CCl4-treated Mice Research Articles

Inositol-Requiring Enzyme 1 Alpha Endoribonuclease Specific Inhibitor STF-083010 Alleviates Carbon Tetrachloride Induced Liver Injury and Liver Fibrosis in Mice.

Accumulating data demonstrated that hepatic endoplasmic reticulum (ER) stress was involved in the pathogenesis of liver fibrosis. Long-term chronic hepatocyte death contributed to liver fibrosis initiation and progression. Previous researches reported that ER stress sensor inositol-requiring enzyme 1 alpha (IRE1α) was first activated in the process of liver fibrosis. STF-083010 was an IRE1α RNase specific inhibitor. This study aimed to explore the effects of STF-083010 on carbon tetrachloride (CCl4)-induced liver injury and subsequent liver fibrosis. Mice were intraperitoneally (i.p.) injected with CCl4 (0.15 ml/kg) for 8 weeks. In STF-083010+CCl4 group, mice were injected with STF-083010 (30 mg/kg, i.p.), twice a week, beginning from the 6th week after CCl4 injection. CCl4 treatment markedly enhanced the levels of serum ALT, TBIL, DBIL and TBA, and STF-083010 had obviously extenuated CCl4-induced exaltation of ALT, DBIL, and TBA levels. CCl4-induced hepatic hydroxyproline and collagen I, major indicators of liver fibrosis, were alleviated by STF-083010. Additionally, CCl4-induced α-smooth muscle actin, a marker for hepatic stellate cells activation, was obviously attenuated in STF-083010-treated mice. Moreover, CCl4-induced upregulation of inflammatory cytokines was suppressed by STF-083010. Mechanistic exploration found that hepatic miR-122 was downregulated in CCl4-treated mice. Hepatic MCP1, CTGF, P4HA1, Col1α1, and Mmp9, target genes of miR-122, were upregulated in CCl4-treated mice. Interestingly, STF-083010 reversed CCl4-induced hepatic miR-122 downregulation. Correspondingly, STF-083010 inhibited CCl4-induced upregulation of miR-122 target genes. This study provides partial evidence that STF-083010 alleviated CCl4-induced liver injury and thus protected against liver fibrosis associated with hepatic miR-122.

Identification of a Novel Bone Marrow Cell-Derived Accelerator of Fibrotic Liver Regeneration Through Mobilization of Hepatic Progenitor Cells in Mice.

Granulocyte colony stimulating factor (G-CSF) has been reported to ameliorate impaired liver function in patients with advanced liver diseases through mobilization and proliferation of hepatic progenitor cells (HPCs). However, the underlying mechanisms remain unknown. We previously showed that G-CSF treatment increased the number of bone marrow (BM)-derived cells migrating to the fibrotic liver following repeated carbon tetrachloride (CCl4 ) injections into mice. In this study, we identified opioid growth factor receptor-like 1 (OGFRL1) as a novel BM cell-derived accelerator of fibrotic liver regeneration in response to G-CSF treatment. Endogenous Ogfrl1 was highly expressed in the hematopoietic organs such as the BM and spleen, whereas the liver contained a relatively small amount of Ogfrl1 mRNA. Among the peripheral blood cells, monocytes were the major sources of OGFRL1. Endogenous Ogfrl1 expression in both the peripheral blood monocytes and the liver was decreased following repeated CCl4 injections. An intrasplenic injection of cells overexpressing OGFRL1 into CCl4 -treated fibrotic mice increased the number of HPC and stimulated proliferation of hepatic parenchymal cells after partial resection of the fibrotic liver. Furthermore, overexpression of OGFRL1 in cultured HPC accelerated their differentiation as estimated by increased expression of liver-specific genes such as hepatocyte nuclear factor 4α, cytochrome P450, and fatty acid binding protein 1, although it did not affect the colony forming ability of HPC. These results indicate a critical role of OGFRL1 in the mobilization and differentiation of HPC in the fibrotic liver, and administration of OGFRL1-expressing cells may serve as a potential regenerative therapy for advanced liver fibrosis. Stem Cells 2019;37:89-101.

Noncanonical farnesoid X receptor signaling inhibits apoptosis and impedes liver fibrosis.

BackgroundHepatocyte is particularly vulnerable to apoptosis, a hallmark of many liver diseases. Although pro-apoptotic mechanisms have been extensively explored, less is known about the hepatocyte-specific anti-apoptotic molecular events and it lacks effective approach to combat hepatocyte apoptosis. We investigated the anti-apoptotic effect and mechanism of farnesoid X receptor (FXR), and strategies of how to target FXR for inhibiting apoptosis implicated in liver fibrosis.MethodsSensitivity to apoptosis was compared between wild type and Fxr−/− mice and in cultured cells. Cell-based and cell-free assays were employed to identify the binding protein of FXR and to uncover the mechanism of its anti-apoptotic effect. Overexpression of FXR by adenovirus-FXR was employed to determine its anti-fibrotic effect in CCl4-treated mice. Specimens from fibrotic patients were collected to validate the relevance of FXR on apoptosis/fibrosis.FindingsFXR deficiency sensitizes hepatocytes to death receptors (DRs)-engaged apoptosis. FXR overexpression, but not FXR ligands, inhibits apoptosis both in vitro and in vivo. Apoptotic stimuli lead to drastic reduction of FXR protein levels, a prerequisite for DRs-engaged apoptosis. Mechanistically, FXR interacts with caspase 8 (CASP8) in the cytoplasm, thus preventing the formation of death-inducing signaling complex (DISC) and activation of CASP8. Adenovirus-FXR transfection impedes liver fibrosis in CCl4-treated mice. Specimens from fibrotic patients are characterized with reduced FXR expression and compromised FXR/CASP8 colocalization.InterpretationFXR represents an intrinsic apoptosis inhibitor in hepatocytes and can be targeted via restoring its expression or strengthening FXR/CASP8 interaction for inhibiting hepatocytes apoptosis in liver fibrosis.FundNational Natural Science Foundation of China.

Targeting hepatic miR-221/222 for therapeutic intervention of nonalcoholic steatohepatitis in mice.

BackgroundEffective targeting therapies for common chronic liver disease nonalcoholic steatohepatitis (NASH) are in urgent need. MicroRNA-targeted therapeutics would be potentially an effective treatment strategy of hepatic diseases. Here we investigated the functional role of miR-221/222 and the therapeutic effects of antimiRs-221/222 in NASH mouse models.MethodsWe generated the miR-221/222flox/flox mice on a C57BL/6 J background and the hepatic miR-221/222 knockout (miR-221/222-LKO) mice. The mice were challenged with the methionine and choline deficient diet (MCDD) or chronic carbon tetrachloride (CCl4) treatment to generate experimental steatohepatitis models. Adenovirus-mediated re-expression of miR-221/222 was performed on the MCDD-fed miR-221/222-LKO mice. The MCDD and control diet-fed mice were treated with locked nucleic acid (LNA)-based antimiRs of miR-221/222 to evaluate the therapeutic effects. Histological analysis, RNA-seq, quantitative PCR and Western blot of liver tissues were carried out to study the hepatic lipid accumulation, inflammation and collagen deposition in mouse models.FindingsHepatic deletion of miR-221/222 resulted in significant reduction of liver fibrosis, lipid deposition and inflammatory infiltration in the MCDD-fed and CCl4-treated mouse models. The hepatic steatosis and fibrosis were dramatically aggravated by miR-221/222 re-expression in MCDD-fed miR-221/222-LKO mice. AntimiRs of miR-221/222 could effectively reduce the MCDD-mediated hepatic steatosis and fibrosis. Systematically mechanistic study revealed that hepatic miR-221/222 controlled the expression of target gene Timp3 and promoted the progression of NASH.InterpretationOur findings demonstrate that miR-221/222 are crucial for the regulation of lipid metabolism, inflammation and fibrosis in the liver. LNA-antimiRs targeted miR-221/222 could reduce steatohepatitis with prominent antifibrotic effect in NASH mice.FundThis work is supported by the Natural Science Foundation of China (81530020, 81390352 to Dr. Ning and 81522032 to Dr. Cao and 81670793 to Dr. Jiang); National Key Research and Development Program (No. 2016YFC0905001 and 2017YFC0909703 to Dr. Cao); the Shanghai Rising-Star Program (15QA1402900 to Dr. Cao); Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant (20171905 to Dr. Jiang).

Protective effects of dietary kaempferol glycoside components from unripe soybean (Edamame, Glycine max L. Merrill. 'Jindai') leaves and their serous metabolite on carbon tetrachloride-induced liver injury mice.

The study investigated the protective effects of kaempferol galactoside (KG) components in mice, which were separated from Jindai soybean leaves (JDL) and mainly composed by two kaempferol galactosides. Further, KG-related metabolites in serum of mice were identified by Tof-MS. Results showed that both JDL and KG prevented the CCl4-induced increases in serum aspartate aminotransferase and serum alanine aminotransferase. Additionally, mice treated with KG had significantly decreased TBARS and TNF-alpha levels, compared to CCl4-treated mice. Serous analysis showed that kaempferol, glucuronidated kaempferol and methylated kaempferol with a glucuronic acid moiety were identified in the serum of mice fed unripe soybean leaves or kaempferol galactosides isolated from the leaves. The results indicated that kaempferol 3-O-galactoside connected to other glycosides via galactose might be hydrolyzed in the gastro-intestinal tract and/or epithelium cells to release kaempferol, followed by glucuronidation and/or methylation in the liver to contribute to a reduction in liver injury. The use of raw leaves containing kaempferol galactosides as food materials may contribute to a reduction in oxidation-related diseases.

Protective Effect of Eckol against Acute Hepatic Injury Induced by Carbon Tetrachloride in Mice.

Several in vitro studies have shown the potential hepatoprotective properties of eckol, a natural phlorotannin derived from the brown alga. However, the in vivo hepatoprotective potential of eckol has not been determined. In this study, we performed an in vivo study to investigate the protective effect of eckol and its possible mechanisms on the carbon tetrachloride (CCl4)-induced acute liver injury model in mice. Results revealed that eckol pre-treatment at the dose of 0.5 and 1.0 mg/kg/day for 7 days significantly suppressed the CCl4-induced increases of alanine transaminase (ALT) and aspartate aminotransferase (AST) levels in serum and meliorated morphological liver injury. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) analysis showed that the number of positive apoptotic hepatocytes in the eckol-treated group was lower than that in the CCl4 model group. Western blotting analysis also demonstrated the enhanced expression of bcl-2 and suppressed expression of cleaved caspase-3 by eckol. The CCl4-induced oxidative stress in liver was significantly ameliorated by eckol, which was characterized by reduced malondialdehyde (MDA) formations, and enhanced superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activities and glutathione (GSH) content. Moreover, the CCl4-induced elevations of pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 were markedly suppressed in the eckol-treated group. However, eckol enhanced the level of IL-10, a potent anti-inflammatory cytokine, and recruited CD11c+ dendritic cells into the liver tissues of CCl4-treated mice. These results indicated that eckol has the protective effect on CCl4-induced acute liver injury via multiple mechanisms including anti-apoptosis, anti-oxidation, anti-inflammation and immune regulation.

MicroRNA-224 down-regulates Glycine N-methyltransferase gene expression in Hepatocellular Carcinoma

Glycine N-methyltransferase (GNMT) is a tumor suppressor for HCC. It is down-regulated in HCC, but the mechanism is not fully understood. MicroRNA-224 (miR-224) acts as an onco-miR in HCC. This study is the first to investigate miR-224 targeting the coding region of GNMT transcript. The GNMT-MT plasmid containing a miR-224 binding site silent mutation of the GNMT coding sequence can escape the suppression of miR-224 in HEK293T cells. Expression of both exogenous and endogenous GNMT was suppressed by miR-224, while miR-224 inhibitor enhanced GNMT expression. miR-224 counteracts the effects of GNMT on the reduction of cell proliferation and tumor growth. The levels of miR-224 and GNMT mRNA showed a significant inverse relationship in tumor specimens from HCC patients. Utilizing CCl4-treated hepatoma cells and mice as a cell damage of inflammatory or liver injury model, we observed that the decreased expression levels of GNMT were accompanied with the elevated expression levels of miR-224 in hepatoma cells and mouse liver. Finally, hepatic AAV-mediated GNMT also reduced CCl4-induced miR-224 expression and liver fibrosis. These results indicated that AAV-mediated GNMT has potential liver protection activity. miR-224 can target the GNMT mRNA coding sequence and plays an important role in GNMT suppression during liver tumorigenesis.

Protective effect of free phenolics from Lycopus lucidus Turcz. root on carbon tetrachloride-induced liver injury in vivo and in vitro.

Protective effect of free phenolics from Lycopus lucidus Turcz. root (FPLR) on CCl4-induced hepatotoxicity in vivo and in vitro was first evaluated. Oral administration of FPLR (100 mg/kg bw) to mice significantly reduced the CCl4-induced elevation of serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, triacylglycerols, total cholesterol, and total bilirubin. FPLR also increased the hepatic GSH contents and antioxidant enzyme activities of SOD and CAT and decreased the hepatic MDA level. Histopathological examinations further confirmed that the FPLR could protect the liver from CCl4-induced damage. Further research indicated that FPLR prevented the DNA fragmentation caused by CCl4 based on TUNEL assay. Moreover, immunohistochemistry staining demonstrated that pretreatment with FPLR significantly inhibited the elevation of hepatic TNF-α, IL-6, IL-8, iNOS, COX-2, and Caspase-3 in CCl4-treated mice. In vitro experiments showed that FPLR remarkably reduced BRL hepatocyte apoptosis and damage caused by CCl4 treatment. These findings indicate that FPLR could be developed as a functional food or medication for therapeutic purpose and prevention of hepatic injury.

Dose-Dependent Antifibrotic Effect of Chrysin on Regression of Liver Fibrosis: The Role in Extracellular Matrix Remodeling.

Liver fibrosis represents an overaccumulation of extracellular matrix (ECM). This study was designed to investigate the effect of chrysin on established ECM overproduction in carbon tetrachloride (CCl4) mouse liver fibrosis. Experimental fibrosis was induced by intraperitoneal injection of 2 mL/kg CCl4 twice a week, for 7 weeks. Mice were orally treated with 3 doses of chrysin (5,7-dihydroxyflavone). For the assessment of the spontaneous reversion of fibrosis, CCl4-treated mice were investigated after 2 weeks of recovery time. Silymarin was used as a standard of liver protection. In fibrotic livers, the results showed the upregulation of collagen I (Col I) and tissue inhibitors of metalloproteinase 1 (TIMP-1) and modulation of matrix metalloproteinases (MMPs), which led to an altered ECM enriched in Col, confirmed as well by electron microscopy investigations. Treatment with chrysin significantly reduced ultrastructural changes, downregulated Col I, and restored TIMP-1/MMP balance, whereas in the group observed for the spontaneous regression of fibrosis, they remained in the same pattern with fibrotic livers. In this study, we have shown chrysin efficacy to attenuate dose-dependent CCl4-stimulated liver ECM accumulation by regulation of MMP/TIMP imbalance and inhibition of Col production. We have shown the dose-dependent chrysin efficiency in attenuation of CCl4-induced liver ECM accumulation by regulation of MMP/TIMP imbalance and inhibition of Col production. Our findings suggest that chrysin oral administration may introduce a new strategy for treating liver fibrosis in humans.

Nephroprotective Effect of Hydroalcoholic Extract Allium jesdianum Boiss against Carbon Tetrachloride Induced Nephrotoxicity via Stress Oxidative in Mice

Background: Nephrotoxicity is one of the most common renal problems that especially occur when the body is exposed to drugs or chemical reagents. Allium jesdianum Boiss is the largest and most important plants genus of onion family that possesses many pharmacological effects. The present study was undertaken to investigate the effect Allium jesdianum Boiss in the treatment of nephrotoxicity induced by carbon tetrachloride (CCl4). Methods: Forty two male mice were randomly divided into six groups; control, sham, CCl4 (1 ml/Kg, i.p) single dose, Allium (500, 1000 and 2000 mg/kg) + CCl4. Blood urea nitrogen (BUN) and serum creatinine (Cr) evaluated in serum. Glutathione (GSH), catalase (CAT), malondialdehyde (MDA) and reactive oxygen species (ROS) were analyzed in kidney tissue homogenate and done the microscopic studies of renal tissue. Results: The results indicated a significant increase of serum BUN and Cr as well as MDA and ROS levels and decrease of GSH and CAT in CCl4 treated mice when compared with the control group (p < 0.001), whereas all studied endpoints were significantly altered in pretreatment with Allium extract when compared with CCl4 treated mice (p < 0.001). Renal histopathology indicated normal appearances reduced in CCl4 treated mice and Allium extract administration improved changes in renal tissue. Conclusion: Administration of the hydroalcoholic extract of Allium jesdianum Boiss could prevent nephrotoxicity induced by CCl4. The protective potential may involve the powerful antioxidant of this plant by eliminating free radicals induced by CCl4.

Celecoxib Does Not Protect against Fibrosis and Inflammation in a Carbon Tetrachloride-Induced Model of Liver Injury.

The cyclooxygenase-2 (COX-2) selective inhibitor celecoxib is widely used in the treatment of pain and inflammation. Celecoxib has been explored as a possible treatment of liver fibrosis with contradictory results, depending on the model. The present study reports the effect of celecoxib in a 5-week carbon tetrachloride (CCl4)-induced liver fibrosis mouse model. Celecoxib alone and in combination with inhibitors of the enzyme-soluble epoxide hydrolase (sEH), as well as a dual inhibitor that targets both COX-2 and sEH, were administered via osmotic minipump to mice receiving intraperitoneal injections of CCl4 Collagen deposition was elevated in the mice treated with both celecoxib and CCl4 compared with the control or CCl4-only groups, as assessed by trichrome staining. Histopathology revealed more extensive fibrosis and cell death in the animals treated with both celecoxib and CCl4 compared with all other experimental groups. Although some markers of fibrosis, such as matrix metalloprotease, were unchanged or lowered in the animals treated with both celecoxib and CCl4, overall, hepatic fibrosis was more severe in this group. Cotreatment with celecoxib and an inhibitor of sEH or treatment with a dual inhibitor of COX-2 and sEH decreased the elevated levels of fibrotic markers observed in the group that received both celecoxib and CCl4 Oxylipid analysis revealed that celecoxib reduced the level of prostaglandin E2 relative to the CCl4 only group. Overall, celecoxib treatment did not decrease liver fibrosis in CCl4-treated mice.

Antioxidant and hepatoprotective effects of purified Rhodiola rosea polysaccharides

In this study, two polysaccharide fractions (RRP1: Mw = 5.5 kDa, and RRP2: Mw = 425.7 kDa) were isolated from Rhodiola rosea to investigate their antioxidation and hepatoprotective effects. Physicochemical analysis showed that RRP1 was composed of mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose with a relative molar ratio of 0.69:0.11:0.15:1:0.51:7.5 and RRP2 was consisted of mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose (relative molar ratio = 0.15:0.19:1.01:0.18:0.47:1). Periodate oxidation and Smith degradation analysis revealed that, in RRP1, part of the arabinose and glucose residues were 1 → 3,6/1 → 3/1 → 2,3/1 → 3,4/1 → 2,4/1 → 2,3,4-linked, and the mannose, rhamnose and galactose residues were 1 → 2,6/1 → 6/1 → 2/1→/1 → 4,6/1 → 4-linked. In RRP2, the rhamnose, glucose and galactose residues were linked by 1 → 3,6/1 → 3/1 → 2,3/1 → 3,4/1 → 2,4/1 → 2,3,4 linkages, and the arabinose and mannose residues were 1 → 2/1 → 6/1 → 4-linked. The methylation analysis confirmed the structure information of the two fractions. Importantly, fraction RRP1 demonstrated stronger antioxidative activities than RRP2 by scavenging DPPH, hydroxyl and superoxide anion radicals in vitro. Correspondently, RRP1 showed more significant effects than RRP2 on decreasing the levels of ALT, AST and MDA, and increasing the GSH, SOD and CAT levels in the CCl4-treated mice. These data demonstrated that the polysaccharide RRP1 could be developed as a promising candidate for preventing and treating liver damage induced by toxic chemicals.

MicroRNA-125b Promotes Hepatic Stellate Cell Activation and Liver Fibrosis by Activating RhoA Signaling

miR-125b is frequently dysregulated in different diseases. Activation of hepatic stellate cells (HSCs) is a critical event during liver fibrogenesis. However, the function and its underlying mechanism of miR-125b in HSC activation and liver fibrosis are still unknown. Here, we showed that miR-125b was upregulated in HSCs, but not in hepatocytes, during hepatic fibrogenesis in vivo and upon culture activation in vitro. Inhibition of miR-125b suppressed the expression of profibrogenic genes in culture-activated primary HSCs and reduced the basal and transforming growth factor β (TGF-β)-induced alpha-smooth muscle actin (α-SMA) expression and cell contraction of the immortalized HSC cell line. In contrast, ectopic expression of miR-125b promoted α-SMA expression and HSC contraction. Moreover, antagonizing miR-125b in vivo significantly alleviated liver fibrosis in CCl4-treated mice. Mechanistically, overexpression of miR-125b in HSCs enhanced RhoA activity by directly targeting StAR-related lipid transfer (START) domain containing 13 (Stard13), a RhoA-specific GTPase-activating protein, whereas knockdown of miR-125b abrogated RhoA activation. Furthermore, inhibition of RhoA or its downstream molecules, Mrtf-A and Srf, attenuated the miR-125b-induced α-SMA expression and HSC contraction. Therefore, our findings identify a miR-125b-Stard13-RhoA-α-SMA signaling cascade in HSCs and highlight its importance in hepatic fibrosis.

Therapeutic effects of serum extracellular vesicles in liver fibrosis

ABSTRACTThe lack of approved therapies for hepatic fibrosis seriously limits medical management of patients with chronic liver disease. Since extracellular vesicles (EVs) function as conduits for intercellular molecular transfer, we investigated if EVs from healthy individuals have anti-fibrotic properties. Hepatic fibrogenesis or fibrosis in carbon tetrachloride (CCl4)- or thioacetic acid-induced liver injury models in male or female mice were suppressed by serum EVs from normal mice (EVN) but not from fibrotic mice (EVF). CCl4-treated mice undergoing EVN therapy also exhibited reduced levels of hepatocyte death, inflammatory infiltration, circulating AST/ALT levels and hepatic or circulating pro-inflammatory cytokines. Hepatic histology, liver function tests or circulating proinflammatory cytokine levels were unaltered in control mice receiving EVN. As determined using PKH26-labelled EVN, principal target cells included hepatic stellate cells (HSC; a normally quiescent fibroblastic cell that undergoes injury-induced activation and produces fibrosis during chronic injury) or hepatocytes which showed increased EVN binding after, respectively, activation or exposure to CCl4. In vitro, EVN decreased proliferation and fibrosis-associated molecule expression in activated HSC, while reversing the inhibitory effects of CCl4 or ethanol on hepatocyte proliferation. In mice, microRNA-34c, -151-3p, -483-5p, -532-5p and -687 were more highly expressed in EVN than EVF and mimics of these microRNAs (miRs) individually suppressed fibrogenic gene expression in activated HSC. A role for these miRs in contributing to EVN actions was shown by the ability of their corresponding antagomirs to individually and/or collectively block the therapeutic effects of EVN on activated HSC or injured hepatocytes. Similarly, the activated phenotype of human LX-2 HSC was attenuated by serum EVs from healthy human subjects and contained higher miR-34c, -151-3p, -483-5p or -532-5p than EVs from hepatic fibrosis patients. In conclusion, serum EVs from normal healthy individuals are inherently anti-fibrogenic and anti-fibrotic, and contain microRNAs that have therapeutic actions in activated HSC or injured hepatocytes.Abbreviations: ALT: alanine aminotransferase; AST: aspartate aminotransferase; CCl4: carbon tetrachloride; CCN2: connective tissue growth factor; E: eosin; EGFP: enhanced green fluorescent protein; EVs: extracellular vesicles; EVF: serum EVs from mice with experimental hepatic fibrosis; EVN: serum EVs from normal mice; H: hematoxylin; HSC: hepatic stellate cell; IHC: immunohistochemistry; IL: interleukin; MCP-1: monocyte chemotactic protein-1; miR: microRNA; mRNA: messenger RNA; NTA: nanoparticle tracking analysis; PCNA: proliferating cell nuclear antigen; qRT-PCR: quantitative real-time polymerase chain reaction; SDS-PAGE: sodium dodecyl sulphate – polyacrylamide gel electrophoresis; αSMA: alpha smooth muscle actin; TAA: thioacetic acid; TG: transgenic; TGF-β: transforming growth factor beta; TEM: transmission electron microscopy; TNFα: tumour necrosis factor alpha.

Protective effect of rutin against carbon tetrachloride-induced oxidative stress, inflammation and apoptosis in mouse kidney associated with the ceramide, MAPKs, p53 and calpain activities

Rutin, a natural flavonoid, possess beneficial health effects. However, its renoprotective effect against carbon tetrachloride (CCl4) induced injury and the underlying mechanism is not clarified. The current study aims is to identify the therapeutic effects of rutin on oxidative stress, inflammation and apoptosis in mouse kidney exposed to CCl4. ICR mice received CCl4 with or without rutin co-administration for one week. Compared with the control group, mice receiving CCl4 alone showed kidney injury as evidenced by elevation in serum biochemical markers, inflammation, caspase-3 activity and apoptosis in kidney, while rutin administration significantly attenuated these pathophysiological changes. Exploration of the underlying mechanisms of its action demonstrated that rutin reduced the ROS, calpain and ceramide levels in mouse kidneys. Rutin significantly decreased the p53, TNF-α, IL-1β activities and mitogen-activated protein kinase (MAPK) phosphorylation in the kidneys. In addition, rutin increased the levels of Bcl-2 protein and reduced levels protein of Bax. Rutin also inhibited the release of cytochrome C from mitochondria in kidneys of the CCl4-treated mice. Taken together, rutin ameliorates CCl4-induced oxidative stress, inflammation and apoptosis through regulating the ceramide, MAPK, p53 and calpain activities and thereby suppressing apoptosis by the mitochondrial pathway.

Metformin attenuates motility, contraction, and fibrogenic response of hepatic stellate cells in vivo and in vitro by activating AMP-activated protein kinase.

AIMTo investigate the effect of metformin on activated hepatic stellate cells (HSCs) and the possible signaling pathways involved.METHODSA fibrotic mouse model was generated by intraperitoneal injection of carbon tetrachloride (CCl4) and subsequent treatment with or without metformin. The level of fibrosis was detected by hematoxylin-eosin staining, Sirius Red staining, and immunohistochemistry. The HSC cell line LX-2 was used for in vitro studies. The effect of metformin on cell proliferation (CCK8 assay), motility (scratch test and Transwell assay), contraction (collagen gel contraction assay), extracellular matrix (ECM) secretion (Western blot), and angiogenesis (ELISA and tube formation assay) was investigated. We also analyzed the possible signaling pathways involved by Western blot analysis.RESULTSMice developed marked liver fibrosis after intraperitoneal injection with CCl4 for 6 wk. Metformin decreased the activation of HSCs, reduced the deposition of ECM, and inhibited angiogenesis in CCl4-treated mice. Platelet-derived growth factor (PDGF) promoted the fibrogenic response of HSCs in vitro, while metformin inhibited the activation, proliferation, migration, and contraction of HSCs, and reduced the secretion of ECM. Metformin decreased the expression of vascular endothelial growth factor (VEGF) in HSCs through inhibition of hypoxia inducible factor (HIF)-1α in both PDGF-BB treatment and hypoxic conditions, and it down-regulated VEGF secretion by HSCs and inhibited HSC-based angiogenesis in hypoxic conditions in vitro. The inhibitory effects of metformin on activated HSCs were mediated by inhibiting the Akt/mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK) pathways via the activation of adenosine monophosphate-activated protein kinase (AMPK).CONCLUSIONMetformin attenuates the fibrogenic response of HSCs in vivo and in vitro, and may therefore be useful for the treatment of chronic liver diseases.

Mogroside IVE attenuates experimental liver fibrosis in mice and inhibits HSC activation through downregulating TLR4-mediated pathways

Liver fibrosis has been emphasized as a serious threat to human health. There is currently no effective clinical drug treatment. Although mogrosides (MGs) have extensive pharmacological effects with minimal toxicity, their effects on liver function, inflammation, matrix metalloproteinases and hepatic stellate cell (HSC) activation remain to be researched. In the current study, we investigated whether mogroside IVE (MGIVE), a main compound isolated from MGs, provided protection against liver fibrosis in mice. MGIVE (25mg/kg) significantly reduced carbon tetrachloride (CCl4)-induced inflammatory infiltration, pro-inflammatory cytokine release, and myeloperioxide (MPO) activity, as well as improved liver function in CCl4-treated mice. Additionally, MGIVE also significantly impaired CCl4-induced increases in liver fibrotic marker expression, such as collagen type I and hypoxia inducible factor-1α (HIF-1α). Further investigation indicated that the possible molecular target of MGIVE is the toll-like receptor 4 (TLR4)-mediated pathway, and MGIVE treatment significantly prevented CCl4-induced transforming growth factor-β1 (TGF-β1) overexpression and the phosphorylation of mitogen activated protein kinase (MAPK) in vivo. In vitro tests of HSCs or RAW 264.7 cells challenged with TGF-β1 or lipopolysaccharide (LPS) demonstrated that TLR4 expression partly mediated the anti-fibrotic effects of MGIVE. In conclusion, supplementation with MGIVE may attenuate liver fibrosis through inhibiting the TLR4 signaling pathway, including MyD88 and MAPKs, as well as HIF-1α. MGIVE may act as a therapeutic potential drug for the treatment of liver fibrosis via the TLR4/HIF-1α cohort signaling pathway.

TIPE2 attenuates liver fibrosis by reversing the activated hepatic stellate cells

TIPE2, the tumor necrosis factor (TNF)-α-induced protein 8-like 2 (TNFAIP8L2), plays an important role in regulating inflammation and immune homeostasis. Recent studies discovered that TIPE-2 involved in the development of several tumors and other proliferative diseases. The purpose of this study was to explore the function of TIPE-2 in the activation and proliferation in HSC-T6 cells. Our study showed low expression of TIPE-2 in primary HSCs from CCl4-treated mice and activated HSC-T6 cells. Functionally, over-expression of TIPE-2 by GV141-TIPE-2 hindered the HSC-T6 cells activation and proliferation and expressions of β-Catenin, Cmyc, Cyclin D1. However, inhibition TIPE-2 expression by TIPE-2 siRNA showed the opposite effect. These observations revealed that TIPE-2 held a protective effect on liver fibrosis and could be a potential therapeutic target.

MiR-185 Inhibits Fibrogenic Activation of Hepatic Stellate Cells and Prevents Liver Fibrosis.

Recent studies have shown the effect of microRNAs on HSC activation and transformation, which is essential for the pathogenesis of liver fibrosis. In our study, we explored the role of miR-185 in liver fibrosis. Plasma miR-185 was detected in hepatitis B virus-related liver fibrosis patients (S2/3, n = 10) by Illumina HiSeq sequencing, and healthy volunteers were selected (n = 8) as the control group. We found that the plasma miR-185 level in fibrosis patients was significantly downregulated. CCl4-induced fibrosis tissues in mouse livers and TGF-β1-activated HSCs also presented downregulated miR-185 concomitant with an increased expression of RHEB and RICTOR. To explore the correlations, LX-2 cells were transiently transfected with miR-185 mimics. The expression levels of α-SMA, collagen I, and collagen III were decreased as well as RHEB and RICTOR. Inhibition of endogenous miR-185 increased fibrogenic activity. Furthermore, dual-luciferase reporter assays indicated that miR-185 inhibited the expression of RHEB and RICTOR by directly targeting their 3′ UTRs. Moreover, silencing RHEB and RICTOR suppressed α-SMA and collagen expression levels. In conclusion, miR-185 prevents liver fibrogenesis by inhibiting HSC activation via inhibition of RHEB and RICTOR. These results provide new insights into the mechanisms behind the anti-fibrotic effect of miR-185.

CM-101: Type I Collagen-targeted MR Imaging Probe for Detection of Liver Fibrosis.

Purpose To evaluate the biodistribution, metabolism, and pharmacokinetics of a new type I collagen-targeted magnetic resonance (MR) probe, CM-101, and to assess its ability to help quantify liver fibrosis in animal models. Materials and Methods Biodistribution, pharmacokinetics, and stability of CM-101 in rats were measured with mass spectrometry. Bile duct-ligated (BDL) and sham-treated rats were imaged 19 days after the procedure by using a 1.5-T clinical MR imaging unit. Mice were treated with carbon tetrachloride (CCl4) or with vehicle two times a week for 10 weeks and were imaged with a 7.0-T preclinical MR imaging unit at baseline and 1 week after the last CCl4 treatment. Animals were imaged before and after injection of 10 µmol/kg CM-101. Change in contrast-to-noise ratio (ΔCNR) between liver and muscle tissue after CM-101 injection was used to quantify liver fibrosis. Liver tissue was analyzed for Sirius Red staining and hydroxyproline content. The institutional subcommittee for research animal care approved all in vivo procedures. Results CM-101 demonstrated rapid blood clearance (half-life = 6.8 minutes ± 2.4) and predominately renal elimination in rats. Biodistribution showed low tissue gadolinium levels at 24 hours (<3.9% injected dose [ID]/g ± 0.6) and 10-fold lower levels at 14 days (<0.33% ID/g ± 12) after CM-101 injection with negligible accumulation in bone (0.07% ID/g ± 0.02 and 0.010% ID/g ± 0.004 at 1 and 14 days, respectively). ΔCNR was significantly (P < .001) higher in BDL rats (13.6 ± 3.2) than in sham-treated rats (5.7 ± 4.2) and in the CCl4-treated mice (18.3 ± 6.5) compared with baseline values (5.2 ± 1.0). Conclusion CM-101 demonstrated fast blood clearance and whole-body elimination, negligible accumulation of gadolinium in bone or tissue, and robust detection of fibrosis in rat BDL and mouse CCl4 models of liver fibrosis. © RSNA, 2017 Online supplemental material is available for this article.