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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.

Background and aims Studies of the identity and pathophysiology of fibrogenic HSCs have been hampered by a lack of genetic tools that permit specific and inducible fate-mapping of these cells in vivo. Here, by single-cell RNA sequencing of nonparenchymal cells from mouse liver, we identified transcription factor 21 (Tcf21) as a unique marker that restricted its expression to quiescent HSCs. Approach and results Tracing Tcf21+ cells by Tcf21-CreER (Cre-Estrogen Receptor fusion protein under the control of Tcf21 gene promoter) targeted ~10% of all HSCs, most of which were located at periportal and pericentral zones. These HSCs were quiescent under steady state but became activated on injuries, generating 62%-67% of all myofibroblasts in fibrotic livers and ~85% of all cancer-associated fibroblasts (CAFs) in liver tumors. Conditional deletion of Transforming Growth Factor Beta Receptor 2 (Tgfbr2) by Tcf21-CreER blocked HSC activation, compromised liver fibrosis, and inhibited liver tumor progression. Conclusions In conclusion, Tcf21-CreER-targeted perivenous stellate cells are the main source of myofibroblasts and CAFs in chronically injured livers. TGF-β signaling links HSC activation to liver fibrosis and tumorigenesis.

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.

Transient receptor potential vanilloid 3 (TRPV3) is a member of the TRP superfamily. Previous studies have demonstrated that TRPV3 is associated with myocardial fibrosis. However, the role of TRPV3 in hepatic fibrosis and its underlying mechanisms are still unclear. This study aimed to elucidate the underlying effects of TRPV3 on hepatic fibrosis at multiple biological levels. First, immunohistochemical staining was performed to examine TRPV3 expression in human hepatic cirrhosis tissues. Then, we established a CCl4-induced hepatic fibrosis mouse model. The TRPV3 selective agonist drofenine and its inhibitor, forsythoside B, were intraperitoneally injected to investigate the relationship between TRPV3 and liver fibrosis progression. Finally, in vitro studies were performed using hepatic stellate cells (HSCs) to discover the potential molecular biological mechanisms. Immunohistochemistry revealed TRPV3 overexpression in liver cirrhosis. In the liver fibrosis groups, TRPV3 inhibitor treatment significantly reduced liver fibrosis, while TRPV3 agonist exacerbated its progression. In HSCs, knocking down TRPV3 with siRNA impaired DNA synthesis and cell proliferation and increased cell apoptosis. Furthermore, we found that knockdown of TRPV3 could reduce the lectin like oxidized lowdensity lipoprotein receptor-1 (LOX-1) protein levels. Our research suggests that lower expression or functional levels of TRPV3 can ameliorate the inflammatory response and fibrotic tissue proliferation.

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-β1 (TGF-β1) 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.

Polycystic liver disease (PLD) is a hereditary liver disease in which the number of cysts increases over time, causing various abdominal symptoms and poor quality of life. Although effective treatment for PLD has not been established, we recently reported that long-term exercise ameliorated liver cyst formation and fibrosis with the activation of AMP-activated protein kinase (AMPK) in polycystic kidney (PCK) rats, a PLD model. Therefore, the aim of this study was to investigate whether metformin, an indirect AMPK activator, was effective in PCK rats. PCK rats were randomly divided into a control (Con) group and a metformin-treated (Met) group. The Met group was treated orally with metformin in drinking water. After 12 wk, liver function, histology, and signaling cascades of PLD were examined in the groups. Metformin did not affect the body weight or liver weight, but it reduced liver cyst formation, cholangiocyte proliferation, and fibrosis around the cyst. Metformin increased the phosphorylation of AMPK and tuberous sclerosis complex 2 and decreased the phosphorylation of mammalian target of rapamycin, S6, and extracellular signal-regulated kinase and the expression of cystic fibrosis transmembrane conductance regulator, aquaporin I, transforming growth factor-β, and type 1 collagen without changes in apoptosis or collagen degradation factors in the liver. Metformin slows the development of cyst formation and fibrosis with the activation of AMPK and inhibition of signaling cascades responsible for cellular proliferation and fibrosis in the liver of PCK rats.

Wnt signaling dysfunction and gut dysbiosis may lead to liver fibrosis, yet the underlying mechanisms are not well elucidated. This study demonstrated the role of RSPO4, a Wnt signaling agonist, in liver fibrogenesis and its impact on the gut microbiome. RSPO4 gene in CCl4-induced fibrotic-liver rats was knockout by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, with healthy rats served as the control. Tissue samples and hepatic stellate cells (HSCs) isolated from rats were examined for curative effect of RSPO4-CRISPR treatment. Fecal sample were collected and analyzed with 16 S rRNA sequencing. We found RSPO4-CRISPR relieved liver fibrosis in rats and reversed HSC activation. Further, results showed RSPO4-CRISPR tended to restore the microflora composition. Significance species between groups were identified. Bacteroides and Escherichia-Shigella were the key microbes in the model and negative group, whereas Lactobacillus, Romboutsia, and Lachnospiraceae NK4A136 group were abundant in the control. Notably, Bacteroidales S24-7 group and Ruminococcaceae UCG-005 were the significantly enriched in CRISPR group. We show that the microbiome of rats treated with RSPO4-CRISPR presents a trend towards the restoration of the original condition. Our findings pave a new way to evaluate the curative effect of liver fibrosis treatment., Yu et al. demonstrates the role of RSPO4, a Wnt signaling agonist, in liver fibrogenesis and its impact on the gut microbiome. By knocking out RSPO4 gene in fibrotic-liver rat model, they show that it relieved liver fibrosis in rats and restored the microflora composition, highlighting its potential in liver fibrosis treatment.

Normally, there are multiple microRNAs involved in the pathogenesis of liver fibrosis. In our work, we aimed at identifying the role of miR-34c in the hepatic stellate cell (HSC) activation and liver fibrosis and its potential mechanism. Our results have shown that during natural activation of HSC, the level of miR-34c was increased significantly whereas acyl-CoA synthetase long-chain family member-1(ACSL1), which is a key enzyme can affect fatty acid(FA) synthesis, was decreased. A double fluorescence reporter assay further confirmed that ACSL1 is a direct target gene of miR-34c. Moreover, the inhibition of miR-34C can attenuate the synthesis of collagen in HSC-T6. In our rescue assay, ACSL1 expression was 1.49-fold higher compared to normal control cells which were transfected with the miR-34c inhibitor in a stable low expression ACSL1 cell line. While at the same time, α-SMA and Col1α expression decreased by 18.22% and 2.58%, respectively. Moreover, we performed an in vivo model using dimethylnitrosamine (DMN) in conjunction with the miR-34c agomir, combined with the treatment of DMN and the miR-34c agomir can increase liver fibrosis. Meanwhile, the degree of hepatic fibrosis was increased and lipid droplets reduced dramatically in rats and HSC-T6 cell treated with miR-34c mimics alone compared to untreated groups. Our results indicate that miR-34c plays an essential role in liver fibrosis by targeting ACSL1 closely associated with lipid droplets, and it might be used as a potential therapeutic target.

Hepatic fibrosis is the wound healing response upon the liver tissue damage caused by multiple stimuli. Targeting activated hepatic stellate cells (HSCs), the major extracellular matrix (ECM)-producing cells within the damaged liver, has been regarded as one of the main treatments for hepatic fibrosis. In the present study, we performed preliminary bioinformatics analysis attempting to identify possible factors related to hepatic fibrosis and found that lncRNA G protein-coupled receptor 137B (Gpr137b-ps) and C-X-C motif chemokine ligand 14 (CXCL14) showed to be markedly upregulated within carbon tetrachloride (CCl4)-caused hepatic fibrotic mice tissue samples and activated HSCs. CXCL14 The silencing of lncRNA Gpr137b-ps or CXCL14 alone could significantly improve CCl4-induced fibrotic changes in mice liver in vivo and collagen I and III release by HSCs and HSC proliferation in vitro. miR-200a-3p directly targeted lncRNA Gpr137b-ps and CXCL14, respectively. LncRNA Gpr137b-ps relieved miR-200a-3p-induced inhibition on CXCL14 expression via acting as a ceRNA. In HSCs, the effects of lncRNA Gpr137b-ps silencing on collagen I and III release by HSCs and HSC proliferation were significantly reversed by miR-200a-3p inhibition, and the effects of miR-200a-3p inhibition were reversed by CXCL14 silencing. In conclusion, we demonstrated a lncRNA Gpr137b-ps/miR-200a-3p/CXCL14 axis that modulates HSC activation and might exert an effect on the pathogenesis of liver fibrosis.

Hepatic stellate cell (HSC) activation plays an important role in the pathogenesis of liver fibrosis, and epithelial-mesenchymal transition (EMT) is suggested to potentially promote HSC activation. Superoxide dismutase 3 (SOD3) is an extracellular antioxidant defense against oxidative damage. Here, we found downregulation of SOD3 in a mouse model of liver fibrosis induced by carbon tetrachloride (CCl4 ). SOD3 deficiency induced spontaneous liver injury and fibrosis with increased collagen deposition, and further aggravated CCl4 -induced liver injury in mice. Depletion of SOD3 enhanced HSC activation marked by increased α-smooth muscle actin and subsequent collagen synthesis primarily collagen type I in vivo, and promoted transforming growth factor-β1 (TGF-β1)-induced HSC activation in vitro. SOD3 deficiency accelerated EMT process in the liver and TGF-β1-induced EMT of AML12 hepatocytes, as evidenced by loss of E-cadherin and gain of N-cadherin and vimentin. Notably, SOD3 expression and its pro-fibrogenic effect were positively associated with sirtuin 1 (SIRT1) expression. SOD3 deficiency inhibited adenosine monophosphate-activated protein kinase (AMPK) signaling to downregulate SIRT1 expression and thus involving in liver fibrosis. Enforced expression of SIRT1 inhibited SOD3 deficiency-induced HSC activation and EMT, whereas depletion of SIRT1 counteracted the inhibitory effect of SOD3 in vitro. These findings demonstrate that SOD3 deficiency contributes to liver fibrogenesis by promoting HSC activation and EMT process, and suggest a possibility that SOD3 may function through modulating SIRT1 via the AMPK pathway in liver fibrosis.

Nonalcoholic fatty liver disease is a chronic condition involving steatosis, steatohepatitis and fibrosis, and its progression remains unclear. Although the tetraspanin transmembrane 4 L six family member 5 (TM4SF5) is involved in hepatic fibrosis and cancer, its role in nonalcoholic steatohepatitis (NASH) progression is unknown. We investigated the contribution of TM4SF5 to liver pathology using transgenic and KO mice, diet- or drug-treated mice, in vitro primary cells, and in human tissue. TM4SF5-overexpressing mice exhibited nonalcoholic steatosis and NASH in an age-dependent manner. Initially, TM4SF5-positive hepatocytes and liver tissue exhibited lipid accumulation, decreased Sirtuin 1 (SIRT1), increased sterol regulatory-element binding proteins (SREBPs) and inactive STAT3 via suppressor of cytokine signaling (SOCS)1/3 upregulation. In older mice, TM4SF5 promoted inflammatory factor induction, SIRT1 expression and STAT3 activity, but did not change SOCS or SREBP levels, leading to active STAT3-mediated ECM production for NASH progression. A TM4SF5-associated increase in chemokines promoted SIRT1 expression and progression to NASH with fibrosis. Suppression of the chemokine CCL20 reduced immune cell infiltration and ECM production. Liver tissue from high-fat diet- or CCl4 -treated mice and human patients exhibited TM4SF5-dependent steatotic or steatohepatitic livers with links between TM4SF5-mediated SIRT1 modulation and SREBP or SOCS/STAT3 signaling axes. TM4SF5-mediated STAT3 activation in fibrotic NASH livers increased collagen I and laminin γ2. Both collagen I α1 and laminin γ2 suppression resulted in reduced SIRT1 and active STAT3, but no change in SREBP1 or SOCS, and abolished CCl4 -mediated mouse liver damage. TM4SF5-mediated signaling pathways that involve SIRT1, SREBPs and SOCS/STAT3 promoted progression to NASH. Therefore, TM4SF5 and its downstream effectors may be promising therapeutic targets to treat nonalcoholic fatty liver disease. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

BACKGROUND AND AIMS: Liver fibrosis (LF) is a central pathological process that occurs in most types of chronic liver diseases. Advanced LF causes cirrhosis, hepatocellular carcinoma, and liver failure. However, the exact molecular mechanisms underlying the initiation and progression of LF remain largely unknown. APPROACH AND RESULTS: This study was designed to investigate the role of protein kinase D3 (PKD3; gene name Prkd3) in the regulation of liver homeostasis. We generated global Prkd3 knockout (Prkd3(−/−)) mice and myeloid-cell–specific Prkd3 knockout (Prkd3(ΔLysM)) mice, and we found that both Prkd3(−/−) mice and Prkd3(ΔLysM) mice displayed spontaneous LF. PKD3 deficiency also aggravated CCl(4)-induced LF. PKD3 is highly expressed in hepatic macrophages (HMs), and PKD3 deficiency skewed macrophage polarization toward a profibrotic phenotype. Activated profibrotic macrophages produced transforming growth factor beta that, in turn, activates hepatic stellate cells to become matrix-producing myofibroblasts. Moreover, PKD3 deficiency decreased the phosphatase activity of SH2-containing protein tyrosine phosphatase-1 (a bona-fide PKD3 substrate), resulting in sustained signal transducer and activator of transcription 6 activation in macrophages. In addition, we observed that PKD3 expression in HMs was down-regulated in cirrhotic human liver tissues. CONCLUSIONS: PKD3 deletion in mice drives LF through the profibrotic macrophage activation.

Cirrhotic cardiomyopathy is a critical factor that causes morbidity and mortality in crucial conditions such as liver transplantation. In animal model, the common pathophysiologic mechanisms of cirrhotic cardiomyopathy are similar to those associated with bile duct ligation (BDL). Overproduction of inflammatory and oxidant markers plays a crucial role in cirrhotic cardiomyopathy. Spermidine, a multifunctional polyamine, is known for its antioxidant and anti-inflammatory effects. In this study, we investigated the effects of spermidine on development of cirrhotic cardiomyopathy in BDL rats. Rats were randomly housed in 6 groups. Except the normal and sham groups, BDL was performed for all the control and spermidine groups. Seven days after operation, 3 different doses of spermidine (5, 10 and 50 mg/kg) were administrated until day 28, in spermidine groups. At the end of the fourth week, the electrocardiography (ECG) and papillary muscle isolation were performed. The serum level of tumor necrosis factor-a (TNF-α), interleukin-1β (IL-1β), and IL-10 and cardiac level of superoxide dismutase, glutathione (GSH). and malondialdehyde (MDA) were assessed. Furthermore, the nuclear factor-κB (NF-κB) expression was assessed by western blot. Cardiac histopathological changes were monitored. The serum levels of magnesium (Mg) and potassium (K) were investigated. Control group, exhibited exaggerated signs of cirrhotic cardiomyopathy in comparison with the sham group. Co-administration of spermidine at the dose of 10 mg/kg in BDL rats significantly improved the cardiac condition, reduced the inflammatory mediators, and increased antioxidant enzymes. In addition, the histopathologic findings were in accordance with the other results of the study. Besides, there was no significant alteration in serum levels of Mg and K. This study demonstrates that spermidine at the dose of 10 mg/kg significantly improved the cirrhotic cardiomyopathy in BDL model in rats.

Non-alcoholic steatohepatitis (NASH) is a recognized risk factor for liver fibrosis and malignancies, and is associated with features of metabolic syndrome, such as obesity and insulin resistance (IR). We previously demonstrated that the disturbance of connexin 32 (Cx32), a gap junctional protein of hepatocytes, exacerbated NASH in Cx32 dominant-negative transgenic (Cx32ΔTg) rats fed methionine choline-deficient diet (MCDD). MCDD is well-established means of inducing NASH in rodents; however, the Cx32ΔTg-MCDD NASH model does not reproduce obesity and IR. In this study, we aimed to establish an improved NASH model. Eight-week-old male Cx32ΔTg and wild-type (Wt) rats received a high-fat diet (HFD) with dimethylnitrosamine (DMN) for 12 weeks. The HFD with DMN led to gains in body, liver, and visceral fat weights in both genotypes. IR was significantly greater in Cx32ΔTg than in Wt rats. Elevation of serum hepatic enzymes (AST, ALT), inflammatory cytokine expressions (Tnfα, Il-6, Tgf-β1, Il-1β, Timp2, and Col1a1), steatohepatitis, and fibrosis were significantly greater in Cx32ΔTg as compared with Wt rats. Regarding carcinogenesis, the number and area of glutathione S-transferase placental form (GST-P)-positive preneoplastic hepatic foci were significantly increased in Cx32ΔTg versus Wt rats. Moreover, activation of NF-κB and JNK contributed to the progression of NASH in Cx32ΔTg rats. These results suggest that Cx32 dysfunction promoted the progression of NASH, metabolic syndrome, and carcinogenesis. Therefore, the novel Cx32ΔTg–HFD–DMN NASH model may be a rapid and useful tool for evaluating the progression of NASH.

Background in liver cirrhosis, a marked splanchnic vasodilation causes an increase in portal blood flow, contributing to the development of portal hypertension. Aim To evaluate if, in experimental cirrhosis, a different vascular reactivity exists between splenic and mesenteric components of the splanchnic circulation. Methods Liver cirrhosis was induced in Sprague Dawley rats by common bile duct ligation. in sections of splenic and superior mesenteric arteries, cumulative dose-response curves were obtained. mRNA expression of endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), and prostaglandin I-2 synthase (PTGIS) was evaluated. Results in cirrhotic rats, mesenteric but not splenic arteries showed a significant increase in endothelium-dependent relaxation to acetylcholine. in control and cirrhotic rats, COX inhibition alone did not significantly change the response of mesenteric arteries to acetylcholine; after inhibiting also NOS, the relaxation was completely abolished in control but only partially decreased in cirrhotic rats. After the inhibition of COX and NOS, the relaxation to acetylcholine was similarly decreased in splenic arteries from control and cirrhotic animals. the contraction induced by phenylephrine of both mesenteric and splenic arteries was decreased in cirrhotic rats. PTGIS mRNA expression did not differ in splenic and mesenteric arteries from control and cirrhotic rats; in cirrhotic rats, eNOS and iNOS mRNA expression was increased in mesenteric but not in splenic vascular bed. Conclusion in cirrhotic rats, a decreased splenic arterial response to vasoconstrictors, rather than an increased response to vasodilators, contributes to splanchnic vasodilation, while in mesenteric arteries also an increased response to vasodilators secondary to, but not only, eNOS and iNOS overexpression, plays a role.

Angiotensin II (Ang II) has been reported to aggravate hepatic fibrosis by inducing NADPH oxidase (NOX)-dependent oxidative stress. Alamandine (ALA) protects against fibrosis by counteracting Ang II via the MAS-related G-protein coupled (MrgD) receptor, though the effects of alamandine on hepatic fibrosis remain unknown. Autophagy activated by reactive oxygen species (ROS) is a novel mechanism of hepatic fibrosis. However, whether autophagy is involved in the regulation of Ang II-induced hepatic fibrosis still requires investigation. We explored the effect of alamandine on hepatic fibrosis via regulation of autophagy by redox balance modulation. In vivo, alamandine reduced CCl4-induced hepatic fibrosis, hydrogen peroxide (H2O2) content, protein levels of NOX4 and autophagy impairment. In vitro, Ang II treatment elevated NOX4 protein expression and ROS production along with up-regulation of the angiotensin converting enzyme (ACE)/Ang II/Ang II type 1 receptor (AT1R) axis. These changes resulted in the accumulation of impaired autophagosomes in hepatic stellate cells (HSCs). Treatment with NOX4 inhibitor VAS2870, ROS scavenger N-acetylcysteine (NAC), and NOX4 small interfering RNA (siRNA) inhibited Ang II-induced autophagy and collagen synthesis. Alamandine shifted the balance of renin–angiotensin system (RAS) toward the angiotensin converting enzyme 2 (ACE2)/alamandine/MrgD axis, and inhibited both Ang II-induced ROS and autophagy activation, leading to attenuation of HSCs migration or collagen synthesis. In summary, alamandine attenuated liver fibrosis by regulating autophagy induced by NOX4-dependent ROS.

Background & Aims C-C motif chemokine receptor 2 (CCR2) has been recognized as a promising target for the treatment of liver fibrosis. PC3-secreted microprotein (PSMP)/microseminoprotein (MSMP) is a novel chemotactic cytokine and its receptor is CCR2. In the present study we investigated the expression and role of PSMP in liver fibrosis/cirrhosis. Methods PSMP expression was studied in patients with fibrosis/cirrhosis and in 3 murine models of liver fibrosis, including mice treated with carbon tetrachloride (CCl4), bile-duct ligation, or a 5-diethoxycarbonyl-1,4-dihydrocollidine diet. The role of PSMP was evaluated in Psmp-/- mice and after treatment with a PSMP antibody in wild-type mice. The direct effects of PSMP on macrophages and hepatic stellate cells were studied in vitro. Results In this study, we found that PSMP was highly expressed in fibrotic/cirrhotic tissues from patients with different etiologies of liver disease and in the 3 experimental mouse models of fibrosis. Damage-associated molecular pattern molecules HMGB-1 and IL-33 induced hepatocytes to produce PSMP. PSMP deficiency resulted in a marked amelioration of hepatic injury and fibrosis. In CCl4-induced hepatic injury, the infiltration of macrophages and CCR2+ monocytes into the liver was significantly decreased in Psmp-/- mice. Consistent with the decreased levels of intrahepatic macrophages, proinflammatory cytokines were significantly reduced. Moreover, adeno-associated virus-8 vectors successfully overexpressing human PSMP in Psmp-/- mouse livers could reverse the attenuation of liver injury and fibrosis induced by CCl4 in a CCR2-dependent manner. Treatment with a specific PSMP-neutralizing antibody, 3D5, prevented liver injury and fibrosis induced by CCl4 in mice. At the cellular level, PSMP directly promoted M1 polarization of macrophages and activation of LX-2 cells. Conclusion PSMP enhances liver fibrosis through its receptor, CCR2. PSMP is a potentially attractive therapeutic target for the treatment of patients with liver fibrosis. Lay summary Our present study identifies the essential role of the protein PSMP for the development and progression of liver fibrosis in humans and mice. PSMP promotes liver fibrosis through inflammatory macrophage infiltration, polarization and production of proinflammatory cytokines, as well as direct activation of hepatic stellate cells via its receptor CCR2. A PSMP antibody can significantly reduce liver fibrosis development in vivo. These findings indicate that PSMP is a potential therapeutic target and its antibody is a potential therapeutic agent for the treatment of liver fibrosis.

With the spread of hexavalent chromium [Cr(VI)] contamination, risk of exposure in non-occupational populations is increasing. The liver is the main target organ for Cr(VI) accumulation; however, the effect of long-term Cr(VI) exposure on liver toxicity is largely unknown. In this study, we investigated the effect of chronic Cr(VI) exposure on liver fibrosis and its possible mechanism. Mice were injected with Cr(VI) for two months, and our results showed Cr(VI) treatment caused liver toxicity characterized by liver structure disorganization, liver dysfunction, and antioxidant enzyme system inhibition. The development of liver fibrosis was also found via the emergence of collagen fibril deposition, increased expression of extracellular matrix-related genes, activation of hepatic stellate cells (HSCs) and increase the expression levels of Hedgehog (Hh) signaling pathway-related molecules. To demonstrate the role of Hh signaling in the regulation of Cr(VI)-induced liver fibrosis, genetically modified mice with heterozygous deficiency of Shh (Shh+/-) were used. In the Shh+/- mice, Hh signaling, HSCs activation and liver fibrosis development were all ameliorated. In conclusion, we demonstrated that Cr(VI)-induced liver fibrosis development resulted from Hh pathway-mediated HSCs activation. Our findings strongly suggest that inhibition of Hh pathway may help in the development of new strategies for Cr(VI)-associated liver fibrosis.

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 (CCl

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 CCl

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

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-β1 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.

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.

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.

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.

Introduction and objectives Liver fibrosis is a major characteristic of most chronic liver diseases which leads to accumulation of extracellular matrix (ECM) proteins. Hedgehog (Hh) pathway activated by Gli genes participated in the pathogenesis of liver fibrosis. However, the regulatory role of miR-125b in liver fibrosis via targeting Gli genes remains unknown. Materials and methods RT-qPCR and western blot were employed to the expression levels of mRNA and protein, respectively. The fibrosis level of liver tissue was determined by Masson's trichrome staining. The interaction between miR-125b and Gli3 was tested by luciferase reporter assay. In addition, LX2 cells were activated and CCl4-induced rat model was used in this study. Results miR-125b was significantly declined in serum samples of the clinical liver fibrosis patient, activated LX2 cells and the liver tissues of the CCl4-induced rat model. Furthermore, in cellular level, the alpha-smooth muscle actin (α-SMA) and Albumin expressions were ascending and descending in LX2 cells, respectively, with the decline of miR-125b. However, when transfecting with miR-125b mimic, the expressions of α-SMA and Albumin was reversed and Gli3 expression was notably repressed in LX2 cells. The target interaction between miR-125b and Gli3 was determined by dual-luciferase assays. It was further discovered that the changes of α-SMA, Albumin, and Gli3 were similar to the expression trend in LX2 cells with miR-125b mimic transfection. Conclusion These results suggested that miR-125b might be protective against liver fibrosis via regulating Gli3 and it might be a promising target in the development of novel therapies to treat pathological fibrotic disorders.

In the liver microenvironment, interactions among diverse types of hepatic cells are involved in liver fibrosis. In fibrotic tissues, exosomes act as transporters in intercellular communication. Long non-coding RNAs (lncRNAs) are involved in the activation of hepatic stellate cells (HSCs), which are participants in liver fibrosis. However, the functions of exosomal lncRNAs in liver fibrosis induced by arsenite are undefined. The purposes of the present study were (a) to determine if lncRNAs secreted from human hepatic (L-02) cells exposed to arsenite are shuttled to hepatic stellate LX-2 cells and (b) to establish their effects on LX-2 cells. In mice, MALAT1 was overexpressed in the progression of liver fibrosis induced by arsenite as well as in L-02 cells exposed to arsenite. Co-cultures with arsenite-treated L-02 cells induced the activation of LX-2 cells and overexpression of MALAT1. Arsenite-treated L-02 cells transported MALAT1 into LX-2 cells. Downregulation of MALAT1, which reduced the MALAT1 levels in exosomes derived from arsenite-treated L-02 cells, inhibited the activation of LX-2 cells. Additionally, exosomal MALAT1 derived from arsenite-treated L-02 cells promoted the activation of LX-2 cells via microRNA-26b regulation of COL1A2. Furthermore, circulating exosomal MALAT1 was up-regulated in people exposed to arsenite. In sum, exosomes derived from arsenite-treated hepatic cells transferred MALAT1 to HSCs, which induced their activation. These findings support the concept that, during liver fibrosis induced by arsenite, exosomal lncRNAs are involved in cell-cell communication. Funding Statement: This work was supported by the Natural Science Foundations of China (81730089, 81430077); the National Key Research and Development Program of China (2016YFE0204900); the open project of Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment and Jiangsu Collaborative Innovation Center For Cancer Personalized Medicine, Nanjing Medical University, China (JX21817902/009); the open project of the Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Guizhou Medical University, China (GMU-2018-HJZ01); and the Priority Academic Program Development of Jiangsu Higher Education Institutions (2018). Declaration of Interests: The authors declare they have no competing financial interests. Ethics Approval Statement: Animals were treated humanely and with regard for alleviation of suffering according to a protocol approved by the Institutional Animal Care and Use committee (IACUC) of Nanjing Medical University.

BACKGROUND Portal hypertension (PHT) is primarily caused by an increase in resistance to portal outflow and secondarily by an increase in splanchnic blood flow. Vascular hyporeactivity both in systemic circulation and in the mesenteric artery plays a role in the hyperdynamic circulatory syndrome. AIM To explore gender differences and the role of endogenous sex hormones in PHT and vascular reactivity of mesenteric arterioles in rats. METHODS Cirrhosis and PHT were established by subcutaneous injection of carbon tetrachloride (CCl4) in both male and female integral and castrated rats (ovariectomized [OVX] in female rats, orchiectomy [ORX] in male rats). The third-order branch of the mensenteric artery was divided and used to measure vascular reactivity to vasoconstrictors. RESULTS No significant difference in portal pressure was observed between integral and castrated male PHT rats (15.2 ± 2.1 mmHg vs 16.7 ± 2.7 mmHg, P > 0.05). The portal pressure in integral female PHT rats was lower than that in OVX female PHT rats (12.7 ± 2.7 mmHg vs 16.5 ± 2.4 mmHg, P < 0.05). In PHT rats, the concentration response curves of the mesenteric arterioles to norepinephrine were shifted to the right, and the maximal responses (Emax) values were decreased and effective concentrations causing half maximum responses (EC50) values were increased, compared to those of non-PHT rats, both in male and female rats. Compared to non-PHT integral male rats, the sensitivity of the mesenteric arterioles of non-PHT ORX male rats to norepinephrine was decreased (P > 0.05). However, there was no difference between integral and ORX male rats with PHT. In integral female PHT rats, the concentration response curves were shifted to the left (P < 0.05), and the Emax values were increased and EC50 values were decreased compared to OVX female PHT rats. CONCLUSION Clear gender differences were observed in mesenteric vascular reactivity in CCl4-induced cirrhotic and PHT rats. Conservation of estrogen can retain the sensitivity of the mesenteric arterioles to vasoconstrictors and has a protective effect on splanchnic vascular function in PHT.

Background Clinically, liver fibrosis and cholestasis are two major disease entities, ultimately leading to hepatic failure. Although autophagy plays a substantial role in the pathogenesis of these diseases, its precise mechanism has not been determined yet. Materials and methods Mouse models of liver fibrosis or cholestasis were obtained after the serial administration of thioacetamide (TAA) or surgical bile duct ligation (BDL), respectively. Then, after obtaining liver specimens at specific time points, we compared the expression of makers related to apoptosis (cleaved caspases), inflammation (CD68), necrosis (high-mobility group box 1), phospho-c-Jun N-terminal kinase (p-JNK), and autophagy (microtubule-associated protein light chain 3B and p62) in the fibrotic or cholestatic mouse livers, by polymerase chain reaction, Western blot analysis, immunohistochemistry, and immunofluorescence. Results Although cholestatic livers exhibited the tendency of progressively increasing the expression of most apoptosis-related markers (cleaved caspases), it was not prominent when it was compared with the tendency found in the livers of TAA-treated mice. Contrastingly, the necrosis-related factor (high-mobility group box 1) was significantly increased in the livers of BDL mice over time, reaching their peak values on day 7 after BDL. In addition, the inflammation-related factor (CD68) was highly expressed in BDL mice compared with TAA-treated mice over time. Autophagy marker studies indicated that autophagy was upregulated in fibrotic livers, whereas it was downregulated in cholestatic livers. We also observed mild to moderate activation of p-JNK in the livers of TAA-treated mice, whereas significantly higher p-JNK activation was detected in the livers of BDL mice. Conclusions Unlike TAA-treated mice, BDL mice exhibited higher expression of the markers related with inflammation and necrosis, especially including p-JNK, while maintaining low levels of autophagic process. Therefore, obstructive cholestasis is characterized by higher p-JNK activation, which could be related with marked necrotic cell death resulting from extensive inflammation and little chance of compensatory autophagy.

BACKGROUND The role of bone marrow-derived mesenchymal stem cells (BM-MSCs) in liver fibrosis remains poorly understood. This study aimed to use a mouse model of carbon tetrachloride (CCL₄)-induced liver fibrosis to investigate the effects of BM-MSCs during liver hypoxia and the involvement of the transforming growth factor beta 1 (TGF-s1) and SMADs pathway. MATERIAL AND METHODS Thirty C57BL/6 mice were randomly divided into the control group (n=10), the model group (n=10), and the BM-MSC-treated model group (n=10). In the model group, liver fibrosis was induced by intraperitoneal injection of CCl₄. BM-MSCs were transplanted after 12 weeks of CCl₄ treatment. The serum biochemical parameters and histological changes in the liver, using histochemical stains, were investigated. The expression of collagen type I (collagen I), alpha-smooth muscle actin (alpha-SMA), TGF-s1, SMAD3, SMAD7, hypoxia-inducible factor 1 alpha (HIF-1alpha), and vascular endothelial grow factor (VEGF) were assessed by immunohistochemistry and quantitative real-time polymerase chain (RT-qPCR) reaction. RESULTS Treatment with BM-MSCs reduced the expression of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) compared with the model group, and reduced liver fibrosis determined histologically using hematoxylin and eosin (H&E) and Masson's trichrome staining compared with the model group. The area of liver fibrosis decreased after BM-MSCs treatment (p

Background Monocyte-derived fibrocytes play an important role in the progression of fibrosis in the skin, lungs, heart and kidney. However, the contribution of fibrocytes to liver fibrosis is unclear. The aim of this study was to investigate whether fibrocytes contributed to fibrosis progression in the livers of carbon tetrachloride (CCl4)-treated mice. Methods C57BL/6J mice were divided into 4 groups: normal control group, CCl4-treated group, CCl4 + control liposome-treated group, and CCl4 + clodronate liposome-treated group. For the elimination of systemic monocyte and monocyte-derived fibrocyte, one group was treated with clodronate liposome, and another group with control liposome as a control. After 4 weeks of treatment, hepatic mononuclear cells were subjected to immunofluorescent (IF) staining and fluorescence-activated cell sorter (FACS) analysis to detect fibrocytes. Measurement of collagen-positive Sirius red stained area and collagen-I mRNA expression in the liver were performed to evaluate the degree of liver fibrosis quantitatively. Results In the liver of the CCl4-treated and CCl4 + control liposome-treated groups, the number of fibrocytes, the area positive for Sirius red staining and collagen-I mRNA expression significantly increased compared with those in the normal control group. In the liver of the CCl4 + clodronate liposome-treated group, few fibrocytes was observed as in the normal control group, but Sirius red staining positive area and collagen-I mRNA expression were increased and equivalent to the CCl4-treated and CCl4 + control liposome-treated groups. Conclusion Monocyte-derived fibrocytes play a minimal role in CCl4-induced liver fibrosis. Cells other than fibrocytes such as hepatic stellate cells play a central role in liver fibrosis.

Introduction and aim Stevia has exhibited antioxidant, antihyperglycemic, antihypertensive and anti-inflammatory properties in several in vivo and in vitro models. The objective of this study was to investigate the ability of an aqueous extract of stevia (AES) to prevent experimental cirrhosis in rats and to explore its mechanism of action. Materials and methods Liver cirrhosis was induced by administering carbon tetrachloride (CCl4) (400 mg/kg by i.p. injection 3 times a week for 12 weeks); AES was administered (100 mg/kg by gavage daily) during the CCl4 treatment. Fibrosis was evaluated with histological, biochemical and molecular approaches, and liver damage was assessed with standardized procedures. The profibrotic pathways were analyzed by western blotting, qRT-PCR and immunohistochemistry. Results and conclusions Chronic CCl4 administration increased nuclear factor kappa B (NF-κB) and proinflammatory cytokine production as well as oxidative parameters such as lipid peroxidation and 4-hydroxynonenal levels, whereas GSH and nuclear factor-E2-related factor 2 (Nrf2) levels were decreased. CCl4 induced profibrogenic mediator expression, hepatic stellate cell (HSC) activation and, consequently, extracellular matrix production. AES exhibited antioxidant, anti-inflammatory and antifibrotic properties, probably because of its capacity to induce Nrf2 expression, reduce NF-κB expression and block several profibrogenic signaling pathways, subsequently inhibiting HSC activation and preventing fibrosis induced by chronic CCl4 administration.

Non-selective β-blockers have largely been used for prophylaxis of bleeding from gastroesophageal varices, but their hepatic effects and their influence on the development of varices has yet to be clarified. This study examined whether carvedilol would reduce acute and chronic liver injury in rats in comparison to propranolol. Experiment (1) Investigated the effects of carvedilol (1.2 mg/kg) and propranolol (4.0 mg/kg) administered daily for 7 days by gavage on paracetamol (1500 mg/kg i.p.) -induced acute liver injury in rats. Experiment (2) Investigated the effects of carvedilol (1.2 mg/kg) and propranolol (4.0 mg/kg) by gavage daily for 8 weeks on CCl

Background Previous research suggested that insulin-like growth factor binding protein related protein 1 (IGFBPrP1), as a novel mediator, contributes to hepatic fibrogenesis. Matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) play an essential role in hepatic fibrogenesis by regulating homeostasis and remodeling of the extracellular matrix (ECM). However, the interaction between IGFBPrP1 and MMP/TIMP is not clear. The present study was to knockdown IGFBPrP1 to investigate the correlation between IGFBPrP1 and MMP/TIMP in hepatic fibrosis. Methods Hepatic fibrosis was induced by thioacetamide (TAA) in mice. Knockdown of IGFBPrP1 expression by ultrasound-targeted microbubble destruction-mediated CMB-shRNA-IGFBPrP1 delivery, or inhibition of the Hedgehog (Hh) pathway by cyclopamine treatment, was performed in TAA-induced liver fibrosis mice. Hepatic fibrosis was determined by hematoxylin and eosin and Sirius red staining. Hepatic expression of IGFBPrP1, α-smooth muscle actin (α-SMA), transforming growth factor β 1 (TGFβ1), collagen I, MMPs/TIMPs, Sonic Hedgehog (Shh), and glioblastoma family transcription factors (Gli1) were investigated by immunohistochemical staining and Western blotting analysis. Results We found that hepatic expression of IGFBPrP1, TGFβ1, α-SMA, and collagen I were increased longitudinally in mice with TAA-induced hepatic fibrosis, concomitant with MMP2/TIMP2 and MMP9/TIMP1 imbalance and Hh pathway activation. Knockdown of IGFBPrP1 expression, or inhibition of the Hh pathway, reduced the hepatic expression of IGFBPrP1, TGFβ1, α-SMA, and collagen I and re-established MMP2/TIMP2 and MMP9/TIMP1 balance. Conclusions Our findings suggest that IGFBPrP1 knockdown attenuates liver fibrosis by re-establishing MMP2/TIMP2 and MMP9/TIMP1 balance, concomitant with the inhibition of hepatic stellate cell activation, down-regulation of TGFβ1 expression, and degradation of the ECM. Furthermore, the Hh pathway mediates IGFBPrP1 knockdown-induced attenuation of hepatic fibrosis through the regulation of MMPs/TIMPs balance.

Objectives: To measure the expression of matrix metalloproteinase (MMP)-2, tissue inhibitor of matrix metalloproteinase inhibitor (TIMP)-2, and CD147 in mice with chronic liver injury induced by carbon tetrachloride after treatment with the traditional Chinese medicine (TCM) “Compound T11”. Method: Sixty male ICR mice were divided randomly into 6 groups of 10: control (C), model (M), low-dose treatment (LT; 50 mg/mL of Compound T11), medium-dose treatment (MT, 100 mg/mL), high-dose treatment (HT, 150 mg/mL), and positive drug treatment (YT, 67.5 mg/mL). Each group was modeled for 7 weeks. Groups M, LT, MT, HT, and YT were injected (s.c.) with 20% carbon tetrachloride diluted with olive oil, and group C was given olive oil in the same way twice a week. After modeling, the treatment groups were administered Compound T11 at the concentrations shown above by oral gavage daily for 2 weeks, while group C was given 0.5% carboxymethyl cellulose sodium. After the final treatment, mice were killed and their liver tissues were excised. Immunohistochemical staining was performed to measure the protein expression of MMP-2, TIMP-2, and CD147, and western blotting was used to measure the protein expression of MMP-2, TIMP-2, CD147, and α-smooth muscle actin (SMA). MMP-2, TIMP-2, and CD147 mRNA expression was determined by quantitative fluorescence real-time PCR. Results: Compound T11 increased the protein expression of MMP-2 and CD147 and decreased the protein expression of TIMP-2 and α-SMA. Conclusions: Treatment of chronic liver injury by TCM Compound T11 may be associated with changes to the expression of MMP-2 and CD147, and the inhibition of TIMP-2 expression.

Type 3 innate lymphoid cell (ILC3) has recently emerged as a crucial effector in inflammatory and fibrotic diseases. The present study was designed to determine the roles of ILC3 in liver fibrosis. By flow cytometry, we documented increased frequencies of peripheral ILC3 (Lin−CD127+CD117+CD294− lymphocytes) in patients, especially at the advanced stage of hepatitis B virus (HBV)-related chronic liver diseases, and demonstrated their correlations with disease progression. The in vitro fibrogenic effects by ILC3 were determined by co-culture experiments with LX-2 (a human hepatic stellate cell (HSC) line). The data indicate that pathogenic ILC3 can directly promote LX-2 fibrogenesis in non-contact manners by producing interleukin (IL)-17A and IL-22. Additionally, they had indirect fibrogenic effects by producing IL-22 to suppress interferon (IFN)-γ (a well-known anti-fibrotic cytokine) production by other immune cells. In carbon tetrachloride (CCl4)-induced wild-type mouse liver fibrosis models, we also documented significantly increased frequencies of both non-natural killer (NK) ILC (Lin−CD127+ lymphocytes) and ILC3 (Lin−CD127+RORγt+ lymphocytes) in liver and spleen specimens. Furthermore, the ILC3 from fibrotic mice contained more IL-17A+ILC3 and IL-22+ILC3 subsets than those from normal and less-fibrotic mice. The in vivo effects of ILC3 in liver fibrogenesis were further determined using RAG-1−/− mice with ILC depletion and further adoptive transfer of ILC3 from wild-type mice. The immunohistochemical staining of liver specimens showed the beneficial effects by ILC depletion and the detrimental effects by ILC3 transfer in CCl4-induced mouse liver fibrosis models. Collectively, ILC3 plays a pro-fibrotic role in liver fibrosis progression.

Mao Mu,1,* Shi Zuo,2,* Rong-Min Wu,3 Kai-Sheng Deng,1 Shuang Lu,1 Juan-Juan Zhu,1 Gao-Liang Zou,1 Jing Yang,1 Ming-Liang Cheng,1 Xue-Ke Zhao1 1Department of Infectious Diseases, The Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou, China; 2Department of Hepatobiliary Surgery, The Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou, China; 3Department of Ultrasonography, The Maternity Hospital of Guizhou, Guiyang, Guizhou, China *These authors contributed equally to this work Purpose: Liver fibrosis is a worldwide health issue. Development of effective new drugs for treatment of this disease is of great importance. This study investigated the therapeutic effects of ferulic acid on liver fibrosis invitro and invivo.Materials and methods: Human hepatic stellate cell line (HSC) LX-2 was used for invitro assays. Transforming growth factor β1 (TGF-β1) was used to induce hepatic fibrosis in LX-2 cells. Western blot was used to detect protein levels of collagen I, fibronectin, α-smooth muscle actin (SMA), p-Smad2, p-Smad3, p-p38, and p-JNK. Gene expression was measured by RT-qPCR. Fluorescence staining was used to determine localization of Smad4. CCl4-induced hepatic fibrosis in SD rats was used as an invivo model. Histological features were detected by hematoxylin and eosin staining. Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hexadecenoic acid (HA), and hydroxyproline (Hyp) were measured by ELISA.Results: TGF-β1 treatment significantly increased levels of collagen I, fibronectin, α-SMA, p-Smad2, p-Smad3, and Smad4 in LX-2 cells. Ferulic acid improved TGF-β1-induced hepatic fibrosis via regulation of the TGF-β1/Smad pathway. Consistent with invitro data, CCl4 caused severe hepatic fibrosis in SD rats, as determined by ALT, AST, HA, and Hyp upregulation. Protein levels of p-Smad2 and p-Smad3 in liver tissues were significantly increased following treatment with CCl4. All CCL4-induced changes were markedly attenuated by ferulic acid treatment.Conclusion: Ferulic acid potently improved hepatic fibrosis via inhibition of the TGF-β1/Smad pathway invitro and invivo. These findings provided evidence for potential use of ferulic acid to treat or prevent liver fibrosis. Keywords: ferulic acid, TGF-β1, CCl4, hepatic fibrosis, Smad signaling pathway&nbsp

As a specialized intraparenchymal vascular conduit, hepatic sinusoids play a key role in liver microcirculation. This study aimed to explore the three-dimensional (3D) morphological changes of cirrhotic sinusoids by serial histological sections.Cirrhosis was induced by tail vein injection of albumin in Wistar rats with a positive antibody. A total of 356 serial histological sections were prepared from liver tissue blocks of normal and cirrhotic rats. The optical microscope images were registered and reconstructed, and 3D reconstructions of the fine structures of fibrous tissues and sinusoids were subsequently visualized.The fibrosis area of the cirrhotic sample was 6-16 times that of the normal sample (P0.001). Cirrhosis led to obvious changes in the distribution and morphology of sinusoids, which were mainly manifested as dilation, increased quantity and disordered distribution. Compared with normal liver, cirrhotic liver has a significantly increased volume ratio, number and volume of sinusoids (1.63-, 0.53-, and 1.75-fold, respectively, P0.001). Furthermore, the samples were further divided into three zones according to the oxygen supply, and there were significant differences in the morphology of the sinusoids in the normal and cirrhotic samples (P0.05). In particular, morphological parameters of the cirrhotic sinusoids near the portal area were obviously greater than those in the normal liver (P0.05).3D morphological structures of hepatic sinusoids were reconstructed, and the adaptive microstructure changes of cirrhotic sinusoids were accurately measured, which has an important implications for the study of hepatic microcirculation and pathological changes of 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.

Nutritional ketosis as a therapeutic tool has been extended to the treatment of metabolic diseases, including obesity, type 2 diabetes, and nonalcoholic fatty liver disease (NAFLD). The purpose of this study was to determine whether dietary administration of the ketone ester (KE) R,S-1,3-butanediol diacetoacetate (BD-AcAc(2)) attenuates markers of hepatic stellate cell (HSC) activation and hepatic fibrosis in the context of high-fat diet (HFD)-induced obesity. Six-week-old male C57BL/6J mice were placed on a 10-wk ad libitum HFD (45% fat, 32% carbohydrates, 23% proteins). Mice were then randomized to one of three groups (n = 10 per group) for an additional 12 wk: 1) control (CON), continuous HFD; 2) pair-fed (PF) to KE, and 3) KE (HFD + 30% energy from BD-AcAc(2), KE). KE feeding significantly reduced histological steatosis, inflammation, and total NAFLD activity score versus CON, beyond improvements observed for calorie restriction alone (PF). Dietary KE supplementation also reduced the protein content and gene expression of profibrotic markers (α-SMA, COL1A1, PDGF-β, MMP9) versus CON (P < 0.05), beyond reductions observed for PF versus CON. Furthermore, KE feeding increased hepatic markers of anti-inflammatory M2 macrophages (CD163) and also reduced proinflammatory markers [tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and cellular communication network factor 1 (CCN1)] versus CON and PF (P ≤ 0.05), in the absence of changes in markers of total hepatic macrophage content (F4/80 and CD68; P > 0.05). These data highlight that the dietary ketone ester BD-AcAc(2) ameliorates histological NAFLD and inflammation and reduces profibrotic and proinflammatory markers. Future studies to further explore potential mechanisms are warranted. NEW & NOTEWORTHY To our knowledge, this is the first study focusing on hepatic outcomes in response to dietary ketone ester feeding in male mice with HFD-induced NAFLD. Novel findings include that dietary ketone ester feeding ameliorates NAFLD outcomes via reductions in histological steatosis and inflammation. These improvements were beyond those observed for caloric restriction alone. Furthermore, dietary ketone ester feeding was associated with greater reductions in markers of hepatic fibrogenesis and inflammation compared with control and calorie-restricted mice.

Nonalcoholic steatohepatitis (NASH) could progress to hepatic fibrosis in the absence of effective control. The purpose of our experiment was to investigate the protective effect of drinking water with a high concentration of hydrogen, namely, hydrogen-rich water (HRW), on mice with nonalcoholic fatty liver disease to elucidate the mechanism underlying the therapeutic action of molecular hydrogen. The choline-supplemented, l-amino acid-defined (CSAA) or the choline-deficient, l-amino acid-defined (CDAA) diet for 20 wk was used to induce NASH and fibrosis in the mice model and simultaneously treated with the high-concentration 7-ppm HRW for different periods (4 wk, 8 wk, and 20 wk). Primary hepatocytes were stimulated by palmitate to mimic liver lipid metabolism during fatty liver formation. Primary hepatocytes were cultured in a closed vessel filled with 21% O

Background & aims Splenomegaly is usually taken as a consequence of liver cirrhosis. However, as a risk factor for cirrhosis, the impacts of spleen-liver axis on the development of cirrhosis are largely unknown. This study focused on the impacts of splenomegaly on the development of cirrhosis and assessment of the effects of celecoxib, a selective COX-2 inhibitor, on the splenomegaly and cirrhotic liver. Materials and methods Liver cirrhosis was induced by thioacetamide (TAA). Sixty rats were randomly divided into control, TAA-16w, TAA + celecoxib groups and normal, TAA + sham, TAA + splenectomy groups. Hepatic stellate cells (HSCs) or hepatocytes were co-cultured with splenocytes from those groups. Results Splenocytes of cirrhotic rats stimulated the HSCs activation and induced hepatocyte apoptosis via enhancing oxidative stress. The hepatic levels of NOX-4 and the in situ O2− were profoundly reduced in TAA + splenectomy group by 50.6% and 18.5% respectively, p Conclusion Splenomegaly contributed to the development of liver cirrhosis through enhancing oxidative stress in liver. Celecoxib could effectively ameliorate liver cirrhosis via reducing inflammatory cytokines and immune cells derived from spleen and suppressing oxidative stress.

BACKGROUND AND OBJECTIVE Numerous experimental studies have shown various pharmacological activities including geraniol's cancer prevention agent and antioxidant capacity. The goal of this investigation is to mark the prospective defensive role of geraniol in rat's carbon tetrachloride (CCl4) instigated in liver fibrosis. MATERIALS AND METHODS Liver fibrosis was prompted by subcutaneous injections of CCl4, twice week by week and for about a month. Simultaneously, geraniol (200 mg kg-1) was orally regulated every day. Post-Hoc-Test were carried out where p

Activation of hepatic stellate cells (HSCs), characterized by development of a robust actin cytoskeleton and expression of abundant extracellular matrix (ECM) proteins, such as type 1 collagen (COL.1), is a central cellular and molecular event in liver fibrosis. It has been demonstrated that HSCs express both myocardin and myocardin-related transcription factor-A (MRTF-A). However, the biological effects of myocardin and MRTF-A on HSC activation and liver fibrosis, as well as the molecular mechanism under the process, remain unclear. Here, we report that myocardin and MRTF-A's expression and nuclear accumulation are prominently increased during the HSC activation process, accompanied by robust activation of actin cytoskeleton dynamics. Targeting myocardin and MRTF-A binding and function with a novel small molecule, CCG-203971, led to dose-dependent inhibition of HSC actin cytoskeleton dynamics and abrogated multiple functional features of HSC activation (i.e., HSC contraction, migration and proliferation) and decreased COL.1 expression in vitro and liver fibrosis in vivo. Mechanistically, blocking the myocardin and MRTF-A nuclear translocation pathway with CCG-203971 directly inhibited myocardin/MRTF-A-mediated serum response factor (SRF), and Smad2/3 activation in the COL.1α2 promoter and indirectly abrogated actin cytoskeleton-dependent regulation of Smad2/3 and Erk1/2 phosphorylation and their nuclear accumulation. Finally, there was no effect of CCG-203971 on markers of inflammation, suggesting a direct effect of the compound on HSCs and liver fibrosis. These data reveal that myocardin and MRTF-A are two important cotranscriptional factors in HSCs and represent entirely novel therapeutic pathways that might be targeted to treat liver fibrosis.