Analysis of culture and RNA isolation methods for precision-cut liver slices from cirrhotic rats.

Precision-cut liver slices (PCLS) are increasingly used as a model to investigate anti-fibrotic therapies. However, many studies use PCLS from healthy animals treated with pro-fibrotic stimuli in culture, which reflects only the early stages of fibrosis. The effects of different culture conditions on PCLS from cirrhotic animals has not been well characterized and there is no consensus on optimal methods. In this study, we report a method for the collection and culture of cirrhotic PCLS and compare the effect of common culture conditions on viability, function, and gene expression. Additionally, we compared three methods of RNA isolation and identified a protocol with high yield and purity. We observed significantly increased albumin production when cultured with insulin-transferrin-selenium and dexamethasone, and when incubated on a rocking platform. Culturing with insulin-transferrin-selenium and dexamethasone maintained gene expression closer to the levels in fresh slices. However, despite stable viability and function up to 4 days, we found significant changes in expression of key genes by day 2. Interestingly, we also observed that cirrhotic PCLS maintain viability in culture longer than slices from healthy animals. Due to the influence of matrix stiffness on fibrosis and hepatocellular function, it is important to evaluate prospective anti-fibrotic therapies in a platform that preserves tissue biomechanics. PCLS from cirrhotic animals represent a promising tool for the development of treatments for chronic liver disease.

IFNγ-IL12 axis regulates intercellular crosstalk in metabolic dysfunction-associated steatotic liver disease.

Obesity is a major cause of metabolic dysfunction-associated steatohepatitis (MASH) and is characterized by inflammation and insulin resistance. Interferon-γ (IFNγ) is a pro-inflammatory cytokine elevated in obesity and modulating macrophage functions. Here, we show that male mice with loss of IFNγ signaling in myeloid cells (Lyz-IFNγR2-/-) are protected from diet-induced insulin resistance despite fatty liver. Obesity-mediated liver inflammation is also attenuated with reduced interleukin (IL)-12, a cytokine primarily released by macrophages, and IL-12 treatment in vivo causes insulin resistance by impairing hepatic insulin signaling. Following MASH diets, Lyz-IFNγR2-/- mice are rescued from developing liver fibrosis, which is associated with reduced fibroblast growth factor (FGF) 21 levels. These results indicate critical roles for IFNγ signaling in macrophages and their release of IL-12 in modulating obesity-mediated insulin resistance and fatty liver progression to MASH. In this work, we identify the IFNγ-IL12 axis in regulating intercellular crosstalk in the liver and as potential therapeutic targets to treat MASH.

Establishment of diagnostic model and identification of diagnostic markers between liver cancer and cirrhosis based on multi-chip and machine learning.

OBJECTIVE: Most cases of hepatocellular carcinoma (HCC) arise as a consequence of cirrhosis. In this study, our objective is to construct a comprehensive diagnostic model that investigates the diagnostic markers distinguishing between cirrhosis and HCC. METHODS: Based on multiple GEO datasets containing cirrhosis and HCC samples, we used lasso regression, random forest (RF)-recursive feature elimination (RFE) and receiver operator characteristic analysis to screen for characteristic genes. Subsequently, we integrated these genes into a multivariable logistic regression model and validated the linear prediction scores in both training and validation cohorts. The ssGSEA algorithm was used to estimate the fraction of infiltrating immune cells in the samples. Finally, molecular typing for patients with cirrhosis was performed using the CCP algorithm. RESULTS: The study identified 137 differentially expressed genes (DEGs) and selected five significant genes (CXCL14, CAP2, FCN2, CCBE1 and UBE2C) to construct a diagnostic model. In both the training and validation cohorts, the model exhibited an area under the curve (AUC) greater than 0.9 and a kappa value of approximately 0.9. Additionally, the calibration curve demonstrated excellent concordance between observed and predicted incidence rates. Comparatively, HCC displayed overall downregulation of infiltrating immune cells compared to cirrhosis. Notably, CCBE1 showed strong correlations with the tumour immune microenvironment as well as genes associated with cell death and cellular ageing processes. Furthermore, cirrhosis subtypes with high linear predictive scores were enriched in multiple cancer-related pathways. CONCLUSION: In conclusion, we successfully identified diagnostic markers distinguishing between cirrhosis and hepatocellular carcinoma and developed a novel diagnostic model for discriminating the two conditions. CCBE1 might exert a pivotal role in regulating the tumour microenvironment, cell death and senescence.

Clinical and genetic risk factors for progressive fibrosis in metabolic dysfunction-associated steatotic liver disease.

BACKGROUND: Fibrosis-4 (FIB4) is a recommended noninvasive test to assess hepatic fibrosis among patients with metabolic dysfunction-associated steatotic liver disease (MASLD). Here, we used FIB4 trajectory over time (ie, "slope" of FIB4) as a surrogate marker of liver fibrosis progression and examined if FIB4 slope is associated with clinical and genetic factors among individuals with clinically defined MASLD within the Million Veteran Program Cohort. METHODS: In this retrospective cohort study, FIB4 slopes were estimated through linear regression for participants with clinically defined MASLD and FIB4 <2.67 at baseline. FIB4 slope was correlated with demographic parameters and clinical outcomes using logistic regression and Cox proportional hazard models. FIB4 slope as a quantitative phenotype was used in a genome-wide association analysis in ancestry-specific analysis and multiancestry meta-analysis using METAL. RESULTS: FIB4 slopes, generated from 98,361 subjects with MASLD (16,045 African, 74,320 European, and 7996 Hispanic), showed significant associations with sex, ancestry, and cardiometabolic risk factors (p < 0.05). FIB4 slopes also correlated strongly with hepatic outcomes and were independently associated with time to cirrhosis. Five genetic loci showed genome-wide significant associations (p < 5 × 10-8) with FIB4 slope among European ancestry subjects, including 2 known (PNPLA3 and TM6SF2) and 3 novel loci (TERT 5.1 × 10-11; LINC01088, 3.9 × 10-8; and MRC1, 2.9 × 10-9). CONCLUSIONS: Linear trajectories of FIB4 correlated significantly with time to progression to cirrhosis, with liver-related outcomes among individuals with MASLD and with known and novel genetic loci. FIB4 slope may be useful as a surrogate measure of fibrosis progression.

Glycolysis in hepatic stellate cells coordinates fibrogenic extracellular vesicle release spatially to amplify liver fibrosis.

Liver fibrosis is characterized by the activation of perivascular hepatic stellate cells (HSCs), the release of fibrogenic nanosized extracellular vesicles (EVs), and increased HSC glycolysis. Nevertheless, how glycolysis in HSCs coordinates fibrosis amplification through tissue zone-specific pathways remains elusive. Here, we demonstrate that HSC-specific genetic inhibition of glycolysis reduced liver fibrosis. Moreover, spatial transcriptomics revealed a fibrosis-mediated up-regulation of EV-related pathways in the liver pericentral zone, which was abrogated by glycolysis genetic inhibition. Mechanistically, glycolysis in HSCs up-regulated the expression of EV-related genes such as Ras-related protein Rab-31 (RAB31) by enhancing histone 3 lysine 9 acetylation on the promoter region, which increased EV release. Functionally, these glycolysis-dependent EVs increased fibrotic gene expression in recipient HSC. Furthermore, EVs derived from glycolysis-deficient mice abrogated liver fibrosis amplification in contrast to glycolysis-competent mouse EVs. In summary, glycolysis in HSCs amplifies liver fibrosis by promoting fibrogenic EV release in the hepatic pericentral zone, which represents a potential therapeutic target.

[Relationships between Cxcl12, Tweak, Notch1, and Yap mRNA Expression Levels in Molecular Mechanisms of Liver Fibrogenesis].

Current data on the molecular mechanisms of liver fibrosis and cirrhosis fail to fully explain all stages of their development. Interactions between individual genes and signaling pathways are known to play an important role in their functions. However, data on their relationships are insufficient and often contradictory. For the first time, mRNA expression of Notch1, Notch2, Yap1, Tweak (Tnfsf12), Fn14 (Tnfrsf12a), Ang, Vegfa, Cxcl12 (Sdf), Nos2, and Mmp-9 was studied in detail at several stages of thioacetamide-induced liver fibrosis in Wistar rats. A factor analysis isolated three factors, which combined highly correlated target genes. The first factor included four genes: Cxcl12 (r = 0.829, p < 0.05), Tweak (r = 0.841, p < 0.05), Notch1 (r = 0.848, p < 0.05), and Yap1 (r = 0.921, p < 0.05). The second factor described the correlation between Mmp-9 (r = 0.791, p < 0.05) and Notch2 (r = 0.836, p < 0.05). The third factor included Ang (r = 0.748, p < 0.05) and Vegfa (r = 0.679, p < 0.05). The Nos2 and Fn14 genes were not included in any of the factors. The gene grouping by mRNA expression levels made it possible to assume a pathogenetic relationship between their products in the development of fibrotic changes due to liver toxicity.

Do iron homeostasis biomarkers mediate the associations of liability to type 2 diabetes and glycemic traits in liver steatosis and cirrhosis: a two-step Mendelian randomization study.

BACKGROUND: Previous studies, including Mendelian randomization (MR), have demonstrated type 2 diabetes (T2D) and glycemic traits are associated with increased risk of metabolic dysfunction-associated steatotic liver disease (MASLD). However, few studies have explored the underlying pathway, such as the role of iron homeostasis. METHODS: We used a two-step MR approach to investigate the associations of genetic liability to T2D, glycemic traits, iron biomarkers, and liver diseases. We analyzed summary statistics from various genome-wide association studies of T2D (n = 933,970), glycemic traits (n ≤ 209,605), iron biomarkers (n ≤ 246,139), MASLD (n ≤ 972,707), and related biomarkers (alanine aminotransferase (ALT) and proton density fat fraction (PDFF)). Our primary analysis was based on inverse-variance weighting, followed by several sensitivity analyses. We also conducted mediation analyses and explored the role of liver iron in post hoc analysis. RESULTS: Genetic liability to T2D and elevated fasting insulin (FI) likely increased risk of liver steatosis (ORliability to T2D: 1.14 per doubling in the prevalence, 95% CI: 1.10, 1.19; ORFI: 3.31 per log pmol/l, 95% CI: 1.92, 5.72) and related biomarkers. Liability to T2D also likely increased the risk of developing liver cirrhosis. Genetically elevated ferritin, serum iron, and liver iron were associated with higher risk of liver steatosis (ORferritin: 1.25 per SD, 95% CI 1.07, 1.46; ORliver iron: 1.15 per SD, 95% CI: 1.05, 1.26) and liver cirrhosis (ORserum iron: 1.31, 95% CI: 1.06, 1.63; ORliver iron: 1.34, 95% CI: 1.07, 1.68). Ferritin partially mediated the association between FI and liver steatosis (proportion mediated: 7%, 95% CI: 2-12%). CONCLUSIONS: Our study provides credible evidence on the causal role of T2D and elevated insulin in liver steatosis and cirrhosis risk and indicates ferritin may play a mediating role in this association.

From Inflammation to Oncogenesis: Tracing Serum DCLK1 and miRNA Signatures in Chronic Liver Diseases.

Chronic liver diseases, fibrosis, cirrhosis, and HCC are often a consequence of persistent inflammation. However, the transition mechanisms from a normal liver to fibrosis, then cirrhosis, and further to HCC are not well understood. This study focused on the role of the tumor stem cell protein doublecortin-like kinase 1 (DCLK1) in the modulation of molecular factors in fibrosis, cirrhosis, or HCC. Serum samples from patients with hepatic fibrosis, cirrhosis, and HCC were analyzed via ELISA or NextGen sequencing and were compared with control samples. Differentially expressed (DE) microRNAs (miRNA) identified from these patient sera were correlated with DCLK1 expression. We observed elevated serum DCLK1 levels in fibrosis, cirrhosis, and HCC patients; however, TGF-ß levels were only elevated in fibrosis and cirrhosis. While DE miRNAs were identified for all three disease states, miR-12136 was elevated in fibrosis but was significantly increased further in cirrhosis. Additionally, miR-1246 and miR-184 were upregulated when DCLK1 was high, while miR-206 was downregulated. This work distinguishes DCLK1 and miRNAs' potential role in different axes promoting inflammation to tumor progression and may serve to identify biomarkers for tracking the progression from pre-neoplastic states to HCC in chronic liver disease patients as well as provide targets for treatment.

Chronic liver fibrosis induction in aging causes significant ultra-structural deterioration in liver and alteration on immune response gene expressions in liver-spleen axis.

The relationship between damage to the liver and spleen by aging and the immune response status in these two organs, which are anatomically and immunologically interconnected, is unknown. The authors investigated the histopathological, ultrastructural, and immunological effects of aging in young and aged fibrotic mice by using an experimental model. Four groups were planned, with 10 mice in each experimental group. The levels of fibrosis and ultrastructural destruction in the liver were determined by α-SMA staining and TEM analysis. Expression levels of immunity genes (Il2, Il4, Il6, Il10, Il12, Il17, Tnf, Ifng, Tgfb1, Gata3, Rorc, Tbx21, Foxp3, Ccl2, Ccr2, Cxcr3, Pf4, Cxcl10) were carried out by qRT-PCR. While structural disorders were detected in the mitochondria of aged healthy group, cellular destruction in the fibrosis-induced elderly group was at a dramatic level. Fibrosis induction in aged mice caused an elevation in the expression of chemokines (CCl2, CXCL10, CCR2) and cytokine (IL-17a) genes that induce autoinflammatory response in the liver. Unlike the cellular pathology and genes activated in fibrosis in youth and the natural occurrence of fibrosis with aging, induction of fibrosis during aging causes deterioration in the liver and expression of genes responsible for autoimmunity in both the liver and spleen.

Hypoalbuminemia contributes to ascites formation via sodium and water retention: Evidence from clinical date and albumin deficient mice.

Albumin infusions improve circulatory and renal function in patients with decompensated cirrhosis. However, there is no convincing evidence that hypoalbuminemia contributes to ascites formation in liver cirrhosis. The aim of our study is to determine the exact role of hypoalbuminemia in the formation of ascites caused by liver cirrhosis and its underlying mechanism. Clinical profiles of patients with liver cirrhosis retrospectively analyzed. The details of albumin involved in ascites formation were investigated in rat model and murine model. Statistical analysis demonstrated hypoalbuminemia was an independent risk factor for ascites formation in patients with liver cirrhosis (OR = 0.722, P < 0.001). In carbon tetrachloride (CCl4)-induced rat model of liver cirrhosis, a significant reduction in serum albumin was observed in rats with ascites (13.37 g/L) compared with rats without ascites (21.43 g/L, P < 0.001). In thioacetamide (TAA)-treated mice, ascites amount of heterozygous albumin (Alb+/-) mice (112.0 mg) was larger than that of wild-type (Alb+/+) mice (58.46 mg, P < 0.001). In CCl4-induced chronic liver injury, ascites amounts of Alb+/- or Alb+/+ mice were 80.00 mg or 48.46 mg (P = 0.001). Further study demonstrated 24-h urinary sodium excretion in Alb+/- mice was lower than that of Alb+/+ mice in TAA/CCl4-induce murine models of liver cirrhosis. Additionally, serum sodium concentration of Alb+/- mice was lower than that of Alb+/+ mice. In cirrhotic mice, higher level of antidiuretic hormone was observed in Alb+/- mice compared with the control; and renal aquaporin (AQP2) expression in Alb+/- mice was significantly higher than that of WT mice. These revealed hypoalbuminemia contributed to the occurrence of ascites in liver cirrhosis through sodium and water retention.

Microarray analysis demonstrates up-regulation of the endothelin-1 gene with compensatory down-regulation of the ETA receptor gene in human portal vein.

High blood pressure in the portal vein, portal hypertension (PH), is the final common pathway in liver cirrhosis regardless of aetiology. Complications from PH are the major cause of morbidity and mortality in these patients. Current drug therapy to reduce portal pressure is mainly limited to ß-adrenergic receptor blockade but approximately 40% of patients do not respond. Our aim was to use microarray to measure the expression of ∼20,800 genes in portal vein from patients with PH undergoing transplantation for liver cirrhosis (PH, n=12) versus healthy vessels (control, n=9) to identify potential drug targets to improve therapy. Expression of 9,964 genes above background was detected in portal vein samples. Comparing PH veins versus control (adjusted P-value < 0.05, fold change > 1.5) identified 548 up-regulated genes and 1,996 down-regulated genes. The 2,544 differentially expressed genes were subjected to pathway analysis. We identified 49 significantly enriched pathways. The endothelin pathway was ranked the tenth most significant, the only vasoconstrictive pathway to be identified. ET-1 gene (EDN1) was significantly up-regulated, consistent with elevated levels of ET-1 peptide previously measured in PH and cirrhosis. ETA receptor gene (EDNRA) was significantly down-regulated, consistent with an adaptive response to increased peptide levels in the portal vein but there was no change in the ETB gene (EDNRB). The results provide further support for evaluating the efficacy of ETA receptor antagonists as a potential therapy in addition to ß-blockers in patients with PH and cirrhosis.

KDM4C represses liver fibrosis by regulating H3K9me3 methylation of ALKBH5 and m6A methylation of snail1 mRNA.

OBJECTIVE: We aimed to disclose the molecular mechanism of snail1 in liver fibrosis. METHODS: Carbon tetrachloride (CCl4) was used to induce a liver fibrosis model in mice whereby serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were evaluated, and liver pathological alternations were assessed. Rat hepatic stellate cells (HSC-T6) were irritated with transforming growth factor (TGF)-ß1, followed by assessment of cell viability and migration. The levels of snail1, ALKBH5, and lysine specific demethylase 4C (KDM4C) were quantified by immunohistochemistry, western blot, or reverse transcription-quantitative polymerase chain reaction, in addition to α-smooth muscle actin (SMA), anti-collagen type I α1 (COL1A1), vimentin, and E-cadherin. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation and RNA stability were evaluated to determine the relationship between ALKBH5 and snail1. Changes in KDM4C-bound ALKBH5 promoter and enrichment of histone H3 lysine 9 trimethylation (H3K9me3) at the ALKBH5 promoter were determined using chromatin immunoprecipitation. RESULTS: In fibrosis mice, snail1 was upregulated while ALKBH5 and KDM4C were downregulated. KDM4C overexpression reduced serum ALT and AST levels, liver injury, and α-SMA, COL1A1 and VIMENTIN expressions but increased E-cadherin expression. However, the aforementioned trends were reversed by concurrent overexpression of snail1. In HSC-T6 cells exposed to TGF-ß1, ALKBH5 overexpression weakened cell viability and migration, downregulated α-SMA, COL1A1 and VIMENTIN, upregulated E-CADHERIN, and decreased m6A modification of snail1 and its mRNA stability. KDM4C increased ALKBH5 expression by lowering H3K9me3 level, but inhibited HSC-T6 cell activation by regulating the ALKBH5/snail1 axis. CONCLUSION: KDM4C decreases H3K9me3 methylation to upregulate ALKBH5 and subsequently inhibits snail1, ultimately impeding liver fibrosis.

Stiffness sensing via Piezo1 enhances macrophage efferocytosis and promotes the resolution of liver fibrosis.

Tissue stiffening is a predominant feature of fibrotic disorders, but the response of macrophages to changes in tissue stiffness and cellular context in fibrotic diseases remains unclear. Here, we found that the mechanosensitive ion channel Piezo1 was up-regulated in hepatic fibrosis. Macrophages lacking Piezo1 showed sustained inflammation and impaired spontaneous resolution of early liver fibrosis. Further analysis revealed an impairment of clearance of apoptotic cells by macrophages in the fibrotic liver. Macrophages showed enhanced efferocytosis when cultured on rigid substrates but not soft ones, suggesting stiffness-dependent efferocytosis of macrophages required Piezo1 activation. Besides, Piezo1 was involved in the efficient acidification of the engulfed cargo in the phagolysosomes and affected the subsequent expression of anti-inflammation genes after efferocytosis. Pharmacological activation of Piezo1 increased the efferocytosis capacity of macrophages and accelerated the resolution of inflammation and fibrosis. Our study supports the antifibrotic role of Piezo1-mediated mechanical sensation in liver fibrosis, suggesting that targeting PIEZO1 to enhance macrophage efferocytosis could induce fibrosis regression.

Egr2 drives the differentiation of Ly6Chi monocytes into fibrosis-promoting macrophages in metabolic dysfunction-associated steatohepatitis in mice.

Metabolic dysfunction-associated steatohepatitis (MASH), previously called non-alcoholic steatohepatitis (NASH), is a growing concern worldwide, with liver fibrosis being a critical determinant of its prognosis. Monocyte-derived macrophages have been implicated in MASH-associated liver fibrosis, yet their precise roles and the underlying differentiation mechanisms remain elusive. In this study, we unveil a key orchestrator of this process: long chain saturated fatty acid-Egr2 pathway. Our findings identify the transcription factor Egr2 as the driving force behind monocyte differentiation into hepatic lipid-associated macrophages (hLAMs) within MASH liver. Notably, Egr2-deficiency reroutes monocyte differentiation towards a macrophage subset resembling resident Kupffer cells, hampering hLAM formation. This shift has a profound impact, suppressing the transition from benign steatosis to liver fibrosis, demonstrating the critical pro-fibrotic role played by hLAMs in MASH pathogenesis. Long-chain saturated fatty acids that accumulate in MASH liver emerge as potent inducers of Egr2 expression in macrophages, a process counteracted by unsaturated fatty acids. Furthermore, oral oleic acid administration effectively reduces hLAMs in MASH mice. In conclusion, our work not only elucidates the intricate interplay between saturated fatty acids, Egr2, and monocyte-derived macrophages but also highlights the therapeutic promise of targeting the saturated fatty acid-Egr2 axis in monocytes for MASH management.

Finding key genes (UBE2T, KIF4A, CDCA3, and CDCA5) co-expressed in hepatitis, cirrhosis and hepatocellular carcinoma based on multiple bioinformatics techniques.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Hepatitis B virus (HBV) is one of the major causes of liver cirrhosis (LC) and HCC. Therefore, the discovery of common markers for hepatitis B or LC and HCC is crucial for the prevention of HCC. METHODS: Expressed genes for to chronic active hepaititis B (CAH-B), LC and HCC were obtained from the GEO and TCGA databases, and co-expressed genes were screened using Protein-protein interaction (PPI) networks, least absolute shrinkage and selection operator (LASSO), random forest (RF) and support vector machine - recursive feature elimination (SVM-RFE). The prognostic value of genes was assessed using Kaplan-Meier (KM) survival curves. Columnar line plots, calibration curves and receiver operating characteristic (ROC) curves of individual genes were used for evaluation. Validation was performed using GEO datasets. The association of these key genes with HCC clinical features was explored using the UALCAN database ( https://ualcan.path.uab.edu/index.html ). RESULTS: Based on WGCNA analysis and TCGA database, the co-expressed genes (565) were screened. Moreover, the five algorithms of MCODE (ClusteringCoefficient, MCC, Degree, MNC, and DMNC) was used to select one of the most important and most closely linked clusters (the top 50 genes ranked). Using, LASSO regression model, RF model and SVM-RFE model, four key genes (UBE2T, KIF4A, CDCA3, and CDCA5) were identified for subsequent research analysis. These 4 genes were highly expressed and associated with poor prognosis and clinical features in HCC patients. CONCLUSION: These four key genes (UBE2T, KIF4A, CDCA3, and CDCA5) may be common biomarkers for CAH-B and HCC or LC and HCC, promising to advance our understanding of the molecular basis of CAH-B/LC/HCC progression.

Sphingosine d18:1 promotes nonalcoholic steatohepatitis by inhibiting macrophage HIF-2α.

Non-alcoholic steatohepatitis (NASH) is a severe type of the non-alcoholic fatty liver disease (NAFLD). NASH is a growing global health concern due to its increasing morbidity, lack of well-defined biomarkers and lack of clinically effective treatments. Using metabolomic analysis, the most significantly changed active lipid sphingosine d18:1 [So(d18:1)] is selected from NASH patients. So(d18:1) inhibits macrophage HIF-2α as a direct inhibitor and promotes the inflammatory factors secretion. Male macrophage-specific HIF-2α knockout and overexpression mice verified the protective effect of HIF-2α on NASH progression. Importantly, the HIF-2α stabilizer FG-4592 alleviates liver inflammation and fibrosis in NASH, which indicated that macrophage HIF-2α is a potential drug target for NASH treatment. Overall, this study confirms that So(d18:1) promotes NASH and clarifies that So(d18:1) inhibits the transcriptional activity of HIF-2α in liver macrophages by suppressing the interaction of HIF-2α with ARNT, suggesting that macrophage HIF-2α may be a potential target for the treatment of NASH.

Unraveling the hepatic stellate cells mediated mechanisms in aging's influence on liver fibrosis.

Aging enhances numerous processes that compromise homeostasis and pathophysiological processes. Among these, activated HSCs play a pivotal role in advancing liver fibrosis. This research delved into how aging impacts liver fibrosis mechanisms. The study involved 32 albino rats categorized into four groups: Group I (young controls), Group II (young with liver fibrosis), Group III (old controls), and Group IV (old with liver fibrosis). Various parameters including serum ALT, adiponectin, leptin, and cholesterol levels were evaluated. Histopathological analysis was performed, alongside assessments of TGF-ß, FOXP3, and CD133 gene expressions. Markers of fibrosis and apoptosis were the highest in group IV. Adiponectin levels significantly decreased in Group IV compared to all other groups except Group II, while cholesterol levels were significantly higher in liver fibrosis groups than their respective control groups. Group III displayed high hepatic expression of desmin, α-SMA, GFAP and TGF- ß and in contrast to Group I. Increased TGF-ß and FOXP3 gene expressions were observed in Group IV relative to Group II, while CD133 gene expression decreased in Group IV compared to Group II. In conclusion, aging modulates immune responses, impairs regenerative capacities via HSC activation, and influences adipokine and cholesterol levels, elevating the susceptibility to liver fibrosis.

Gallbladder dysfunction caused by MYPT1 ablation triggers cholestasis-induced hepatic fibrosis in mice.

BACKGROUND: The incidence of gallbladder diseases is as high as 20%, but whether gallbladder diseases contribute to hepatic disorders remains unknown. METHODS: Here, we established an animal model of gallbladder dysfunction and assessed the role of a diseased gallbladder in cholestasis-induced hepatic fibrosis (CIHF). RESULTS: Mice with smooth muscle-specific deletion of Mypt1, the gene encoding the main regulatory subunit of myosin light chain phosphatase (myosin phosphatase target subunit 1 [MYPT1]), had apparent dysfunction of gallbladder motility. This dysfunction was evidenced by abnormal contractile responses, namely, inhibited cholecystokinin 8-mediated contraction and nitric oxide-resistant relaxation. As a consequence, the gallbladder displayed impaired bile filling and biliary tract dilation comparable to the alterations in CIHF. Interestingly, the mutant animals also displayed CIHF features, including necrotic loci by the age of 1 month and subsequently exhibited progressive fibrosis and hyperplastic/dilated bile ducts. This pathological progression was similar to the phenotypes of the animal model with bile duct ligation and patients with CIHF. The characteristic biomarker of CIHF, serum alkaline phosphatase activity, was also elevated in the mice. Moreover, we observed that the myosin phosphatase target subunit 1 protein level was able to be regulated by several reagents, including lipopolysaccharide, exemplifying the risk factors for gallbladder dysfunction and hence CIHF. CONCLUSIONS: We propose that gallbladder dysfunction caused by myosin phosphatase target subunit 1 ablation is sufficient to induce CIHF in mice, resulting in impairment of the bile transport system.

Hepatocyte-specific loss of DDB1 attenuates hepatic steatosis but aggravates liver inflammation and fibrosis in MASH.

BACKGROUND: MASH is a common clinical disease that can lead to advanced liver conditions, but no approved pharmacotherapies are available due to an incomplete understanding of its pathogenesis. Damaged DNA binding protein 1 (DDB1) participates in lipid metabolism. Nevertheless, the function of DDB1 in MASH is unclear. METHODS: Clinical liver samples were obtained from patients with MASH and control individuals by liver biopsy. Hepatocyte-specific Ddb1-knockout mice and liver Hmgb1 knockdown mice were fed with a methionine-and choline-deficient diet to induce MASH. RESULTS: We found that the expression of DDB1 in the liver was significantly decreased in MASH models. Hepatocyte-specific ablation of DDB1 markedly alleviated methionine-and choline-deficient diet-induced liver steatosis but unexpectedly exacerbated inflammation and fibrosis. Mechanistically, DDB1 deficiency attenuated hepatic steatosis by downregulating the expression of lipid synthesis and uptake genes. We identified high-mobility group box 1 as a key candidate target for DDB1-mediated liver injury. DDB1 deficiency upregulated the expression and extracellular release of high-mobility group box 1, which further increased macrophage infiltration and activated HSCs, ultimately leading to the exacerbation of liver inflammation and fibrosis. CONCLUSIONS: These data demonstrate the independent regulation of hepatic steatosis and injury in MASH. These findings have considerable clinical implications for the development of therapeutic strategies for MASH.