Liver-targeted nanoparticles delivering nitric oxide reduce portal hypertension in cirrhotic rats.

Nitric oxide (NO) is a small vasodilator playing a key role in the pathogenesis of portal hypertension. Here, we assessed the potential therapeutic effect of a NO donor targeted to the liver by poly(beta-amino ester) nanoparticles (pBAE NPs) in experimental cirrhosis. Retinol-functionalized NO donor pBAE NPs (Ret pBAE NPs) were synthetized with the aim of actively targeting the liver. Administration of Ret pBAE NPs resulted in uptake and transfection by the liver and spleen. NPs were not found in other organs or the systemic circulation. Treatment with NO donor Ret pBAE NPs (30 mg/ kg body weight) significantly decreased aspartate aminotransferase, lactate dehydrogenase and portal pressure (9.75 ± 0.64 mmHg) compared to control NPs (13.4 ± 0.53 mmHg) in cirrhotic rats. There were no effects on mean arterial pressure and cardiac output. Liver-targeted NO donor NPs reduced collagen fibers and steatosis, activation of hepatic stellate cells and mRNA expression of profibrogenic and proinflammatory genes. Finally, Ret pBAE NPs displayed efficient transfection in human liver slices. Overall, liver-specific NO donor NPs effectively target the liver and mitigated inflammation and portal hypertension in cirrhotic rats. The use of Ret pBAE may prove to be an effective therapeutic strategy to treat advanced liver disease.

Changes in Macrophage Subpopulations in Rat Liver at Different Stages of Experimental Fibrosis.

The number, phenotypic composition, and functional properties of macrophages in the liver of Wistar rats change depending on the stages of fibrosis induced by thioacetamide. In the sinusoidal capillaries of the liver of control rats, CD68+ wing-shaped cells were mainly detected. The number of CD68+ cells at the stages of fibrosis before the process of its transformation into cirrhosis was 2-fold higher (p=0.0000) than in the control. At later terms of the experiment, no significant differences were found. Immunohistochemical method revealed two morphologically different groups of CD68+ cells differing in shape and localization. At all stages of the experiment, round and elongated CD206+ cells of were detected in the sinusoidal capillaries. At the stage of cirrhosis (13 weeks), the number of CD206+ cells was higher than during the third week of the experiment by 3.21 times (p=0.0000). Later, a decrease in the number of CD206+ cells was observed. At the same time, in the portal zones and connective tissue septa around the false hepatic lobules, round CX3CR1+ cells were noted. By the end of the experiment (17 weeks), their number exceeded that on the third week of the experiment by 5.66 times (p=0.0000).

Experimental Liver Cirrhosis Inhibits Restenosis after Balloon Angioplasty.

The effect of liver cirrhosis on vascular remodeling in vivo remains unknown. Therefore, this study investigates the influence of cholestatic liver cirrhosis on carotid arterial remodeling. A total of 79 male Sprague Dawley rats underwent bile duct ligation (cirrhotic group) or sham surgery (control group) and 28 days later left carotid artery balloon dilatation; 3, 7, 14 and 28 days after balloon dilatation, the rats were euthanized and carotid arteries were harvested. Histological sections were planimetrized, cell counts determined, and systemic inflammatory parameters measured. Up to day 14 after balloon dilatation, both groups showed a comparable increase in neointima area and degree of stenosis. By day 28, however, both values were significantly lower in the cirrhotic group (% stenosis: 20 ± 8 vs. 42 ± 10, p = 0.010; neointimal area [mm2]: 0.064 ± 0.025 vs. 0.138 ± 0.025, p = 0.024). Simultaneously, cell density in the neointima (p = 0.034) and inflammatory parameters were significantly higher in cirrhotic rats. This study demonstrates that cholestatic liver cirrhosis in rats substantially increases neointimal cell consolidation between days 14 and 28. Thereby, consolidation proved important for the degree of stenosis. This may suggest that patients with cholestatic cirrhosis are at lower risk for restenosis after coronary intervention.

Soluble Guanylyl Cyclase Activator BI 685509 Reduces Portal Hypertension and Portosystemic Shunting in a Rat Thioacetamide-Induced Cirrhosis Model.

Portal hypertension (PT) commonly occurs in cirrhosis. Nitric oxide (NO) imbalance contributes to PT via reduced soluble guanylyl cyclase (sGC) activation and cGMP production, resulting in vasoconstriction, endothelial cell dysfunction, and fibrosis. We assessed the effects of BI 685509, an NO-independent sGC activator, on fibrosis and extrahepatic complications in a thioacetamide (TAA)-induced cirrhosis and PT model. Male Sprague-Dawley rats received TAA twice-weekly for 15 weeks (300-150 mg/kg i.p.). BI 685509 was administered daily for the last 12 weeks (0.3, 1, and 3 mg/kg p.o.; n = 8-11 per group) or the final week only (Acute, 3 mg/kg p.o.; n = 6). Rats were anesthetized to measure portal venous pressure. Pharmacokinetics and hepatic cGMP (target engagement) were measured by mass spectrometry. Hepatic Sirius Red morphometry (SRM) and alpha-smooth muscle actin (αSMA) were measured by immunohistochemistry; portosystemic shunting was measured using colored microspheres. BI 685509 dose-dependently increased hepatic cGMP at 1 and 3 mg/kg (3.92 ± 0.34 and 5.14 ± 0.44 versus 2.50 ± 0.19 nM in TAA alone; P < 0.05). TAA increased hepatic SRM, αSMA, PT, and portosystemic shunting. Compared with TAA, 3 mg/kg BI 685509 reduced SRM by 38%, αSMA area by 55%, portal venous pressure by 26%, and portosystemic shunting by 10% (P < 0.05). Acute BI 685509 reduced SRM and PT by 45% and 21%, respectively (P < 0.05). BI 685509 improved hepatic and extrahepatic cirrhosis pathophysiology in TAA-induced cirrhosis. These data support the clinical investigation of BI 685509 for PT in patients with cirrhosis. SIGNIFICANCE STATEMENT: BI 685509 is an NO-independent sGC activator that was tested in a preclinical rat model of TAA-induced nodular, liver fibrosis, portal hypertension, and portal systemic shunting. BI 685509 reduced liver fibrosis, portal hypertension, and portal-systemic shunting in a dose-dependent manner, supporting its clinical assessment to treat portal hypertension in patients with cirrhosis.

Asiaticoside conveys an antifibrotic effect by inhibiting activation of hepatic stellate cells via the Jagged-1/Notch-1 pathway.

The aim of this study was to investigate the underlying protective mechanisms of asiaticoside (AS) against liver fibrosis (LF) both in vivo and in vitro. A rat model with carbon tetrachloride (CCl4)-induced liver fibrosis is employed to verify the effect and mechanism of AS on the process of liver fibrosis in vivo experiment. Hematoxylin/eosin and sirius red staining was conducted to assess the severity of liver injury and fibrosis. Further, the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), glutamyl transferase (GGT), and total bilirubin (TBil) were measured. In addition, LX2 cells were cultured for vitro experiment to investigate the influence of AS on hepatic stellate cells (HSCs). Overproduction of α-smooth muscle actin and type I collagen is characteristic of LF and HSCs, as determined by immunohistochemical and Western blot analyses. The expression levels of molecules associated with the Notch signaling pathway (i.e., Notch-1, Jagged-1, and Delta-like-4) were assessed by Western blot analysis. The results revealed that AS attenuated LF, as defined by reduced deposition of collagen, expression of α-smooth muscle actin and collagen type 1, and expression of biochemical parameters (alanine aminotransferase, aspartate aminotransferase, and hydroxyproline). Notably, AS suppressed the expression levels of Notch-1, Jagged-1, and Delta-like-4 in activated HSCs and LF. Collectively, these results demonstrate that AS prevented the progression of LF by modulating the Notch signaling pathway, indicating that AS has potential therapeutic effects against LF.

Experimental VX2 Rabbit Liver Tumor Model in Carbon Tetrachloride-Induced Cirrhosis of the Liver.

Liver cirrhosis is a major underlying factor in the development of hepatocellular carcinoma. Currently, there is an unmet need for midsize experimental vertebrate models that would offer reproducible implantable liver tumors in a cirrhotic liver background. This study establishes a protocol for a syngeneic rabbit model of VX2 liver cancer with underlying liver cirrhosis induced using carbon tetrachloride (CCl4). Male New Zealand white rabbits (n = 3) received CCl4 by intragastric administration once weekly. Concentrations started at 5% v/v CCl4 dissolved in olive oil. CCl4 dosing was progressively increased every week by 2.5% v/v increments for the duration of treatment (16 weeks total). VX2 tumors were then orthotopically implanted into the left hepatic lobe and allowed to grow for 3 weeks. Cross-sectional imaging confirmed the presence of hepatic tumors. Gross and histopathological evaluations showed reproducible tumor growth in the presence of liver cirrhosis in all animals.

Liver cirrhosis: An overview of experimental models in rodents.

The liver, a component of the gastrointestinal tract, is one of the most important organs in the human body. The liver performs over 500 functions to promote physiological homeostasis. In addition, the liver acts as a screen, by metabolizing substances carried by blood coming from the digestive tract before they enter the systemic circulation. This vital function exposes the hepatic tissue to hepatotoxic agents, which can lead to liver damage if the organ's repair and regenerative capacity is insufficient. Several conditions such as persistent exposure to hepatitis C and B viruses, alcohol, and drugs can provoke this disbalance, eventually leading to liver cirrhosis, which is an irreversible and life-threatening condition. This paradigm of irreversibility began to be reconsidered when several studies showed that hepatic fibrosis is potentially reversible after cessation of exposure to the hepatotoxic agent or eradication of the primary disease. In the context of basic research in liver fibrosis and cirrhosis, it is essential to keep in mind that the capacity of the organ to recover spontaneously might be a significant limitation to long-term studies that use experimental models of liver cirrhosis. Here, we review animal models where liver cirrhosis is experimentally induced. We focus on a surgery-based model, i.e., bile duct ligation (BDL), and hepatotoxic drugs such as carbon tetrachloride (CCl4), thioacetamide (TAA), and dimethylnitrosamine (DMN) administrated alone or in association with alcohol, the latter to potentialize the hepatotoxic effect of these agents. Also, we analyze the effects of these approaches, emphasizing the risks, spontaneous reversibility, and outcomes on animal health.

The mouse Anxa6/miR-9-5p/Anxa2 axis modulates TGF-ß1-induced mouse hepatic stellate cell (mHSC) activation and CCl4-caused liver fibrosis.

Chronic liver disease such as hepatic fibrosis is a major cause of morbidity and mortality and has been related to high individual risk of hepatocellular carcinoma (HCC). Hepatic stellate cells (HSCs) activation is a central event of hepatic fibrosis progression. In this study, the up-regulation of lncRNA ANXA2P2 (mouse Anxa6) was found in liver fibrosis. Within CCl4-caused liver fibrosis murine model, Anxa6 knockdown partially ameliorated CCl4-induced hepatic fibrosis and blocked the PI3K/Akt signaling activation. In TGF-ß1-stimulated HSCs, Anxa6 knockdown partially inhibited TGF-ß1-induced HSC activation and blocked the PI3K/Akt signaling activation. Mouse Anxa6 downstream mmu-miR-9-5p directly targeted Anxa2; Anxa6 negatively regulated mmu-miR-9-5p, and mmu-miR-9-5p negatively regulated mouse Anxa2. In TGF-ß1-stimulated HSCs, miR-9-5p inhibitor promoted TGF-ß1-induced HSC activation and PI3K/Akt signaling activation, whereas Anxa2 knockdown exerted opposite effects; Anxa2 knockdown significantly attenuated miR-9-5p inhibitor effects upon TGF-ß1-stimulated HSCs. In conclusion, lncRNA ANXA2P2 (mouse Anxa6) expression is up-regulated in hepatic fibrosis and exerts pro-fibrotic effects on CCl4-caused liver fibrosis model mice and TGF-ß1-stimulated HSCs. The mouse Anxa6/miR-9-5p/Anxa2 axis and the PI3K/Akt pathway might participate in the functions of lncRNA ANXA2P2 (mouse Anxa6) on hepatic fibrosis.

Age-Related Features of Ketanserin Effects in Experimental Liver Cirrhosis.

The effect of ketanserin on inflammation, liver fibrosis, and microviscosity of the plasma and mitochondrial membranes of hepatocytes was studied on young (3 months) and old (9 months) male Wistar rats with experimental liver cirrhosis. Ketanserin reduced inflammation, area of the connective tissue, and liver damage and improved serum biochemical parameters in rats of both age groups; in old rats, the effects were more pronounced than in young animals. In old rats, ketanserin reduced polarity of hepatocyte plasma and mitochondrial membranes in the area of protein-lipid contacts, which determined higher effectiveness of ketanserin during the treatment of liver cirrhosis in aged animals.

Age-Related Features of the Viscosity of Plasma and Mitochondrial Membranes of Hepatocytes in Liver Cirrhosis.

The viscosity of plasma and mitochondrial membranes of hepatocytes was studied in young (3-month-old) and old (9-month-old) male Wistar rats. It was shown that viscosity of hepatocyte plasma and mitochondrial membranes in young rats under optimal vital functions in the area of protein-lipid membrane contacts was significantly lower than in old rats. No age-related differences in the viscosity of lipid-lipid membrane contacts and in the polarity of protein-lipid contacts and lipid layers were found. Liver cirrhosis induced by carbon tetrachloride and ethanol administration was associated with increased fluidity of the plasma and mitochondrial membranes of hepatocytes in rats of both age groups. The decrease in membrane viscosity in young rats occurred due to a decrease of the viscosity in the area of protein-lipid and lipid-lipid contacts, while in old rats in the area of protein-lipid contacts. Carbon tetrachloride and ethanol did not affect the polarity of lipid contacts and lipid layers.

IGF-1 alleviates CCL4-induced hepatic cirrhosis and dysfunction of intestinal barrier through inhibition TLR4/NF-κB signaling mediated by down-regulation HMGB1.

INTRODUCTION AND OBJECTIVES: Cirrhosis has gradually become a serious public health issue, especially the national prevalence of cirrhosis was 29.2% in northwest China. Recent evidence has revealed that intestinal barrier (IB) dysfunction results from and contributes to cirrhosis. Our previous results have indicated that insulin-like growth factors (IGF-1) improved the impaired IB function and downregulated high mobility group protein box-1 (HMGB-1). Nevertheless, the role of the IGF-1/HMGB1 axis in cirrhosis remains largely unknown. MATERIALS AND METHODS: Western blotting and qRT-PCR were used to detect protein and mRNA levels of related genes. The levels of AST, ALT, IL-1ß, and TNF-α were examined using commercial kits. Immunofluorescence was used to evaluate the expression of HMGB1 in tissues. RESULTS: In carbon tetrachloride (CCl4)-treated rat, the levels of AST (380.12 vs. 183.97), ALT (148.12 vs. 53.56), IL-1ß (155.94 vs. 55.60), and TNF-α (155.00 vs. 48.90) were significantly increased compared with the control group, while IGF-1 treatment significantly alleviated CCL4-induced inflammatory response and IB dysfunction by downregulating HMGB1-mediated the TLR4/MyD88/NF-κB signaling pathway. In vitro experiments, HMGB1 treatment promoted inflammatory cytokines secretion and reduced cell viability and tight junctions by activating the TLR4/MyD88/NF-κB signaling pathway in Caco-2 cells, but IGF-1 alleviated these effects. CONCLUSION: Our findings suggest that IGF-1 might serve as a potential therapeutic target for cirrhosis and IB dysfunction via inactivation of the TLR4/MyD88/NF-κB pathway through down-regulation HMGB1.

Impact of lifestyle interventions targeting physical exercise and caloric intake on cirrhosis regression in rats.

In patients, advanced cirrhosis only regresses partially once the etiological agent is withdrawn. Animal models for advanced cirrhosis regression are missing. Lifestyle interventions (LIs) have been shown to improve steatosis, inflammation, fibrosis, and portal pressure (PP) in liver disease. We aimed at characterizing cirrhosis regression after etiological agent removal in experimental models of advanced cirrhosis and to study the impact of different LI on it. Advanced cirrhosis was induced in rats either by carbon tetrachloride (CCl4) or by thioacetamide (TAA) administration. Systemic and hepatic hemodynamics, liver fibrosis, hepatic stellate cell (HSC) activation, hepatic macrophage infiltration, and metabolic profile were evaluated after 48 h, 4 wk or 8 wk of etiological agent removal. The impact of LI consisting in caloric restriction (CR) or moderate endurance exercise (MEE) during the 8-wk regression process was analyzed. The effect of MEE was also evaluated in early cirrhotic and in healthy rats. A significant reduction in portal pressure (PP), liver fibrosis, and HSC activation was observed during regression. However, these parameters remained above those in healthy animals. During regression, animals markedly worsened their metabolic profile. CR although preventing those metabolic disturbances did not further reduce PP, hepatic fibrosis, or HSC activation. MEE also prevented metabolic disturbances, without enhancing, but even attenuating the reduction of PP, hepatic fibrosis, and HSC activation achieved by regression. MEE also worsened hepatic fibrosis in early-TAA cirrhosis and in healthy rats.NEW & NOTEWORTHY We have developed two advanced cirrhosis regression experimental models with persistent relevant fibrosis and portal hypertension and an associated deteriorated metabolism that mimic what happens in patients. LI, despite improving metabolism, did not enhance the regression process in our cirrhotic models. CR did not further reduce PP, hepatic fibrosis, or HSC activation. MEE exhibited a profibrogenic effect in the liver blunting cirrhosis regression. One of the potential explanations of this worsening could be ammonia accumulation.

Yiguanjian decoction inhibits macrophage M1 polarization and attenuates hepatic fibrosis induced by CCl4/2-AAF.

CONTEXT: Our previous studies indicated that Yiguanjian decoction (YGJ) has an anti-hepatic-fibrosis effect and could regulate macrophage status. OBJECTIVE: To elucidate the mechanism of YGJ in regulating macrophages. MATERIALS AND METHODS: Liver cirrhosis was induced by CCl4 for 12 weeks combined with 2-acetylaminofluorene (2-AAF) for the last 4 weeks in male Wistar rats. YGJ (3.56 mg/kg) orally administered in the last 4 weeks, and SORA (1 mg/kg) as control. In vitro, RAW264.7 cells were treated with lipopolysaccharides (LPSs) to induce macrophage polarization to the M1 phenotype, and they were co-cultured with WB-F344 cells and allocated to M group, YGJ group (2 µg/mL) and WIF-1 group (1 µg/mL) with untreated cells as control. The differentiation direction of WB-F344 cell line was observed in the presence or absence of YGJ. Pathology, fibrosis-related cytokines, macrophage polarization-related components, and Wnt signalling pathway components were detected. RESULTS: In vivo, the expression levels of α-SMA, Col (1), OV6, SOX9, EpCAM and M1 macrophage-related components (STAT1, IRF3, IRF5, IRF8, SOCS3) significantly decreased in the YGJ group compared with those in the 2-AAF/CCl4 group (p < 0.01 or 0.05). In vitro, the expression levels of M1 macrophage-related components, including STAT1, NF-κB, IRF3, IRF5, and SOCS3, in RAW264.7 cells decreased significantly in the YGJ group compared with those in the M group (p < 0.05 or p < 0.01). The expression levels of Wnt3A, FZD5, LRP-5/-6, and ß-catenin significantly increased in the YGJ group compared with those in the M group (p < 0.05 or p < 0.01). In addition, the expression levels of Wnt-4/-5A/-5B, and FZD2 significantly decreased in the YGJ group compared with those in the M group (p < 0.05 or p < 0.01). CONCLUSION: This study suggests that the anti-cirrhosis effect of YGJ is associated with its ability to inhibit macrophage M1-polarization, which provides a scientific basis for the clinical application of YGJ.

Anti-Fibrotic and Anti-Angiogenic Activities of Osbeckia octandra Leaf Extracts in Thioacetamide-Induced Experimental Liver Cirrhosis.

Chronic liver inflammation has become a major global health concern. In the absence of clinical surrogate markers to diagnose inflammatory liver disease, the intervention with effective drugs in modern medicine tends to be late. In Sri Lanka, traditional medical practitioners prescribe herbal preparations from Osbeckia octandra for the prevention and treatment of liver disorders. To test the efficacy of such treatments, we have administered thioacetamide (TAA) to male Wistar rats to induce chronic liver damage (disease control; DC) and examined how various leaf extracts: crude leaf suspension (CLS), boiled leaf extract (BLE), sonicated leaf extract (SLE), methanol leaf extract (MLE) and hexane leaf extract (HLE) of O. octandra ameliorate TAA-induced liver disease. The CLS, BLE and SLE treatments in cirrhotic rats significantly attenuated disease-related changes, such as liver weight and hepato-enzymes. The mRNA levels of Tnf-α were significantly decreased by 3.6, 10 and 3.9 times in CLS, BLE and SLE compared to DC. The same treatments resulted in significantly lower (19.5, 4.2 and 2.4 times) α-Sma levels compared to DC. In addition, Tgf-ß1 and Vegf-R2 mRNA expressions were significantly lower with the treatments. Moreover, BLE expressed a strong anti-angiogenic effect. We conclude that CLS, BLE and SLE from O. octandra have potent hepatic anti-fibrotic effects in TAA-induced liver cirrhosis.

Anti-fibrotic activity of sitagliptin against concanavalin A-induced hepatic fibrosis. Role of Nrf2 activation/NF-κB inhibition.

Sitagliptin is known for its anti-diabetic activity though it has other pleiotropic pharmacological actions. Its effect against concanavalin A (Con A)-induced hepatic fibrosis has not been investigated yet. Our target was to test whether sitagliptin can suppress the development of Con A-induced hepatic fibrosis and if so, what are the mechanisms involved? Con A (6 mg/kg) was injected once weekly to male Swiss albino mice for four weeks. Sitagliptin was daily administered concurrently with Con A. Results have shown the potent hepatoprotective activity of sitagliptin against Con A-induced hepatitis and fibrosis. That was evident through the amelioration of hepatotoxicity serum parameters (ALT, AST, ALP, and LDH) and the increase in the level of serum albumin in sitagliptin treated mice. Simultaneously, there was amendment of the Con A-induced hepatic lesions and repression of fibrosis in sitagliptin-treated animals. Hydroxyproline, collagen content and the immuno-expression of the fibrotic markers, TGF-ß and α-SMA were depressed upon sitagliptin treatment. Sitagliptin suppressed Con A-induced oxidative stress and increased antioxidants. RT-PCR analysis showed enhancement of mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its target genes (GCLc, GCLm, NQO-1, HO-1) by sitagliptin. Furthermore, sitagliptin ameliorated the level and immuno-expression of nuclear factor kappa-B (NF-κB) alongside the immuno-expression of the inflammatory cytokine, TNF-α. Taken together, this study demonstrates the hepatoprotective activity of sitagliptin which may be in part related to enhancement of Nrf2 signaling pathway and inhibition of NF-κB which interact inflammatory response in liver. Sitagliptin might be a new candidate to suppress hepatitis-associated fibrosis.

Protective role of coffee supplementation in liver cirrhosis: Study in rats.

Present study was designed to evaluate the effects of coffee on liver function tests and liver antioxidant enzymes in thioacetamide induced liver cirrhosis in rats. Experimental study period was consisted of eighteen weeks divided into two phases. Therefore 24 rats were distributed randomly into four groups (n=6). Group I served as control. In phase I, group II and III received thioacetamide (200mg/kg body weight intraperitoneally twice a week) and group IV received saline for 12 weeks. In phase II, group II received saline while group III and IV received an oral dose of coffee (0.4mg/Kg b.w) daily for 6 weeks. At the end of the study period rats were sacrificed and blood was collected to get serum and liver was homogenized for the determination of antioxidant enzymes. Marked increase in serum total and direct bilirubin, ALT, AST whereas reduced ALP was observed in test group. The reduced tissue SOD activity and increased tissue catalase and tissue MDA activity were also observed in test group. However, coffee consumption in group III in phase II significantly restored liver biomarkers and the tissue antioxidant enzymes SOD, catalase and MDA activities. In conclusion, thioacetamide induced liver cirrhosis can be prevented by coffee supplementation.

Glycyrrhizin Attenuates Portal Hypertension and Collateral Shunting via Inhibition of Extrahepatic Angiogenesis in Cirrhotic Rats.

Portal hypertension develops along with liver cirrhosis then induces the formation of portal-systemic collaterals and lethal complications. Extrahepatic angiogenesis plays an important role. Glycyrrhizin has been found to exhibit anti-angiogenic features, which leads to its extensive use. However, the relevant effects of glycyrrhizin on liver cirrhosis and portal hypertension have not been evaluated. This study thus aimed to investigate the impact of glycyrrhizin on portal hypertension-related derangements in cirrhotic rats. Male Sprague-Dawley rats received bile duct ligation (BDL) to induce cirrhosis or sham operation as control. The rats were subdivided to receive glycyrrhizin (150 mg/kg/day, oral gavage) or vehicle beginning on the 15th day post operation, when BDL-induced liver fibrosis developed. The effects of glycyrrhizin were determined on the 28th day, the typical timing of BDL-induced cirrhosis. Glycyrrhizin significantly reduced portal pressure (p = 0.004). The splanchnic inflow as measured by superior mesenteric arterial flow decreased by 22% (p = 0.029). The portal-systemic collateral shunting degree reduced by 30% (p = 0.024). The mesenteric angiogenesis and phospho-VEGFR2 protein expression were also downregulated (p = 0.038 and 0.031, respectively). Glycyrrhizin did not significantly influence the liver biochemistry data. Although glycyrrhizin tended to reverse liver fibrosis, statistical significance was not reached (p = 0.069). Consistently, hepatic inflow from portal side, hepatic vascular resistance, and liver fibrosis-related protein expressions were not affected. Glycyrrhizin treatment at the stage of hepatic fibrosis still effectively attenuated portal hypertension and portosystemic collateral shunting. These beneficial effects were attributed to, at least in part, the suppression of mesenteric angiogenesis by VEGF signaling pathway downregulation.

Single-Cell Transcriptomic Analysis Reveals a Hepatic Stellate Cell-Activation Roadmap and Myofibroblast Origin During Liver Fibrosis in Mice.

BACKGROUND AND AIMS: HSCs and portal fibroblasts (PFs) are the major sources of collagen-producing myofibroblasts during liver fibrosis, depending on different etiologies. However, the mechanisms by which their dynamic gene expression directs the transition from the quiescent to the activated state-as well as their contributions to fibrotic myofibroblasts-remain unclear. Here, we analyze the activation of HSCs and PFs in CCL4 -induced and bile duct ligation-induced fibrosis mouse models, using single-cell RNA sequencing and lineage tracing. APPROACH AND RESULTS: We demonstrate that HSCs, rather than PFs, undergo dramatic transcriptomic changes, with the sequential activation of inflammatory, migrative, and extracellular matrix-producing programs. The data also reveal that HSCs are the exclusive source of myofibroblasts in CCL4 -treated liver, while PFs are the major source of myofibroblasts in early cholestatic liver fibrosis. Single-cell and lineage-tracing analysis also uncovers differential gene-expression features between HSCs and PFs; for example, nitric oxide receptor soluble guanylate cyclase is exclusively expressed in HSCs, but not in PFs. The soluble guanylate cyclase stimulator Riociguat potently reduced liver fibrosis in CCL4 -treated livers but showed no therapeutic efficacy in bile duct ligation livers. CONCLUSIONS: This study provides a transcriptional roadmap for the activation of HSCs during liver fibrosis and yields comprehensive evidence that the differential transcriptomic features of HSCs and PFs, along with their relative contributions to liver fibrosis of different etiologies, should be considered in developing effective antifibrotic therapeutic strategies.

Conditional depletion of macrophages ameliorates cholestatic liver injury and fibrosis via lncRNA-H19.

Although macrophages are recognized as important players in the pathogenesis of chronic liver diseases, their roles in cholestatic liver fibrosis remain incompletely understood. We previously reported that long noncoding RNA-H19 (lncRNA-H19) contributes to cholangiocyte proliferation and cholestatic liver fibrosis of biliary atresia (BA). We here show that monocyte/macrophage CD11B mRNA levels are increased significantly in livers of BA patients and positively correlated with the progression of liver inflammation and fibrosis. The macrophages increasingly infiltrate and accumulate in the fibrotic niche and peribiliary areas in livers of BA patients. Selective depletion of macrophages using the transgenic CD11b-diphtheria toxin receptor (CD11b-DTR) mice halts bile duct ligation (BDL)-induced progression of liver damage and fibrosis. Meanwhile, macrophage depletion significantly reduces the BDL-induced hepatic lncRNA-H19. Overexpression of H19 in livers using adeno-associated virus serotype 9 (AAV9) counteracts the effects of macrophage depletion on liver fibrosis and cholangiocyte proliferation. Additionally, both H19 knockout (H19-/-) and conditional deletion of H19 in macrophage (H19ΔCD11B) significantly depress the macrophage polarization and recruitment. lncRNA-H19 overexpressed in THP-1 macrophages enhance expression of Rho-GTPase CDC42 and RhoA. In conclusions, selectively depletion of macrophages suppresses cholestatic liver injuries and fibrosis via the lncRNA-H19 and represents a potential therapeutic strategy for rapid liver fibrosis in BA patients.

M2 macrophage accumulation contributes to pulmonary fibrosis, vascular dilatation, and hypoxemia in rat hepatopulmonary syndrome.

Hepatopulmonary syndrome (HPS) markedly increases the mortality of patients. However, its pathogenesis remains incompletely understood. Rat HPS develops in common bile duct ligation (CBDL)-induced, but not thioacetamide (TAA)-induced cirrhosis. We investigated the mechanisms of HPS by comparing these two models. Pulmonary histology, blood gas exchange, and the related signals regulating macrophage accumulation were assessed in CBDL and TAA rats. Anti-polymorphonuclear leukocyte (antiPMN) and anti-granulocyte-macrophage colony stimulating factor (antiGM-CSF) antibodies, clodronate liposomes (CL), and monocyte chemoattractant protein 1 (MCP1) inhibitor (bindarit) were administrated in CBDL rats, GM-CSF, and MCP1 were administrated in bone marrow-derived macrophages (BMDMs). Pulmonary inflammatory cell recruitment, vascular dilatation, and hypoxemia were progressively developed by 1 week after CBDL, but only occurred at 4 week after TAA. Neutrophils were the primary inflammatory cells within 3 weeks after CBDL and at 4 week after TAA. M2 macrophages were the primary inflammatory cells, meantime, pulmonary fibrosis, GM-CSFR, and CCR2 were specifically increased from 4 week after CBDL. AntiPMN antibody treatment decreased neutrophil and macrophage accumulation, CL or the combination of antiGM-CSF antibody and bindarit treatment decreased macrophage recruitment, resulting in pulmonary fibrosis, vascular dilatation, and hypoxemia in CBDL rats alleviated. The combination treatment of GM-CSF and MCP1 promoted cell migration, M2 macrophage differentiation, and transforming growth factor-ß1 (TGF-ß1) production in BMDMs. Conclusively, our results highlight neutrophil recruitment mediates pulmonary vascular dilatation and hypoxemia in the early stage of rat HPS. Further, M2 macrophage accumulation induced by GM-CSF/GM-CSFR and MCP1/CCR2 leads to pulmonary fibrosis and promotes vascular dilatation and hypoxemia, as a result, HPS develops.