were followed-up for a median of 41.9 (IQR:11.0-70.9) months after decompensation. The cause of index decompensation was ascites in 54.8%, variceal bleeding in 35.7% and HE in 9.5% of cases. At time of decompensation, the median MELD-Na was 14 (IQR:11-15). Overall, 33 patients were treated with UDCA for at least 1 year (median dose: 12.5 [IQR:10.7-15.0] mg/kg) and 36% of those (12/33) achieved the Paris-II response criteria.
Over the course of the study, 7 patients (16.7%) achieved recompensation. In a competing risk model, baseline parameters linked to a higher probability of recompensation included a lower MELD-Na (SHR: 0.90; 0.047), a lower Child-Pugh score (SHR: 0.59; p=0.019), lower bilirubin (SHR: 0.44; p=0.005), lower alkaline phosphatase (SHR: 0.96; p=0.001) and variceal bleeding as index decompensation (SHR: 4.37; p=0.069). Interestingly, while 5/12 (42%) UDCA responders recompensated and a response at 1 year was linked to a higher probability of recompensation (SHR: 5.08; p=0.041), 2/21 (10%) patients without UDCA response still eventually achieved recompensation.
Regarding prognostic implications, recompensation was linked to a numerically improved transplant-free survival in a time-dependent Cox regression model (HR: 0.46; p=0.335). Specifically, 3 recompensated patients survived without any further complications or the need for liver transplantation over a follow-up time of 35, 40 and 294 months after recompensation. Nevertheless, the remaining 4 recompensated patients presented with liver-related complications after developing hepatic malignancies and/or portal vein thrombosis (PVT) and two eventually died (18 and 29 months after recompensation). In contrast, of the patients who remained decompensated, 10 underwent liver transplantation (29%) and 17 died (49%).

Conclusion

Patients with decompensated cirrhosis due to PBC may achieve hepatic recompensation under UDCA therapy, particularly if the degree of liver dysfunction at index decompensation is less pronounced. Nonetheless, recompensated patients should be monitored carefully for the development of hepatic malignancy and PVT, as liver transplantation may still be required.

Disclosure

Albert Friedrich Stättermayer: Speaker and/or consultant and/or advisory board member for Boehringer Ingelheim, Gilead, and MSD. Benedikt Simbrunner: Travel support from AbbVie and Gilead. Mattias Mandorfer: Speaker and/or consultant and/or advisory board member for AbbVie, Collective Acumen, Gilead, and W. L. Gore & Associates; Travel support from AbbVie and Gilead. Michael Trauner: Grant support from Albireo, Alnylam, Cymabay, Falk, Gilead, Intercept, MSD, Takeda and UltraGenyx; Honoraria for consulting from Albireo, Boehringer Ingelheim, BiomX, Falk, Genfit, Gilead, Intercept, MSD, Novartis, Phenex, Pliant, Regulus and Shire; Speaker fees from Bristol-Myers Squibb, Falk, Gilead, Intercept and MSD; Travel support from AbbVie, Falk, Gilead and Intercept. Thomas Reiberger: Grant support from Abbvie, Boehringer-Ingelheim, Gilead, Gore, Intercept, MSD, Myr Pharmaceuticals, Philips Healthcare, Pliant, and Siemens; Speaking honoraria from Abbvie, Gilead, Gore, Intercept, Roche, MSD; Consulting/advisory board fee from Abbvie, Bayer, Boehringer-Ingelheim, Gilead, Intercept, MSD, Siemens; Travel support from Abbvie, Boehringer-Ingelheim, Gilead and Roche.

Introduction

Liver fibrosis is primarily caused by the activation of hepatic stellate cells (HSCs), which results from chronic liver damage. Understanding the pathogenesis of HSC activation could identify new therapeutic targets to treat liver fibrosis.

Aims & Methods

In this study, we examined the protective role of the mammalian cleavage factor I 25 kD subunit (CFIm25, NUDT21) in inhibiting hepatic stellate cell activation. CFIm25 expression was measured in liver cirrhosis patients and a CCl4-induced mouse model. Adeno-associated viruses and adenoviruses were used to alter hepatic CFIm25 expression in vivo and in vitro to investigate how CFIm25 functions in liver fibrosis. The underlying mechanisms were explored using RNA-seq and co-IP assays.

Results

Here, we found that CFIm25 expression was drastically decreased in activated murine HSCs and fibrotic liver tissues. CFIm25 overexpression downregulated the expression of genes involved in liver fibrosis, inhibiting the progression of HSC activation, migration and proliferation. These effects resulted from direct activation of the KLF14/PPARγ signaling axis. KLF14 inhibition abrogated the CFIm25 overexpression-mediated reduction in antifibrotic effects.

Conclusion

In conclusion, we reported a protective role for CFIm25 in liver fibrosis. Our findings showed that CFIm25 reversed HSC activation and inhibited CCl4-induced liver fibrosis through the KLF14/PPARγ signaling pathway, which might provide a novel approach for liver fibrosis treatment.

References

1. Hernandez-Gea V, Friedman SL. Pathogenesis of liver fibrosis. Annu Rev Pathol. 2011; 6: 425-56.
2. Bataller R, Brenner DA. Liver fibrosis. The Journal of clinical investigation. 2005; 115: 209-18.
3. Parola M, Pinzani M. Liver fibrosis: Pathophysiology, pathogenetic targets and clinical issues. Mol Aspects Med. 2019; 65: 37-55.
4. Kisseleva T, Brenner D. Molecular and cellular mechanisms of liver fibrosis and its regression. Nature reviews Gastroenterology & hepatology. 2021; 18: 151-66.
5. Schuppan D, Ashfaq-Khan M, Yang AT, Kim YO. Liver fibrosis: Direct antifibrotic agents and targeted therapies. Matrix Biol. 2018; 68-69: 435-51.
6. Higashi T, Friedman SL, Hoshida Y. Hepatic stellate cells as key target in liver fibrosis. Adv Drug Deliv Rev. 2017; 121: 27-42.
7. Tsuchida T, Friedman SL. Mechanisms of hepatic stellate cell activation. Nature reviews Gastroenterology & hepatology. 2017; 14: 397-411.
8. Yin C, Evason KJ, Asahina K, Stainier DY. Hepatic stellate cells in liver development, regeneration, and cancer. The Journal of clinical investigation. 2013; 123: 1902-10.
9. Masamha CP. The emerging roles of CFIm25 (NUDT21/CPSF5) in human biology and disease. Wiley interdisciplinary reviews RNA. 2022: e1757.

Introduction

Liver cirrhosis is a late stage of liver fibrosis characterized by progressive deterioration of liver function, leading to potentially life-threatening complications. Recent studies focusing on bacterial alterations of gut microbiota have shown their association with liver disease pathogenesis, decompensation, and hospitalization rates. Recently, there has been increasing recognition of the potential role of gut fungal changes in liver cirrhosis, however, further investigation in this field is warranted.

Aims & Methods

The aim of this study was to determine gut fungi composition changes in patients with liver cirrhosis and to assess whether these changes are related to liver disease severity. For the analysis were used DNA samples, extracted from the liver cirrhosis patients’ and healthy controls’ stool samples. Internal transcribed spacer (ITS) was amplified and sequenced using MiSeq Illumina sequencing machine. Bioinformatical and statistical analysis were performed to reveal the fungal profile of each study group.

Results

Forty-five patients with liver cirrhosis and 45 healthy controls were included in the study. No significant differences in age or gender were observed between the cirrhosis and control groups. Cirrhosis patients showed significantly reduced fungal richness compared to the control cohort. However, no difference was observed in the Shannon alpha diversity index. No significant alterations in fungal compositions were noted concerning gender and etiology within the cirrhosis group. Patients who had Child-Pugh B and C liver cirrhosis had a significant shift in stool fungi composition compared to those in the Child-Pugh A group. Patients with Child C liver cirrhosis had a significantly increased fungi abundance compared with the Child A group. In accordance with the MELD score, patients with score ≥15, had significantly increased richness and fungal diversity. Furthermore, an elevated abundance of the Candida genus was observed in liver cirrhosis patients group, while Saccharomyces genus was increased in the control group.

Conclusion

Patients with liver cirrhosis had reduced richness in stool fungal composition and an increased abundance of the Candida genus when compared with healthy controls. Moreover, patients with worse liver function had significant shifts in fecal fungal composition, suggesting that fungal alterations may be associated with cirrhosis progression.

Introduction

Recurrent bile duct stones (RBDS) develop in up to 20% of patients after successful endoscopic clearance of initial stones. While anatomical and procedural risk factors are well recognized, the role of bile acid composition and bile microbiota in RBDS has gained attention. Bile acids, critical for cholesterol solubilization and intestinal homeostasis, may undergo deconjugation or biotransformation when exposed to intestinal flora via duodenobiliary reflux, altering their physicochemical properties and promoting lithogenicity.

Aims & Methods

We aimed to investigate whether alterations in bile acid composition and microbial colonization are associated with stone recurrence. A total of 184 patients who underwent biliary drainage at Konyang University Hospital (2019–2022) were enrolled. Patients were categorized into First Diagnosis Group (n=155) and Recurrence Group (n=29). Bile was aspirated after ERCP or PTBD, and 15 bile acids (5 unconjugated, 10 conjugated) were quantified using HPLC-MS/MS. Bile cultures were analyzed for microbial composition. Statistical comparisons were made using independent t-tests and chi-square analysis.

Results

Patients with RBDS were significantly older (77.8 vs. 69.0 years, P=0.005). Total bile acid concentration was lower in the Recurrence Group (4734 vs. 5902 ppm, P=0.083). Conjugated bile acids—including T-CA, T-DCA, G-CA, G-DCA, and G-LCA—were significantly reduced (P<0.05). Taurine-conjugated bile acids showed a marked decline (1326 vs. 1876 ppm, P=0.041). Primary bile acids were lower in the Recurrence Group (3272 vs. 4339 ppm, P=0.035), while the primary-to-secondary bile acid ratio was significantly decreased (3.29 vs. 7.35, P=0.001), indicating a shift toward hydrophobic bile.
Microbiologically, Enterococcus spp. were significantly more prevalent in the Recurrence Group (76% vs. 30%, P<0.001), particularly E. faecalis, E. casseliflavus, and E. gallinarum. Notably, E. faecalis possesses bile salt hydrolase (BSH) activity that can deconjugate bile acids, promoting accumulation of hydrophobic, poorly soluble forms. This aligns with the observed increase in unconjugated LCA and DCA.

Conclusion

Recurrent bile duct stone patients exhibit reduced levels of conjugated and primary bile acids and increased prevalence of bile-resident Enterococcus spp., known for bile salt hydrolase activity. These changes may lead to deconjugation, increased hydrophobicity, and impaired micelle formation, contributing to bile lithogenicity. These findings support the potential utility of bile acid profiling and bile microbiota analysis as predictive and preventive tools in RBDS management.

References

1. Hofmann AF, Mysels KJ. Bile acids as biological surfactants. Colloids Surf. 1987;30(1):145–173.
2. Begley M, Hill C, Gahan CG. Bile salt hydrolase activity in probiotics. Appl Environ Microbiol. 2006;72(3):1729–1738.
3. Choe JW, Lee JM, Hyun JJ, Lee HS. Analysis on microbial profiles and components of bile in patients with recurrent CBD stones after endoscopic removal: a preliminary study. J Clin Med. 2021;10(15):3303.
4. Ryu KH. Gut Microbiota and Pancreatobiliary System. Korean J Gastroenterol. 2020;75(5):231–239. (Korean)
5. Di Ciaula A, Garruti G, Baccetto RL, Molina-Molina E, Bonfrate L, Portincasa P. Bile acid physiology. Ann Hepatol. 2018;16(1):4–14.
6. Wang DQ, Cohen DE, Carey MC. Biliary lipids and cholesterol gallstone disease. J Lipid Res. 2009;50 Suppl:S406–S411.