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1.
Biochem Pharmacol ; 224: 116205, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38615918

ABSTRACT

Nitazoxanide is an FDA-approved antiprotozoal drug. Our previous studies find that nitazoxanide and its metabolite tizoxanide affect AMPK, STAT3, and Smad2/3 signals which are involved in the pathogenesis of liver fibrosis, therefore, in the present study, we examined the effect of nitazoxanide on experimental liver fibrosis and elucidated the potential mechanisms. The in vivo experiment results showed that oral nitazoxanide (75, 100 mg·kg-1) significantly improved CCl4- and bile duct ligation-induced liver fibrosis in mice. Oral nitazoxanide activated the inhibited AMPK and inhibited the activated STAT3 in liver tissues from liver fibrosis mice. The in vitro experiment results showed that nitazoxanide and its metabolite tizoxanide activated AMPK and inhibited STAT3 signals in LX-2 cells (human hepatic stellate cells). Nitazoxanide and tizoxanide inhibited cell proliferation and collagen I expression and secretion of LX-2 cells. Nitazoxanide and tizoxanide inhibited transforming growth factor-ß1 (TGF-ß1)- and IL-6-induced increases of cell proliferation, collagen I expression and secretion, inhibited TGF-ß1- and IL-6-induced STAT3 and Smad2/3 activation in LX-2 cells. In mouse primary hepatic stellate cells, nitazoxanide and tizoxanide also activated AMPK, inhibited STAT3 and Smad2/3 activation, inhibited cell proliferation, collagen I expression and secretion. In conclusion, nitazoxanide inhibits liver fibrosis and the underlying mechanisms involve AMPK activation, and STAT3 and Smad2/3 inhibition.


Subject(s)
Antiprotozoal Agents , Nitro Compounds , Thiazoles , Animals , Mice , Thiazoles/pharmacology , Thiazoles/therapeutic use , Male , Humans , Antiprotozoal Agents/pharmacology , Antiprotozoal Agents/therapeutic use , Cell Line , Hepatic Stellate Cells/drug effects , Hepatic Stellate Cells/metabolism , Liver Cirrhosis/drug therapy , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Liver Cirrhosis/chemically induced , STAT3 Transcription Factor/metabolism , STAT3 Transcription Factor/antagonists & inhibitors , Smad3 Protein/metabolism , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/pathology , Liver Cirrhosis, Experimental/drug therapy , Liver Cirrhosis, Experimental/metabolism , Liver Cirrhosis, Experimental/prevention & control , Mice, Inbred C57BL , Smad2 Protein/metabolism
2.
Bull Exp Biol Med ; 175(2): 279-285, 2023 Jun.
Article in English | MEDLINE | ID: mdl-37477742

ABSTRACT

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).


Subject(s)
Liver Cirrhosis, Experimental , Rats , Animals , Rats, Wistar , Liver Cirrhosis, Experimental/chemically induced , Liver/pathology , Liver Cirrhosis/chemically induced , Liver Cirrhosis/pathology , Macrophages/pathology
3.
J Pharmacol Exp Ther ; 386(1): 70-79, 2023 07.
Article in English | MEDLINE | ID: mdl-37230799

ABSTRACT

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.


Subject(s)
Hypertension, Portal , Liver Cirrhosis, Experimental , Rats , Male , Animals , Soluble Guanylyl Cyclase/pharmacology , Thioacetamide/adverse effects , Rats, Sprague-Dawley , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/drug therapy , Hypertension, Portal/drug therapy , Liver Cirrhosis/chemically induced , Liver Cirrhosis/drug therapy , Liver Cirrhosis/complications , Liver , Cyclic GMP
4.
J Nat Med ; 77(1): 128-136, 2023 Jan.
Article in English | MEDLINE | ID: mdl-36169781

ABSTRACT

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.


Subject(s)
Liver Cirrhosis, Experimental , Rats , Animals , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/drug therapy , Liver Cirrhosis, Experimental/metabolism , Jagged-1 Protein/metabolism , Jagged-1 Protein/pharmacology , Hepatic Stellate Cells , Actins/metabolism , Actins/pharmacology , Liver Cirrhosis/chemically induced , Liver Cirrhosis/drug therapy , Liver , Collagen , Alanine Transaminase
5.
J Vasc Interv Radiol ; 34(3): 404-408.e1, 2023 03.
Article in English | MEDLINE | ID: mdl-36473611

ABSTRACT

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.


Subject(s)
Carcinoma, Hepatocellular , Liver Cirrhosis, Experimental , Liver Neoplasms, Experimental , Liver Neoplasms , Rabbits , Male , Animals , Carbon Tetrachloride/adverse effects , Liver/pathology , Liver Cirrhosis , Liver Neoplasms/pathology , Carcinoma, Hepatocellular/pathology , Liver Neoplasms, Experimental/pathology , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/pathology
6.
Life Sci ; 301: 120615, 2022 Jul 15.
Article in English | MEDLINE | ID: mdl-35526595

ABSTRACT

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.


Subject(s)
Liver Cirrhosis, Experimental , Rodentia , Animals , Carbon Tetrachloride/toxicity , Disease Models, Animal , Liver/pathology , Liver Cirrhosis/pathology , Liver Cirrhosis, Experimental/chemically induced , Models, Theoretical , Thioacetamide/toxicity
7.
Ann Hepatol ; 26: 100560, 2021 Dec.
Article in English | MEDLINE | ID: mdl-34653689

ABSTRACT

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.


Subject(s)
Carbon Tetrachloride Poisoning/complications , Down-Regulation , Gene Expression Regulation , HMGB1 Protein/genetics , Insulin-Like Growth Factor I/therapeutic use , Intestinal Mucosa/metabolism , Liver Cirrhosis, Experimental/genetics , Animals , Caco-2 Cells , Carbon Tetrachloride Poisoning/genetics , Carbon Tetrachloride Poisoning/metabolism , HMGB1 Protein/biosynthesis , Humans , Intestinal Mucosa/pathology , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/therapy , Male , RNA/genetics , Rats
8.
Bull Exp Biol Med ; 171(6): 707-712, 2021 Oct.
Article in English | MEDLINE | ID: mdl-34705170

ABSTRACT

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.


Subject(s)
Carbon Tetrachloride/toxicity , Ethanol/toxicity , Hepatocytes/drug effects , Liver Cirrhosis, Experimental/metabolism , Liver/drug effects , Age Factors , Animals , Cell Membrane/chemistry , Cell Membrane/drug effects , Hepatocytes/metabolism , Hepatocytes/pathology , Liver/metabolism , Liver/pathology , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/pathology , Male , Mitochondria/chemistry , Mitochondria/drug effects , Mitochondrial Membranes/chemistry , Mitochondrial Membranes/drug effects , Rats , Rats, Wistar , Viscosity/drug effects
9.
Am J Physiol Gastrointest Liver Physiol ; 321(6): G603-G616, 2021 12 01.
Article in English | MEDLINE | ID: mdl-34585619

ABSTRACT

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.


Subject(s)
Caloric Restriction , Chemical and Drug Induced Liver Injury/therapy , Energy Intake , Exercise Therapy , Healthy Lifestyle , Liver Cirrhosis, Experimental/therapy , Liver/metabolism , Animals , Carbon Tetrachloride , Chemical and Drug Induced Liver Injury/etiology , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/pathology , Hypertension, Portal/chemically induced , Hypertension, Portal/metabolism , Hypertension, Portal/physiopathology , Hypertension, Portal/therapy , Liver/pathology , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/metabolism , Liver Cirrhosis, Experimental/pathology , Male , Physical Endurance , Rats, Wistar , Risk Reduction Behavior , Thioacetamide , Time Factors
10.
Int Immunopharmacol ; 100: 108088, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34454288

ABSTRACT

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.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Antifibrotic Agents/pharmacology , Chemical and Drug Induced Liver Injury/prevention & control , Liver Cirrhosis, Experimental/prevention & control , Liver/drug effects , NF-E2-Related Factor 2/metabolism , NF-kappa B/metabolism , Sitagliptin Phosphate/pharmacology , Animals , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/pathology , Concanavalin A , Liver/metabolism , Liver/pathology , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/metabolism , Liver Cirrhosis, Experimental/pathology , Male , Mice , NF-E2-Related Factor 2/genetics , Oxidative Stress/drug effects , Signal Transduction , Tumor Necrosis Factor-alpha/metabolism
11.
Pak J Pharm Sci ; 34(2): 591-597, 2021 Mar.
Article in English | MEDLINE | ID: mdl-34275834

ABSTRACT

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.


Subject(s)
Coffee , Dietary Supplements , Liver Cirrhosis, Experimental/metabolism , Alanine Transaminase/metabolism , Alkaline Phosphatase/metabolism , Animals , Aspartate Aminotransferases/metabolism , Bilirubin/metabolism , Catalase/metabolism , Lipid Peroxidation , Liver Cirrhosis/chemically induced , Liver Cirrhosis/metabolism , Liver Cirrhosis, Experimental/chemically induced , Malondialdehyde/metabolism , Rats , Superoxide Dismutase/metabolism , Thioacetamide/toxicity
12.
Hepatology ; 74(5): 2774-2790, 2021 11.
Article in English | MEDLINE | ID: mdl-34089528

ABSTRACT

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.


Subject(s)
Hepatic Stellate Cells/immunology , Liver Cirrhosis, Experimental/immunology , Myofibroblasts/immunology , Animals , Carbon Tetrachloride/administration & dosage , Carbon Tetrachloride/toxicity , Cell Lineage/immunology , Cells, Cultured , Gene Expression Regulation/immunology , Gene Knock-In Techniques , Hepatic Stellate Cells/metabolism , Humans , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/pathology , Male , Mice , Mice, Transgenic , Primary Cell Culture , RNA-Seq , Single-Cell Analysis
13.
Histol Histopathol ; 36(5): 577-586, 2021 May.
Article in English | MEDLINE | ID: mdl-33851410

ABSTRACT

AIM: 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. METHODS: 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. RESULTS: The fibrosis area of the cirrhotic sample was 6-16 times that of the normal sample (P<0.001). 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, P<0.001). 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 (P<0.05). In particular, morphological parameters of the cirrhotic sinusoids near the portal area were obviously greater than those in the normal liver (P<0.05). CONCLUSION: 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.


Subject(s)
Hepatic Veins , Imaging, Three-Dimensional/methods , Liver Cirrhosis/pathology , Animals , Hepatic Veins/anatomy & histology , Hepatic Veins/pathology , Liver/pathology , Liver Cirrhosis, Experimental/chemically induced , Microcirculation , Rats , Rats, Wistar
14.
Biochem Biophys Res Commun ; 558: 86-93, 2021 06 18.
Article in English | MEDLINE | ID: mdl-33906111

ABSTRACT

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.


Subject(s)
Liver Cirrhosis, Experimental/drug therapy , TRPV Cation Channels/antagonists & inhibitors , Animals , Caffeic Acids/pharmacology , Carbon Tetrachloride/toxicity , Cells, Cultured , Disease Models, Animal , Gene Knockdown Techniques , Glucosides/pharmacology , Hepatic Stellate Cells/metabolism , Humans , Immunohistochemistry , Liver Cirrhosis/metabolism , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/metabolism , Male , Mice , Mice, Inbred C57BL , Phenylacetates/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , TRPV Cation Channels/genetics , TRPV Cation Channels/metabolism , Up-Regulation
15.
STAR Protoc ; 2(1): 100353, 2021 03 19.
Article in English | MEDLINE | ID: mdl-33665633

ABSTRACT

Animal models of liver cancer are instrumental in the study of hepatocarcinogenesis and development of novel therapeutic approaches. Here, we describe steps to establish liver cancer in a rat model, via chronic administration of diethylnitrosamine. This causes liver tumors with a sequential progression of hepatitis, cirrhosis, and tumor formation, which closely mimics the development of human liver cancer. This protocol was optimized to significantly increase the incidence of liver tumor formation and reduce the duration of the procedure. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020).


Subject(s)
Carcinogenesis , Diethylnitrosamine/toxicity , Liver Cirrhosis, Experimental , Liver Neoplasms, Experimental , Animals , Carcinogenesis/chemically induced , Carcinogenesis/metabolism , Carcinogenesis/pathology , Humans , Inflammation/chemically induced , Inflammation/metabolism , Inflammation/pathology , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/metabolism , Liver Cirrhosis, Experimental/pathology , Liver Neoplasms, Experimental/chemically induced , Liver Neoplasms, Experimental/metabolism , Liver Neoplasms, Experimental/pathology , Male , Rats , Rats, Sprague-Dawley
16.
Eur J Pharmacol ; 898: 173982, 2021 May 05.
Article in English | MEDLINE | ID: mdl-33647257

ABSTRACT

Liver fibrosis is a compensatory response to the tissue repair process. The activation and proliferation of hepatic stellate cells (HSCs) are thought to be related to the occurrence of hepatic fibrosis. Therefore, inhibiting the activation and proliferation of HSCs is a key step in alleviating liver fibrosis. As a non-specific inhibitor of transient receptor potential melastatin 7 (TRPM7), carvacrol has anti-tumor, anti-inflammatory and anti-hepatic fibrosis activities. This study aimed to explore the protective effect of carvacrol on liver fibrosis and related molecular mechanisms. A CCl4-induced liver fibrosis mouse model and platelet-derived growth factor (PDGF-BB)-activated HSC-T6 cells (a rat hepatic stellate cell line) were employed for in vivo and in vitro experiments. C57BL/6J mice were orally administered different concentrations of carvacrol every day for 6 weeks during the development of CCl4-induced liver fibrosis. The results show that carvacrol could effectively reduce liver damage and the progression of liver fibrosis in mice, which are expressed as fibrotic markers levels were reduced and histopathological characteristics were improved. Moreover, carvacrol inhibited the proliferation and activation of HSC-T6 cells induced by PDGF-BB. In addition, it was found that carvacrol inhibits the expression of TRPM7 and mediated through mitogen-activated protein kinases (MAPK). Collectively, our study shows that carvacrol can reduce liver fibrosis by inhibiting the activation and proliferation of hepatic stellate cells, and the MAPK signaling pathway might be involved in this process.


Subject(s)
Chemical and Drug Induced Liver Injury/prevention & control , Cymenes/pharmacology , Hepatic Stellate Cells/drug effects , Liver Cirrhosis, Experimental/prevention & control , Liver/drug effects , Mitogen-Activated Protein Kinases/metabolism , TRPM Cation Channels/antagonists & inhibitors , Animals , Becaplermin/pharmacology , Carbon Tetrachloride , Cell Line , Cell Proliferation/drug effects , Chemical and Drug Induced Liver Injury/enzymology , Chemical and Drug Induced Liver Injury/etiology , Chemical and Drug Induced Liver Injury/pathology , Collagen/metabolism , Hepatic Stellate Cells/enzymology , Hepatic Stellate Cells/pathology , Liver/enzymology , Liver/pathology , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/enzymology , Liver Cirrhosis, Experimental/pathology , Male , Mice, Inbred C57BL , Rats , Signal Transduction , TRPM Cation Channels/metabolism
17.
Cell Death Dis ; 12(2): 163, 2021 02 08.
Article in English | MEDLINE | ID: mdl-33558482

ABSTRACT

Liver cirrhosis remains major health problem. Despite the progress in diagnosis of asymptomatic early-stage cirrhosis, prognostic biomarkers are needed to identify cirrhotic patients at high risk developing advanced stage disease. Liver cirrhosis is the result of deregulated wound healing and is featured by aberrant extracellular matrix (ECM) remodeling. However, it is not comprehensively understood how ECM is dynamically remodeled in the progressive development of liver cirrhosis. It is yet unknown whether ECM signature is of predictive value in determining prognosis of early-stage liver cirrhosis. In this study, we systematically analyzed proteomics of decellularized hepatic matrix and identified four unique clusters of ECM proteins at tissue damage/inflammation, transitional ECM remodeling or fibrogenesis stage in carbon tetrachloride-induced liver fibrosis. In particular, basement membrane (BM) was heavily deposited at the fibrogenesis stage. BM component minor type IV collagen α5 chain expression was increased in activated hepatic stellate cells. Knockout of minor type IV collagen α5 chain ameliorated liver fibrosis by hampering hepatic stellate cell activation and promoting hepatocyte proliferation. ECM signatures were differentially enriched in the biopsies of good and poor prognosis early-stage liver cirrhosis patients. Clusters of ECM proteins responsible for homeostatic remodeling and tissue fibrogenesis, as well as basement membrane signature were significantly associated with disease progression and patient survival. In particular, a 14-gene signature consisting of basement membrane proteins is potent in predicting disease progression and patient survival. Thus, the ECM signatures are potential prognostic biomarkers to identify cirrhotic patients at high risk developing advanced stage disease.


Subject(s)
Chemical and Drug Induced Liver Injury/metabolism , Collagen Type IV/metabolism , Extracellular Matrix/metabolism , Hepatic Stellate Cells/metabolism , Liver Cirrhosis, Experimental/metabolism , Liver/metabolism , Animals , Carbon Tetrachloride , Cell Line , Cell Proliferation , Chemical and Drug Induced Liver Injury/etiology , Chemical and Drug Induced Liver Injury/genetics , Chemical and Drug Induced Liver Injury/pathology , Collagen Type IV/genetics , Databases, Genetic , Disease Progression , Extracellular Matrix/pathology , Hepatic Stellate Cells/pathology , Liver/pathology , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/genetics , Liver Cirrhosis, Experimental/pathology , Male , Mice, Inbred C57BL , Mice, Knockout , Prognosis , Proteome , Time Factors , Transcriptome
18.
Biomed Pharmacother ; 135: 111181, 2021 Mar.
Article in English | MEDLINE | ID: mdl-33395607

ABSTRACT

Branched-chain amino acids (BCAA) reverse malnutrition and l-carnitine leads to the reduction of hyperammonemia and muscle cramps in cirrhotic patients. BCAA and l-carnitine are involved in glucose and fatty acid metabolism, however their mechanistic activity in cirrhotic liver is not fully understood. We aim to define the molecular mechanism(s) and combined effects of BCAA and l-carnitine using a cirrhotic rat model. Rats were administered carbon tetrachloride for 10 weeks to induce cirrhosis. During the last 6 weeks of administration, cirrhotic rats received BCAA, l-carnitine or a combination of BCAA and l-carnitine daily via gavage. We found that BCAA and l-carnitine treatments significantly improved hepatocellular function associated with reduced triglyceride level, lipid deposition and adipophilin expression, in cirrhotic liver. Lipidomic analysis revealed dynamic changes in hepatic lipid composition by BCAA and l-carnitine administrations. BCAA and l-carnitine globally increased molecular species of phosphatidylcholine. Liver triacylglycerol and phosphatidylcholine hydroperoxides were significantly decreased by BCAA and l-carnitine. Furthermore, serum and liver ATP levels were significantly increased in all treatments, which were attributed to the elevation of mature cardiolipins and mitochondrial component gene expressions. Finally, BCAA and l-carnitine dramatically reduced hepatocellular death. In conclusion, BCAA and l-carnitine treatments attenuate hepatocellular damage through the reduction of lipid peroxides and the overall maintenance of mitochondrial integrity within the cirrhotic liver. These effectiveness of BCAA and l-carnitine support the therapeutic strategies in human chronic liver diseases.


Subject(s)
Amino Acids, Branched-Chain/pharmacology , Carnitine/pharmacology , Chemical and Drug Induced Liver Injury/prevention & control , Fatty Liver/prevention & control , Hepatocytes/drug effects , Lipid Peroxidation/drug effects , Liver Cirrhosis, Experimental/prevention & control , Liver/drug effects , Animals , Carbon Tetrachloride , Cell Death/drug effects , Chemical and Drug Induced Liver Injury/etiology , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/pathology , Fatty Liver/chemically induced , Fatty Liver/metabolism , Fatty Liver/pathology , Hepatocytes/metabolism , Hepatocytes/pathology , Liver/metabolism , Liver/pathology , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/metabolism , Liver Cirrhosis, Experimental/pathology , Male , Mitochondria, Liver/drug effects , Mitochondria, Liver/metabolism , Mitochondria, Liver/pathology , Rats, Wistar
19.
Int J Med Sci ; 18(3): 615-625, 2021.
Article in English | MEDLINE | ID: mdl-33437196

ABSTRACT

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.


Subject(s)
Coenzyme A Ligases/genetics , Hepatic Stellate Cells/pathology , Liver Cirrhosis, Experimental/genetics , Liver/pathology , MicroRNAs/metabolism , Animals , Coenzyme A Ligases/metabolism , Collagen/biosynthesis , Dimethylnitrosamine/administration & dosage , Dimethylnitrosamine/toxicity , Hepatic Stellate Cells/drug effects , Humans , Lipid Droplets/metabolism , Lipid Metabolism/genetics , Liver/cytology , Liver/drug effects , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/pathology , MicroRNAs/agonists , MicroRNAs/antagonists & inhibitors , Rats
20.
Life Sci ; 267: 118944, 2021 Feb 15.
Article in English | MEDLINE | ID: mdl-33359749

ABSTRACT

AIMS: Liver cirrhosis is the main chronic liver disease and is considered a catabolic disease. Cirrhotic patients have a low energy intake and high energy expenditure at rest, leading to metabolic disorders. Malnutrition is associated with complications of cirrhosis and has been shown that a nutritional intervention with increase of energy intake improves the survival of cirrhotic patients. Therefore, our aim was to evaluate the effect of a high sucrose diet in the liver of animals with cirrhosis induced by thioacetamide and investigate the mechanism involved. MAIN METHODS: Male Wistar rats were divided into three groups: Control; Thioacetamide; and Thioacetamide + high sucrose diet. The thioacetamide was administrated (100 mg kg-1) intraperitoneally and the sucrose was offered in drinking water (300 g L-1). KEY FINDINGS: The administration of thioacetamide was associated with fibrosis and inflammatory infiltrate in the liver and increased levels of transaminases enzymes. The high sucrose diet promoted a reduction of theses parameters in cirrhotic rats. The malnutrition observed in cirrhotic rats was attenuated by the high sucrose diet shown by the improvements in weight loss, subcutaneous fat, and caloric intake. The high sucrose diet also attenuated the oxidative stress present in the liver of animals with thioacetamide-induced cirrhosis. SIGNIFICANCE: The high sucrose diet had anti-inflammatory and anti-oxidant effects in the liver of animals with thioacetamide-induced cirrhosis. In addition, the high sucrose diet also improved malnutrition and catabolism present in cirrhosis. Thus, a high sucrose diet may be a therapeutic option for cirrhotic patients in a catabolic state.


Subject(s)
Dietary Sucrose/pharmacology , Liver Cirrhosis/drug therapy , Liver Cirrhosis/metabolism , Animals , Chemical and Drug Induced Liver Injury/drug therapy , Chemical and Drug Induced Liver Injury/metabolism , Diet , Dietary Sucrose/metabolism , Inflammation , Liver/metabolism , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/metabolism , Male , Oxidative Stress/drug effects , Rats , Rats, Wistar , Sucrose/metabolism , Sucrose/pharmacology , Thioacetamide/adverse effects , Thioacetamide/pharmacology
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