ABSTRACT
Liver fibrosis is a reversible wound healing reaction characterized by abnormal accumulation of extracellular matrix (ECM) in response to liver injury. Recent studies have shown that it can be epigenetically regulated, especially by microRNAs (miRNAs). It has been acknowledged that activation of hepatic stellate cells (HSCs) is a pivotal step in the initiation and progression of liver fibrosis. Notably, our results showed that miR-195-3p was increased in HSCs isolated from CCl4-treated mice and that the increase was more pronounced as the degree of liver fibrosis increased. Moreover, treatment of LX-2 cells, a human immortalized hepatic stellate cell line, with TGF-ß1 resulted remarkable upregulation of miR-195-3p. Gain-of-function and loss-of-function experiments have suggested that the increased levels of miR-195-3p inhibit the expression of phosphatase and tension homolog deleted on chromosome 10 (PTEN), a negative regulator of the PI3K/Akt/mTOR signaling pathway in liver fibrosis, thereby contributing to HSC activation and proliferation and promoting the expression of profibrotic genes, such as α-SMA and collagen I, in LX-2 cells, which accelerates the accumulation of fibrous extracellular matrix deposition in the liver, while knockdown of miR-195-3p induced the opposite effect. Taken together, these results provide evidence for the harmful role of miR-195-3p in CCl4-treated mouse liver fibrosis.
Subject(s)
Chemical and Drug Induced Liver Injury/metabolism , Hepatic Stellate Cells/metabolism , Liver Cirrhosis/chemically induced , MicroRNAs/metabolism , PTEN Phosphohydrolase/metabolism , 3' Untranslated Regions , Animals , Carbon Tetrachloride Poisoning/pathology , Cell Line , Gene Expression Regulation/drug effects , Humans , Male , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , PTEN Phosphohydrolase/genetics , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/metabolism , Up-RegulationABSTRACT
AIMS: Liver fibrosis is a growing public health concern without effective medical treatment. Recent reports have indicated that inhibitors of apoptosis proteins (IAPs) were potential targets for idiopathic pulmonary fibrosis therapy. However, their roles have not been well identified in liver fibrosis. METHODS: The expression of IAPs were examined in human liver tissue and experimental mouse models. Liver fibrosis in CCl4-induced mouse models were investigated by Sirius red staining, RT-PCR, Western blotting after hepatocytes-specific cIAP2 knockout or IAPs inhibitor APG-1387 treatment. The underlying molecular mechanism of APG-1387 action was explored by apoptosis analysis, matrix metalloprotein 9 (MMP9) inhibition, neutrophils depletion, and CC Motif Chemokine Ligand 5 (CCL5) gene knockout in vitro and in vivo. FINDINGS: Our study showed that increased expression of cIAP2 was associated with liver fibrosis severity in liver tissues. Deletion of cIAP2 from hepatocytes or degrading cIAPs by APG-1387 ameliorated liver fibrosis induced by CCl4. APG-1387 treatment exhibited increased expression of MMP9 and resulted in higher ratio of MMP9 to tissue inhibitor of metalloproteinase-1. MMP9 was mainly derived from CCL5 chemotactic neutrophils. Further, MMP9 inhibition by CTT peptide, neutrophil depletion by Ly6G antibody or CCL5 deficiency blocked the anti-fibrotic effects of APG-1387 in vivo. SIGNIFICANCE: These results suggested that cIAPs, especially cIAP2, might play a novel role in the pathogenesis of liver fibrosis, and targeting cIAPs represented a promising therapeutic strategy for liver fibrosis by increasing MMP9 expression induced by CCL5 chemotactic neutrophils.
Subject(s)
Baculoviral IAP Repeat-Containing 3 Protein/metabolism , Carbon Tetrachloride Poisoning/metabolism , Gene Expression Regulation, Enzymologic , Liver Cirrhosis/metabolism , Matrix Metalloproteinase 9/biosynthesis , Neutrophils/metabolism , Animals , Baculoviral IAP Repeat-Containing 3 Protein/genetics , Carbon Tetrachloride Poisoning/genetics , Carbon Tetrachloride Poisoning/pathology , Gene Deletion , Humans , Liver Cirrhosis/genetics , Liver Cirrhosis/pathology , Male , Matrix Metalloproteinase 9/genetics , Mice , Mice, Knockout , Neutrophils/pathologyABSTRACT
Chronic liver injury may result in hepatic fibrosis, which can progress to cirrhosis and eventually liver failure. There are no drugs that are specifically approved for treating hepatic fibrosis. The natural product honokiol (HNK), a bioactive compound extracted from Magnolia grandiflora, represents a potential tool in the management of hepatic fibrosis. Though HNK has been reported to exhibit suppressive effects in a rat fibrosis model, the mechanisms accounting for this suppression remain unclear. In the present study, the anti-fibrotic effects of HNK on the liver were evaluated in vivo and in vitro. In vivo studies utilized a murine liver fibrosis model, in which fibrosis is induced by treatment with carbon tetrachloride (CCl4). For in vitro studies, LX-2 human hepatic stellate cells (HSCs) were treated with HNK, and expression of markers of fibrosis, cell viability, the transforming growth factor-ß (TGF-ß1)/SMAD signaling pathway, and autophagy were analyzed. HNK was well tolerated and significantly attenuated CCl4-induced liver fibrosis in vivo. Moreover, HNK decreased HSC activation and collagen expression by downregulating the TGF-ß1/SMAD signaling pathway and autophagy. These results suggest that HNK is a new potential candidate for the treatment of hepatic fibrosis through suppressing both TGF-ß1/SMAD signaling and autophagy in HSCs.
Subject(s)
Autophagy/drug effects , Biphenyl Compounds/pharmacology , Carbon Tetrachloride Poisoning , Hepatic Stellate Cells , Lignans/pharmacology , Liver Cirrhosis , Liver/metabolism , Signal Transduction/drug effects , Smad Proteins/metabolism , Transforming Growth Factor beta1/metabolism , Animals , Carbon Tetrachloride Poisoning/drug therapy , Carbon Tetrachloride Poisoning/metabolism , Carbon Tetrachloride Poisoning/pathology , Hepatic Stellate Cells/metabolism , Hepatic Stellate Cells/pathology , Liver/pathology , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Male , MiceABSTRACT
BACKGROUND Acute chemical liver injury needs to be further explored. The present study aimed to compare the effects of intraperitoneal injection with carbon tetrachloride on acute liver toxicity after 24 h in male and female Kunming mice. MATERIAL AND METHODS In this study, female and male mice were simultaneously divided into 3 different groups. Each group was treated differently, and after 24 h, blood samples were collected to check for changes in the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which were used to assess liver toxicity. Liver samples were used for hematoxylin-eosin staining, and periodic acid Schiff reagent staining was performed to detect the pathological changes of each group. The expression level of biomarker molecules in liver cells was also systematically analyzed. RESULTS Our results showed that, compared with male mice, female mice showed more serious damage: reduced glycogen and higher degree of necrosis, and the levels of heatshock protein 27 (HSP27), heat-shock protein 70 (HSP70), proliferating cell nuclear antigen (PCNA) and B cell lymphoma/lewkmia-2 (Bcl-2) were significantly lower than in the male group (P<0.05 or P<0.01), while the results of Bcl-2-associated X protein (Bax), cysteinyl aspartate specific proteinase 3 (Caspase3), and cytochrome P450 2E1 (CYP2E1) were the opposite (P<0.05 or P<0.01). CONCLUSIONS The findings from this study showed that, compared with male mice, at 24 h after CCl4 toxicity, female mice showed more severe changes of hepatocyte necrosis and PAS-positivity, with significantly reduced expression of HSP27, HSP70, PCNA, and Bcl-2, and significantly increased expression of Bax, caspase-3, and CYP2E1.
Subject(s)
Carbon Tetrachloride Poisoning/diagnosis , Carbon Tetrachloride/toxicity , Chemical and Drug Induced Liver Injury/diagnosis , Animals , Carbon Tetrachloride/administration & dosage , Carbon Tetrachloride Poisoning/etiology , Carbon Tetrachloride Poisoning/pathology , Chemical and Drug Induced Liver Injury/etiology , Chemical and Drug Induced Liver Injury/pathology , Disease Models, Animal , Female , Hepatocytes/drug effects , Hepatocytes/pathology , Humans , Injections, Intraperitoneal , Liver/drug effects , Liver/pathology , Male , Mice , Necrosis/chemically induced , Necrosis/diagnosis , Severity of Illness Index , Sex Factors , Toxicity Tests, Acute/methodsABSTRACT
Zingiber roseum is native to Bangladesh and widely used in folk medicine. This present study was designed to assess the ameliorative potential of Zingiber roseum rhizome extract in carbon tetrachloride (CCl4) induced hepatotoxicity in mice model. Seven phenolic compounds were identified and quantified by HPLC analysis in the plant extract, including quercetin, myricetin, catechin hydrate, trans-ferulic acid, trans-cinnamic acid, (-) epicatechin, and rosmarinic acid. Hepatotoxicity was induced by administrating a single intraperitoneal injection of CCl4 (10 mL/kg) on 7th day of treatment. The results revealed that plant extract at all doses (100, 200 and 400 mg/kg) significantly reduced (p < 0.05) the elevated serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) concentrations, and these effects were comparable to that of standard drug silymarin. Histopathological examination also revealed the evidence of recovery from CCL4 induced cellular damage when pretreated with Z. roseum rhizome extract. The in-vivo hepatoprotective effects were further investigated by the in-silico study of the aforementioned compounds with liver-protective enzymes such as superoxide dismutase (SOD), peroxiredoxin, and catalase. The strong binding affinities (ranging from -7.3359 to -9.111 KCal/mol) between the phenolic compounds (except trans-cinnamic acid) and oxidative stress enzymes inhibit ROS production during metabolism. The compounds were also found non-toxic in computational prediction, and a series of biological activities like antioxidant, anticarcinogen, cardio-protectant, hepato-protectant have been detected.
Subject(s)
Carbon Tetrachloride Poisoning/prevention & control , Chemical and Drug Induced Liver Injury/prevention & control , Polyphenols/chemistry , Polyphenols/pharmacology , Rhizome/chemistry , Zingiberaceae/chemistry , Animals , Carbon Tetrachloride Poisoning/pathology , Catalase/metabolism , Chemical and Drug Induced Liver Injury/enzymology , Chemical and Drug Induced Liver Injury/pathology , Chromatography, High Pressure Liquid , Female , Liver/enzymology , Liver/pathology , Liver Function Tests , Mice , Molecular Docking Simulation , Oxidative Stress/drug effects , Peroxiredoxins/metabolism , Plant Extracts/pharmacology , Protective Agents/pharmacology , Reactive Oxygen Species , Silymarin/therapeutic use , Superoxide Dismutase/metabolismABSTRACT
OBJECTIVE: Dendrobium nobile is a genuine Chinese medicine. Dendrobium nobile Lindl. alkaloids (DNLA) protects against CCl4-induced acute liver injury. This study used RNA-Seq to explore the mechanisms. METHODS: Mice were pretreated with DNLA (10 and 20 mg/kg, po) for 7 days, and subsequently intoxicated with CCl4 (20 µL/kg, ip for 24 h). Liver RNA was extracted and subjected to RNA-Seq. The bioinformatics, including PCA, GO, KEGG, two-dimensional clustering, Ingenuity Pathways Analysis (IPA), and Illumina BaseSpace Correlation Engine (BSCE) were used to analyze the data. qPCR was performed on selected genes to verify RNA-Seq results. RESULTS: DNLA protection against CCl4 hepatotoxicity was confirmed by histopathology. PCA revealed the distinct gene expression patterns between the different treatment groups. GO showed that CCl4 induced the activation, adhesion and proliferation of immune cells. KEGG showed CCl4 induced oxidative stress, diseases and compromised adaptive responses. CCl4 induced differentially expressed genes (DEGs) were identified by DESeq2 with Padj < 0.05 and 2D-clustered with other groups. DNLA reverted CCl4-induced DEGs in a dose-dependent manner. qPCR analysis of S100 g, Sprr1, CCL3/7, Saa2/3, IL1rn, Cox7a2 and Rad15 confirmed RNA-Seq results. IPA showed that CCl4 treatment altered some signaling and metabolic pathways, which were ameliorated or returned to normal following DNLA treatment. The CCl4-activated mitochondrial oxidative phosphorylation was illustrated as an example. IPA Upstream Regulator Analysis further revealed the activated or inhibited molecules and chemicals that are responsible for CCl4-induced DEGs, and DNLA attenuated these changes. BSCE analysis verified that CCl4-induced DEGs were highly correlated with the GEO database of CCl4 hepatotoxicity in rodents, and DNLA dose-dependently attenuated such correlation. CONCLUSION: RNA-Seq revealed CCl4-induced DEGs, disruption of canonical pathways, activation or inhibition of upstream regulators, which are highly correlated with database for CCl4 hepatotoxicity. All these changes were attenuated or returned to normal by DNLA, demonstrating the mechanisms for DNLA to protect against CCl4 hepatotoxicity.
Subject(s)
Alkaloids/chemistry , Alkaloids/therapeutic use , Carbon Tetrachloride Poisoning/drug therapy , Carbon Tetrachloride Poisoning/genetics , Chemical and Drug Induced Liver Injury/drug therapy , Chemical and Drug Induced Liver Injury/genetics , Dendrobium/chemistry , RNA-Seq/methods , Adaptive Immunity/drug effects , Animals , Carbon Tetrachloride Poisoning/pathology , Cell Adhesion/drug effects , Cell Proliferation/drug effects , Chemical and Drug Induced Liver Injury/pathology , Computational Biology , Databases, Genetic , Dose-Response Relationship, Drug , Gene Expression Regulation/drug effects , Liver/chemistry , Liver/pathology , Male , Mice , Mice, Inbred C57BL , Oxidative Phosphorylation/drug effects , Oxidative Stress/drug effectsABSTRACT
SCOPE: The high-fat, high-sucrose, and low-fiber Western diet (WD) is popular in many countries and affects the onset and progression of many diseases. This study is aimed to explore the influence of the WD on chronic liver disease (CLD) and its possible mechanism. METHODS AND RESULTS: C57BL/6 mice are given a control diet (CD) or WD and CLD is induced by intraperitoneally injecting carbon tetrachloride (CCL4 ) twice a week for 8 weeks. The WD aggravated CCL4 -induced chronic liver injury, as evidenced by increased serum transaminase levels, worsened hepatic inflammatory response, and fibrosis. Gut microbiota is disturbed in mice treated with CCL4 +WD (WC group), manifested as the accumulation of Fusobacteria, Streptococcaceae, Streptococcus, Fusobacterium, and Prevotella and the depletion of Firmicutes, Lachnospiraceae, and Roseburia. Additionally, increased hepatic taurocholic acid in the WC group activated sphingosine-1-phosphate receptor 2, which is positively correlated with hepatic fibrosis and inflammation parameters. Mice in the WC group have higher fecal primary bile acid (BA) levels and lower fecal secondary/primary BA ratios. Serum FGF15 levels are also elevated in the WC group, which is positively correlated with hepatic inflammation. CONCLUSION: WD accelerates the progression of CLD which is associated with changes in the gut microbiota and BA metabolism.
Subject(s)
Bile Acids and Salts/metabolism , Chemical and Drug Induced Liver Injury, Chronic/etiology , Diet, Western/adverse effects , Gastrointestinal Microbiome , Animals , Carbon Tetrachloride Poisoning/microbiology , Carbon Tetrachloride Poisoning/pathology , Chemical and Drug Induced Liver Injury, Chronic/metabolism , Chemical and Drug Induced Liver Injury, Chronic/microbiology , Chemical and Drug Induced Liver Injury, Chronic/pathology , Fatty Acids, Volatile/metabolism , Fibroblast Growth Factors/blood , Hepatitis/etiology , Liver Cirrhosis/chemically induced , Liver Cirrhosis/etiology , Liver Cirrhosis/pathology , Male , Mice, Inbred C57BL , Sphingosine-1-Phosphate Receptors/metabolismABSTRACT
Hepatic fibrosis is characterized by abnormal accumulation of extracellular matrix (ECM). Hepatic stellate cells (HSCs) are the primary cells that produce ECM in response to hepatic injury, and transforming growth factor-beta (TGF-ß) has been regarded as the central stimulus responsible for HSC-mediated ECM production. In the present study, we attempted to identify a critical factor in HSC activation and the underlying mechanism. By analyzing online microarray expression profiles, we found that the expression of high-affinity cationic amino acid transporter 1 (CAT1) was upregulated in hepatic fibrosis models and activated HSCs. We isolated and identified mouse HSCs (MHSCs) and found that in these cells, CAT1 was most highly upregulated by TGF-ß1 stimulation in both time- and dose-dependent manners. In vitro, CAT1 overexpression further enhanced, while CAT1 silencing inhibited, the effect of TGF-ß1 in promoting MHSC activation. In vivo, CAT1 silencing significantly improved the hepatic fibrosis induced by both CCl4 and non-alcoholic fatty liver disease (NAFLD). In summary, CAT1 was significantly upregulated in TGF-ß1-activated MHSCs and mice with hepatic fibrosis. CAT1 silencing inhibited TGF-ß1-induced MHSC activation in vitro and fibrogenic changes in vivo. CAT1 is a promising target for hepatic fibrosis treatment that requites further investigation in human cells and clinical practice.
Subject(s)
Calcium Channels/metabolism , Extracellular Matrix/metabolism , Gene Silencing , Hepatic Stellate Cells/metabolism , Liver Cirrhosis/metabolism , TRPV Cation Channels/metabolism , Transforming Growth Factor beta1/metabolism , Animals , Calcium Channels/genetics , Carbon Tetrachloride Poisoning/genetics , Carbon Tetrachloride Poisoning/metabolism , Carbon Tetrachloride Poisoning/pathology , Cell Line , Extracellular Matrix/genetics , Extracellular Matrix/pathology , Hepatic Stellate Cells/pathology , Humans , Liver Cirrhosis/chemically induced , Liver Cirrhosis/genetics , Liver Cirrhosis/pathology , Male , Mice , TRPV Cation Channels/genetics , Transforming Growth Factor beta1/geneticsABSTRACT
Mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-ĸB signaling have been recognized for their causal connection with liver fibrosis. Hence, it is encouraging to discover drugs that can modify the interactions between these signaling cascades. It has been suggested that glucagon-like peptide-1 receptors (GLP-1Rs) might have a role in the observed hepatoprotection of dipeptidyl peptidase-4 inhibitors other than vildagliptin (VLD). Consequently, we aimed to elucidate the mechanisms underlying its potential antifibrotic activity in a CCl4-intoxicated mouse model. VLD increased the percentage of viable CCl4-intoxicated primary rat hepatocytes in vitro. It also attenuated hepatic fibrosis, improved liver function, and prolonged survival of CCl4-intoxicated mice in a dose-dependent manner. This hepatoprotection might be mediated mainly through interference with extracellular signal-regulated protein kinase 1/2 phosphorylation, the most downstream signal of the MAPK pathway. In addition, VLD hepatoprotective activity could be partially mediated through inhibition of p38α phosphorylation and phosphorylation-induced NF-ĸB activation. As a result, VLD downregulated profibrogenic mediators, such as tumor necrosis factor α, transforming growth factor ß, tissue inhibitor of metalloproteinase 1 and platelet-derived growth factor BB. Consequently, decreased expression levels of fibrosis markers, such as hydroxyproline and α smooth muscle actin, were confirmed. VLD showed a strong trend toward increasing the antioxidant defense machinery of fibrotic tissue, and we confirmed that GLP-1Rs were not implicated in the observed hepatoprotection. Since VLD poses little risk of hypoglycemia and is a safe drug for patients with liver injury, it may be a hopeful candidate for adjuvant treatment of liver fibrosis in humans.
Subject(s)
Carbon Tetrachloride Poisoning/drug therapy , Dipeptidyl-Peptidase IV Inhibitors/pharmacology , Liver Cirrhosis/drug therapy , Signal Transduction/drug effects , Vildagliptin/pharmacology , Animals , Carbon Tetrachloride Poisoning/pathology , Cell Survival/drug effects , Dipeptidyl-Peptidase IV Inhibitors/administration & dosage , Dipeptidyl-Peptidase IV Inhibitors/therapeutic use , Dose-Response Relationship, Drug , Inflammation Mediators/metabolism , Liver Cirrhosis/chemically induced , Liver Cirrhosis/pathology , Liver Function Tests , MAP Kinase Signaling System/drug effects , Male , Mice , NF-kappa B/drug effects , Phosphorylation , Primary Cell Culture , Rats , Survival , Vildagliptin/administration & dosage , Vildagliptin/therapeutic use , p38 Mitogen-Activated Protein Kinases/drug effectsABSTRACT
The lack of Lipocalin (LCN2) provokes overwhelming endoplasmic reticulum (ER) stress responses in vitro and in acute toxic liver injury models, resulting in hepatocyte apoptosis. LCN2 is an acute phase protein produced in hepatocytes in response to acute liver injuries. In line with these findings we investigated ER stress responses of Lcn2-/- mice in chronic ER stress using a long-term repetitive carbon tetrachloride (CCl4) injection model. We found chronic CCl4 application to enhance ER stress and unfolded protein responses (UPR), including phosphorylation of eukaryotic initiation factor 2α (eIF2α), increased expression of binding immunoglobulin protein (BiP) and glucose-regulated protein 94 (GRP94). IRE1α/TRAF2/JNK signaling enhanced mitochondrial apoptotic pathways, and showed slightly higher in Lcn2-/- mice compared to the wild type counterparts, leading to increased hepatocyte apoptosis well evidenced by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Hepatocyte injuries were confirmed by significant high serum alanine transaminase (ALT) levels in CCl4-treated Lcn2-/- mice. Lcn2-/- mice furthermore developed mild hepatic steatosis, supporting our finding that ER stress promotes lipogenesis. In a previous report we demonstrated that the pharmacological agent tunicamycin (TM) induced ER stress through altered protein glycosylation and induced high amounts of C/EBP-homologous protein (CHOP), resulting in hepatocyte apoptosis. We compared TM-induced ER stress in wild type, Lcn2-/-, and Chop null (Chop-/-) primary hepatocytes and found Chop-/- hepatocytes to attenuate ER stress responses and resist ER stress-induced hepatocyte apoptosis through canonical eIF2α/GADD34 signaling, inhibiting protein synthesis. Unexpectedly, in later stages of TM incubation, Chop-/- hepatocytes resumed activation of IRE1α/JNK/c-Jun and p38/ATF2 signaling, leading to late hepatocyte apoptosis. This interesting observation indicates Chop-/- mice to be unable to absolutely prevent all types of liver injury, while LCN2 protects the hepatocytes by maintaining homeostasis under ER stress conditions.
Subject(s)
Apoptosis , Carbon Tetrachloride Poisoning/metabolism , Carbon Tetrachloride/toxicity , Endoplasmic Reticulum Stress , Hepatocytes/metabolism , Lipocalin-2/deficiency , Unfolded Protein Response , Animals , Apoptosis/drug effects , Apoptosis/genetics , Carbon Tetrachloride Poisoning/genetics , Carbon Tetrachloride Poisoning/pathology , Endoplasmic Reticulum Stress/drug effects , Endoplasmic Reticulum Stress/genetics , Hepatocytes/pathology , Lipocalin-2/metabolism , Lipogenesis/drug effects , Lipogenesis/genetics , Mice , Mice, Knockout , Signal Transduction/drug effects , Signal Transduction/genetics , Unfolded Protein Response/drug effects , Unfolded Protein Response/geneticsABSTRACT
This study aimed to explore the basic structural features of phosphorylated Pleurotus ostreatus polysaccharide (PPOP) and study the protective effect of PPOP on liver injury induced by carbon tetrachloride in male Kunming mice. The phosphorylated polysaccharide was prepared from the natural polysaccharide extracted from Pleurotus ostreatus (POP). The structures of PPOP and POP were characterized by FT-IR, ESEM spectroscopy, and Congo red test. Chemical composition analysis revealed that PPOP was mainly composed of rhamnose, galacturonic acid, and xylose in a molar ratio of 0.10: 1.98: 1.00. Structural analysis indicated that PPOP had multi-strand structure and the absorption peaks of PO and P-O-C. Furthermore, animal experiments showed that the hepatoprotective effect of PPOP against liver injury was reflected by decreasing the levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total cholesterol, trilaurin, and low-density lipoprotein cholesterol in the serum, increasing the content of high-density lipoprotein cholesterol and albumin in blood, reducing the content of malondialdehyde and promoting the activity of antioxidant enzymes in liver. PPOP exhibited stronger hepatoprotective effect and antioxidant activity in vivo than POP. The final results indicated that PPOP could be used in the treatment of chemical-induced hepatotoxicity based on the above biological research.
Subject(s)
Carbon Tetrachloride Poisoning/drug therapy , Chemical and Drug Induced Liver Injury/drug therapy , Fungal Polysaccharides , Pleurotus/chemistry , Animals , Carbon Tetrachloride Poisoning/metabolism , Carbon Tetrachloride Poisoning/pathology , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/pathology , Fungal Polysaccharides/chemistry , Fungal Polysaccharides/isolation & purification , Fungal Polysaccharides/pharmacology , Male , Mice , Structure-Activity RelationshipABSTRACT
Although the serum-abundant metal-binding protein transferrin (encoded by the Trf gene) is synthesized primarily in the liver, its function in the liver is largely unknown. Here, we generated hepatocyte-specific Trf knockout mice (Trf-LKO), which are viable and fertile but have impaired erythropoiesis and altered iron metabolism. Moreover, feeding Trf-LKO mice a high-iron diet increased their susceptibility to developing ferroptosis-induced liver fibrosis. Importantly, we found that treating Trf-LKO mice with the ferroptosis inhibitor ferrostatin-1 potently rescued liver fibrosis induced by either high dietary iron or carbon tetrachloride (CCl4) injections. In addition, deleting hepatic Slc39a14 expression in Trf-LKO mice significantly reduced hepatic iron accumulation, thereby reducing ferroptosis-mediated liver fibrosis induced by either a high-iron diet or CCl4 injections. Finally, we found that patients with liver cirrhosis have significantly lower levels of serum transferrin and hepatic transferrin, as well as higher levels of hepatic iron and lipid peroxidation, compared with healthy control subjects. Taken together, these data indicate that hepatic transferrin plays a protective role in maintaining liver function, providing a possible therapeutic target for preventing ferroptosis-induced liver fibrosis.
Subject(s)
Ferroptosis/physiology , Iron/metabolism , Liver Cirrhosis/metabolism , Liver/metabolism , Transferrin/physiology , Animals , Carbon Tetrachloride Poisoning/drug therapy , Carbon Tetrachloride Poisoning/metabolism , Carbon Tetrachloride Poisoning/pathology , Cation Transport Proteins/deficiency , Cation Transport Proteins/genetics , Cyclohexylamines/pharmacology , Cytokines/analysis , Erythropoiesis/physiology , Erythropoietin/analysis , Female , Ferroptosis/drug effects , Hepatocytes/metabolism , Homeostasis , Iron Overload/complications , Iron, Dietary/toxicity , Lipid Peroxidation , Liver Cirrhosis/chemically induced , Liver Cirrhosis/drug therapy , Liver Cirrhosis/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle Proteins/analysis , Phenylenediamines/pharmacology , Transferrin/analysisABSTRACT
OBJECTIVES: Curcumol, a guaiane-type sesquiterpenoid hemiketal extracted from the herb Rhizoma Curcumae, exhibits multiple-pharmacological activities. We previously reported that curcumol ameliorated hepatic fibrosis by inhibiting hepatic stellate cell (HSC) activation. In this study, we aimed to investigate the effect of curcumol on HSC migration and adhesion, and reveal its regulation mechanisms. MATERIALS AND METHODS: Cellular viability was determined by Cell Counting Kit-8. Cell migration was detected by boyden chamber and cell scratch experiment. Recombinant human periostin (rh POSTN) and adeno-associated viral (AAV)-GFP-periostin were used to achieve POSTN overexpression in vitro and in vivo, respectively. Nuclear factor kappa B (NF-κB)-p65 overexpression was achieved by using plasmid. ELISA was conducted to detect POSTN level. Immunohistochemistry, qRT-PCR, Western blotting, and immunofluorescence were performed to assess associated factor expression. RESULTS: Curcumol suppressed HSC migration and adhesion, and reduced the secretion and expression of POSTN. By gain of function POSTN in HSCs, using rh POSTN, we found that the inhibition of HSC migration and adhesion by curcumol depended on the decrease of POSTN. Besides, curcumol protection against chronic CCl4-caused hepatic fibrosis could be impaired by POSTN overexpression. Moreover, we showed that curcumol repressed NF-κB signaling and the production of pro-inflammatory factor. Importantly, curcumol down-regulation of POSTN was rescued by knock-in of NF-κB, as well as the inhibition of HSC migration and adhesion. CONCLUSION: These findings reveal the molecular mechanism of curcumol-reduced HSC migration and adhesion, by which points to the possibility of using curcumol based on NF-κB dependent POSTN for the treatment of fibrogenesis.
Subject(s)
Cell Adhesion Molecules/antagonists & inhibitors , Hepatic Stellate Cells/drug effects , Sesquiterpenes/pharmacology , Transcription Factor RelA/antagonists & inhibitors , Animals , Carbon Tetrachloride Poisoning/pathology , Carbon Tetrachloride Poisoning/prevention & control , Cell Adhesion/drug effects , Cell Adhesion Molecules/metabolism , Cell Count , Cell Line , Cell Movement/drug effects , Chemical and Drug Induced Liver Injury/pathology , Chemical and Drug Induced Liver Injury/prevention & control , Down-Regulation/drug effects , Humans , Male , Mice , Mice, Inbred ICR , Recombinant ProteinsABSTRACT
Hepatic fibrosis is a major consequence of chronic liver disease such as non-alcoholic steatohepatitis which is undergoing a dramatic evolution given the obesity progression worldwide, and has no treatment to date. Hepatic stellate cells (HSCs) play a key role in the fibrosis process, because in chronic liver damage, they transdifferentiate from a "quiescent" to an "activated" phenotype responsible for most the collagen deposition in liver tissue. Here, using a diet-induced liver fibrosis murine model (choline-deficient amino acid-defined, high fat diet), we characterized a specific population of HSCs organized as clusters presenting simultaneously hypertrophy of retinoid droplets, quiescent and activated HSC markers. We showed that hypertrophied HSCs co-localized with fibrosis areas in space and time. Importantly, we reported the existence of this phenotype and its association with collagen deposition in three other mouse fibrosis models, including CCl4-induced fibrosis model. Moreover, we have also shown its relevance in human liver fibrosis associated with different etiologies (obesity, non-alcoholic steatohepatitis, viral hepatitis C and alcoholism). In particular, we have demonstrated a significant positive correlation between the stage of liver fibrosis and HSC hypertrophy in a cohort of obese patients with hepatic fibrosis. These results lead us to conclude that hypertrophied HSCs are closely associated with hepatic fibrosis in a metabolic disease context and may represent a new marker of metabolic liver disease progression.
Subject(s)
Carbon Tetrachloride Poisoning , Dietary Fats/adverse effects , Hepatic Stellate Cells , Liver Cirrhosis , Animals , Carbon Tetrachloride Poisoning/metabolism , Carbon Tetrachloride Poisoning/pathology , Dietary Fats/pharmacology , Hepatic Stellate Cells/metabolism , Hepatic Stellate Cells/pathology , Humans , Liver Cirrhosis/chemically induced , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Male , MiceABSTRACT
Carbon tetrachloride (CCl4) is widely used to induce hepatic fibrosis. Therapeutic agents alleviate hepatic fibrosis by inhibiting signal transducer and activator of transcription 3 (STAT3) activation. To understand the direct effects of CCl4 on STAT3 expression in the liver, the present study incubated cultured hepatocytes expressing connective tissue growth factor (CTGF) with CCl4. Rats exposed to CCl4 for 8 weeks exhibited hepatic fibrosis, which was confirmed through the assessment of plasma biomarkers. Isolated liver samples were used to determine the protein levels of CTGF and STAT3 using western blotting. In addition, STAT3 expression was silenced in α mouse liver 12 (AML12) cells using small interfering RNA transfection. In addition, a pharmacological inhibitor, stattic, was used to inhibit STAT3 expression. The incubation of AML12 cells with CCl4 induced a dosedependent increase in CTGF expression and STAT3 activation. Notably, silymarin, an extract from milk thistle, inhibited these changes in AML12 cells and the antioxidant tiron produced similar effects. Silencing of STAT3 reduced the CTGF expression promoted by CCl4 in the hepatocytes. Additionally, similar to tiron, stattic inhibited CTGF expression induced by CCl4. In conclusion, CCl4 may activate STAT3 through oxidative stress to promote CTGF expression, which is one of the main factors contributing to the risk of hepatic fibrosis.
Subject(s)
Carbon Tetrachloride Poisoning/metabolism , Carbon Tetrachloride/toxicity , Connective Tissue Growth Factor/metabolism , Hepatocytes/metabolism , Liver Cirrhosis/metabolism , STAT3 Transcription Factor/metabolism , Animals , Carbon Tetrachloride Poisoning/pathology , Cell Line , Hepatocytes/pathology , Liver Cirrhosis/chemically induced , Liver Cirrhosis/pathology , Male , Rats , Rats, Sprague-DawleyABSTRACT
Schisandrin B (Sch B) and miR-101 family members play critical roles in the pathogenesis of liver fibrosis. However, the relationship between them has not been reported yet. Thus, this study aims to fill this research gap. Results showed that Sch B significantly upregulated the expression of miR-101-5p in HSC-T6 cells. Sch B also increased the expression of miR-101-5p by combined administration of TGF-ß1 and Sch B. Using miR-101-5p inhibitor, we demonstrated that Sch B can target miR-101-5p through the TGF-ß signalling pathway to regulate the proliferation and activation of HSC-T6 cells. A rat model of carbon tetrachloride-induced liver fibrosis was established, and results indicated that Sch B can attenuate liver fibrosis by upregulating the expression of miR-101-5p. In conclusion, Sch B can directly target miR-101 to suppress liver fibrosis. Sch B or miR-101-5p may be used as a therapeutic approach for the prevention and treatment of liver fibrosis.
Subject(s)
Carbon Tetrachloride Poisoning , Lignans/pharmacology , Liver Cirrhosis , MicroRNAs/metabolism , Polycyclic Compounds/pharmacology , Signal Transduction/drug effects , Transforming Growth Factor beta/metabolism , Animals , Carbon Tetrachloride Poisoning/drug therapy , Carbon Tetrachloride Poisoning/metabolism , Carbon Tetrachloride Poisoning/pathology , Cyclooctanes/pharmacology , Humans , Liver Cirrhosis/chemically induced , Liver Cirrhosis/drug therapy , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Male , Rats , Rats, Sprague-DawleyABSTRACT
The active component in cullilawan oil can be synthesized into curcumin analogue product, which has pharmacological activity. The synthesis process by using conventional and microwave methods can produce different isomer products. Different synthesis products and models of animal are used to provide different hepatoprotective effects. The aim of this study was to use the curcumin analogue synthetic products (AKS-k and AKS-m) from cullilawan oil in male mice (Mus musculus L.) liver damage treatment induced by carbon tetrachloride (CCl4). The in vivo method was employed using biochemical of blood and histopathological images of liver cells as indicators. The results showed that the curcumin analogue synthetic product using microwave methods had better pharmacological effects than the conventional method product in terms of the results of blood biochemical analysis and microscopic images of liver cells.
Subject(s)
Chemical and Drug Induced Liver Injury/drug therapy , Cinnamomum/chemistry , Curcumin/analogs & derivatives , Curcumin/pharmacology , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antioxidants/metabolism , Carbon Tetrachloride Poisoning/drug therapy , Carbon Tetrachloride Poisoning/pathology , Chemical and Drug Induced Liver Injury/etiology , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/pathology , Disease Models, Animal , Liver Function Tests/methods , Male , Mice , Plant Bark/chemistry , Plant Oils/chemistryABSTRACT
The in vivo antifibrotic effect of a fucoidan extract (FE) from Sargassum fluitans Borgesen was evaluated in a carbon tetrachloride-induced liver damage model in rats over twelve weeks. Chemical analysis showed the FE to contain carbohydrates, sulfates, uronic acids, protein, phenols, and to have a molecular weight of ~60 kDa. Physiological, biochemical, histological and genetic assays were done. Daily oral administration of FE (50 mg/kg) reduced liver enzymatic activity, liver infiltration of inflammatory cells, collagen fiber deposition and gene expression cytokines such as interleukin beta 1 (IL-ß1), tumor necrosis factor alpha (TNF-α), transforming growth factor beta 1 (TGF-ß1), Smad-3, Smad-2, collagen 1 alpha 1 (col1α1) and tissue inhibitor of metalloproteinase 1 (TIMP-1). It also increased RNA expression of Smad-7 and metalloproteinase 2 and 9 (MMP2 and MMP9). The fucoidan extract exhibited an antifibrotic effect mediated by the inhibiting TGF-ß1/Smad pathway, as well as anti-inflammatory effects.
Subject(s)
Liver Cirrhosis/drug therapy , Plant Extracts/pharmacology , Polysaccharides/chemistry , Sargassum/chemistry , Animals , Carbon Tetrachloride/toxicity , Carbon Tetrachloride Poisoning/drug therapy , Carbon Tetrachloride Poisoning/genetics , Carbon Tetrachloride Poisoning/pathology , Disease Models, Animal , Gene Expression Regulation/drug effects , Humans , Liver/drug effects , Liver Cirrhosis/chemically induced , Liver Cirrhosis/pathology , Plant Extracts/chemistry , Polysaccharides/pharmacology , Rats , Signal Transduction/drug effects , Smad Proteins/genetics , Smad3 Protein/genetics , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta1/geneticsABSTRACT
OBJECTIVES: T-cell immunoglobulin domain and mucin domain-4 (TIM-4) is selectively expressed on antigen-presenting cells (APCs) and modulates various immune responses. However, the role of TIM-4 expressed by Kupffer cells (KCs) in liver fibrosis remains unclear. The present study aimed to explore whether and how TIM-4 expressed by KCs is involved in liver fibrosis. MATERIALS AND METHODS: Mice chronic liver fibrosis models were established and divided into the olive-induced control group, CCL4-induced control group, olive-induced TIM-4 interference group and CCL4-induced TIM-4 interference group. Different techniques were used to monitor the fibrotic effects of TIM-4, including histopathological assays, Western blotting, ELISA and transmission electron microscopy. Additionally, mice liver transplant models were established to determine the fibrotic effects of TIM-4 on fibrosis after liver transplantation (LT). RESULTS: We found that the induction of liver fibrosis by CCL4 was associated with TIM-4 expression in KCs. TIM-4 interference essentially contributed to liver fibrosis resolution. KCs from the TIM-4 interference group had decreased levels of pro-fibrotic markers, reduced TGF-ß1 secretion and inhibited hepatic stellate cell (HSC) differentiation into myofibroblast-like cells. In addition, we used GdCl3 to verify that KCs are the primary source of TGF-ß1 during fibrosis progression. Moreover, KCs from CCL4-induced mice showed increased ROS production, mitophagy activation and TGF-ß1 secretion. However, TIM-4 interference in the KCs inhibited Akt1-mediated ROS production, resulting in the suppression of PINK1, Parkin and LC3-II/I activation and the reduction of TGF-ß1 secretion during liver fibrosis. Additionally, TIM-4 interference potentially attenuated development of fibrosis after LT. CONCLUSIONS: Our findings revealed the underlying mechanisms of TIM-4 interference in KCs to mitigate liver fibrosis.
Subject(s)
Carbon Tetrachloride Poisoning/metabolism , Kupffer Cells/metabolism , Liver Cirrhosis/metabolism , Membrane Proteins/metabolism , Mitophagy/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , Animals , Carbon Tetrachloride/toxicity , Carbon Tetrachloride Poisoning/pathology , Disease Models, Animal , Kupffer Cells/pathology , Liver Cirrhosis/chemically induced , Liver Cirrhosis/pathology , Male , MiceABSTRACT
4-methylumbelliferone (4MU) is an inhibitor of hyaluronan deposition and an active substance of hymecromone, a choleretic and antispasmodic drug. 4MU reported to be anti-fibrotic in mouse models; however, precise mechanism of action still requires further investigation. Here we describe the cellular and molecular mechanisms of 4MU action on CCl4-induced liver fibrosis in mice using NGS transcriptome, Q-PCR and immunohistochemical analysis. Collagen and hyaluronan deposition were prevented by 4MU. The CCl4 stimulated expression of Col1a and αSMA were reduced, while the expression of the ECM catabolic gene Hyal1 was increased in the presence of 4MU. Bioinformatic analysis identified an activation of TGF-beta and Wnt/beta-catenin signaling pathways, and inhibition of the genes associated with lipid metabolism by CCL4 treatment, while 4MU restored key markers of these pathways to the control level. Immunohistochemical analysis reveals the suppression of hepatic stellate cells (HSCs) transdifferentiation to myofibroblasts by 4MU treatment. The drug affected the localization of HSCs and macrophages in the sites of fibrogenesis. CCl4 treatment induced the expression of FSTL1, which was downregulated by 4MU. Our results support the hypothesis that 4MU alleviates CCl4-induced liver fibrosis by reducing hyaluronan deposition and downregulating FSTL1 expression, accompanied by the suppression of HSC trans-differentiation and altered macrophage localization.