CCl4-induced Liver Injury Model Research Articles

Hepatic recruitment of myeloid-derived suppressor cells upon liver injury promotes both liver regeneration and fibrosis

BackgroundThe liver regeneration is a highly complicated process depending on the close cooperations between the hepatocytes and non-parenchymal cells involving various inflammatory cells. Here, we explored the role of myeloid-derived suppressor cells (MDSCs) in the processes of liver regeneration and liver fibrosis after liver injury.MethodsWe established four liver injury models of mice including CCl4-induced liver injury model, bile duct ligation (BDL) model, concanavalin A (Con A)-induced hepatitis model, and lipopolysaccharide (LPS)-induced hepatitis model. The intrahepatic levels of MDSCs (CD11b+Gr-1+) after the liver injury were detected by flow cytometry. The effects of MDSCs on liver tissues were analyzed in the transwell co-culture system, in which the MDSCs cytokines including IL-10, VEGF, and TGF-β were measured by ELISA assay and followed by being blocked with specific antibodies.ResultsThe intrahepatic infiltrations of MDSCs with surface marker of CD11b+Gr-1+ remarkably increased after the establishment of four liver injury models. The blood served as the primary reservoir for hepatic recruitment of MDSCs during the liver injury, while the bone marrow appeared play a compensated role in increasing the number of MDSCs at the late stage of the inflammation. The recruited MDSCs in injured liver were mainly the M-MDSCs (CD11b+Ly6G−Ly6Chigh) featured by high expression levels of cytokines including IL-10, VEGF, and TGF-β. Co-culture of the liver tissues with MDSCs significantly promoted the proliferation of both hepatocytes and hepatic stellate cells (HSCs).ConclusionsThe dramatically and quickly infiltrated CD11b+Gr-1+ MDSCs in injured liver not only exerted pro-proliferative effects on hepatocytes, but also accounted for the activation of profibrotic HSCs.

Novel green-based polyglycerol polymeric nanoparticles loaded with ferulic acid: A promising approach for hepatoprotection

In the pursuit of eco-friendly and sustainable materials, polyglycerol diacid polymers hold immense promise for drug delivery compared to those derived from fossil fuels. Harnessing this potential, we aimed to prepare nanoparticles (NPs) derived from sustainable polymers, loaded with ferulic acid (FA), a natural polyphenolic compound known for its shielding effect against liver-damaging agents, including carbon tetrachloride (CCl4). Glycerol was esterified with renewable monomers, such as succinic acid, adipic acid, and/or FA, resulting in the creation of a novel class of polyglycerol diacid polymers. Characterization via Fourier-transform infrared spectroscopy and nuclear magnetic resonance confirmed the successful synthesis of these polymers with <7 % residual monomers. FA-loaded NPs were fabricated using the newly synthesized polymers. To further augment their potential, the NPs were coated with chitosan. The chitosan-coated NPs boasted an optimal PS of 290 ± 5.03 nm, showing superior physical stability, and a commendable EE% of 58.79 ± 0.43%w/v. The cytotoxicity was examined on fibroblast cells using the SRB assay. In-vivo experiments employing a CCl4-induced liver injury model yielded compelling evidence of the heightened hepatoprotective effects conferred by chitosan-coated particles. This demonstrates the benefits of incorporating sustainable polymers into innovative composites for efficient drug delivery, indicating their potential for creating versatile platforms for various therapeutic applications.

Comparison of the hepatoprotection of intragastric and intravenous cyanidin-3-glucoside administration: focus on the key metabolites and gut microbiota modulation.

Liver injury is a life-threatening condition, and the hepatoprotective potential of cyanidin-3-glucoside (C3G) has been previously demonstrated. However, due to the low bioavailability, it has been doubtful that relatively low concentrations of intact C3G in vivo could account for these bioactivities. In this study, the hepatoprotective effects of intragastric and intravenous administration of C3G were investigated in a CCl4 induced liver injury model. Intragastric C3G administration was more effective than intravenous C3G injection in reducing serum damage biomarkers, oxidative stress, and inflammatory responses, indicating that absorption of C3G into the bloodstream does not fully account for its observed benefits in vivo. Furthermore, intragastric C3G administration modulated the gut microbiota structure and increased the contents of five metabolites in the feces and serum with high inter-individual variation, indicating the key role of the interaction between C3G and the gut microbiota. At equivalent doses, the metabolites cyanidin and protocatechuic acid exhibited greater efficacy than C3G in reducing apoptosis and ROS production by activating the Nrf2 pathway in an AAPH-induced oxidative stress model. To achieve the desired health effects via C3G-rich food intake, more attention should be paid to microbially derived catabolites. Screening of specific metabolite-producing strains will help overcome individual differences and enhance the health-promoting effects of C3G.

Saikosaponin d Alleviates Liver Fibrosis by Negatively Regulating the ROS/NLRP3 Inflammasome Through Activating the ERβ Pathway.

Background and aims: Saikosaponin d (SSd) has a steroidal structure and significant anti-inflammatory effects. The purpose of this study was to explore the mechanism underlying SSd’s inhibitory effects on liver fibrosis. Methods: Wild-type and estrogen receptor knockout (ERKO) mice were treated with CCl4 to establish liver fibrosis mouse models. The effects of SSd on hepatic fibrogenesis were studied in these mouse models. Hepatic stellate cells (HSCs) were activated by H2O2 to investigate the potential molecular mechanisms. The establishment of the models and the degrees of inflammation and liver tissue fibrosis were evaluated by detecting changes in serum liver enzymes and liver histopathology. The expression of α-SMA and TGF-β1 was determined by immunohistochemistry. The expression and significance of NLRP3 inflammasome proteins were explored by RT-PCR and Western blotting analyses. The mitochondrial ROS-related indexes were evaluated by MitoSOX Red. Results: In wild-type and ERKO mice treated with CCl4, the fluorescence expression of mitochondrial ROS was up-regulated, while the mitochondrial membrane potential and ATP content were decreased, suggesting that the mitochondria were damaged. In addition, the expression of NLRP3 inflammatory bodies and fibrosis markers (α-SMA, TGF-β, TIMP-1, MMP-2, and Vimentin) in liver tissue increased. Furthermore, the above indexes showed the same expression trend in activated HSCs. In addition, the peripheral serum ALT and AST levels increased in CCl4-induced liver injury model mice. And HE staining showed a large number of inflammatory cell infiltration in the liver of model mice. Picric acid-Sirius staining and Masson staining showed that there was significant collagen fibrous tissue deposition in mice liver sections. IHC and WB detection confirmed that the expression of α-SMA and TGF-β1 increased. Liver fibrosis scores were also elevated. Then, after SSd intervention, the expression of ROS in wild-type mice and αERKO mice decreased, mitochondrial membrane potential recovered, ATP level increased, NLRP3 inflammasome and fibrosis indexes decreased, liver enzyme levels decreased, and liver pathology showed liver inflammation. The damage and collagen deposition were significantly relieved, the expression of α-SMA and TGF-β1 was decreased, and the fibrosis score was also decreased. More importantly, the effect of SSd in alleviating liver injury and liver fibrosis had no effect on βERKO mice. Conclusion: SSd alleviated liver fibrosis by negatively regulating the ROS/NLRP3 inflammasome through activating the ERβ pathway. By establishing liver fibrosis models using wild-type and ERKO mice, we demonstrated that SSd could alleviate liver fibrosis by inhibiting the ROS/NLRP3 inflammasome axis through activating the ERβ pathway.

Datura stramonium Leaf Extract Exhibits Anti-inflammatory Activity in CCL4-Induced Hepatic Injury Model by Modulating Oxidative Stress Markers and iNOS/Nrf2 Expression.

Background Inflammation is a frequent phenomenon in the pathogenesis of hepatic disorders leading to fibrosis and cirrhosis. Phytopharmaceuticals developed from traditional medicine can provide effective therapeutic alternatives to conventional medications. Datura stramonium (DS) has reported traditional uses in inflammatory diseases. In this study, we have tried to validate its potential as a source of anti-inflammatory agents. Methods Powdered leaf part of DS was extracted using ethyl acetate (EA) to provide the extract (DSL-EA). Lymphocyte and macrophage viability and acute toxicity assays established the safety profile, while nitric oxide (NO) scavenging assay estimated the in vitro anti-inflammatory potential. Noninvasive anti-inflammatory, antidepressant, and antinociceptive activities were monitored using BALB/c mice using low and high doses (150 and 250 mg/kg). Major inflammatory studies were performed on Sprague-Dawley male rats using CCl4-induced liver injury model. Disease induction was initiated by intraperitoneal injections of CCl4 (1 mL/kg of 30% CCl4 in olive oil). The rats were divided into six groups. The anti-inflammatory potential of DSL-EA in low and high doses (150 and 300 mg/kg, respectively) was assessed through hematological, biochemical, liver antioxidant defense, oxidative stress markers, and histological studies as well as the expression of Nrf2 and iNOS. Results DSL-EA exhibited prominent in vitro NO scavenging (IC50: 7.625 ± 0.51 μg/mL) and in vivo anti-inflammatory activity in paw and anal edema models. In CCl4 model, hematological investigations revealed vasotonic effects. Liver functionality was significantly (P < 0.001 − 0.05) improved in DSL-EA-treated rats. The activity level of endogenous antioxidant enzymes in liver tissues was improved in a manner identical to silymarin. The extract reduced the percent concentration of oxidative stress markers in liver tissues. Furthermore, DSL-EA displayed restorative effects on histological parameters (H and E and Masson's trichrome staining). Immunohistochemistry studies showed marked decline in Nrf2 expression, while overexpression of iNOS was also observed in disease control rats. The damage was distinctly reversed by the extract.

Pt(II)-NHC Complex Induces ROS-ERS-Related DAMP Balance to Harness Immunogenic Cell Death in Hepatocellular Carcinoma.

Immunogenic cell death (ICD) can engage a specific immune response and establish a long-term immunity in hepatocellular carcinoma (HCC). Herein, we design and synthesize a series of Pt(II)-N-heterocyclic carbene (Pt(II)-NHC) complexes derived from 4,5-diarylimidazole, which show strong anticancer activities in vitro. Among them, 2c displays much higher anticancer activities than cisplatin and other Pt(II)-NHC complexes, especially in HCC cancer cells. In addition, we find that 2c is a type II ICD inducer, which can successfully induce endoplasmic reticulum stress (ERS) accompanied by reactive oxygen species (ROS) generation and finally lead to the release of damage-associated molecular patterns (DAMPs) in HCC cells. Importantly, 2c shows a great anti-HCC potential in a vaccination mouse model and leads to the in vivo immune cell activation in the CCl4-induced liver injury model.

4-OI Attenuates Carbon Tetrachloride-Induced Hepatic Injury via Regulating Oxidative Stress and the Inflammatory Response.

The liver is an important metabolic organ, and acute liver injury (ALI) is potentially lethal. Itaconate, a metabolic intermediate from the tricarboxylic acid cycle, showed emerging anti-oxidative and anti-inflammation properties, and an accumulating protective effect in multiple diseases, but its role in ALI still needs to be further explored. Here we established an ALI model induced by carbon tetrachloride in mice. Our results showed that 4-Octyl itaconate (OI), a derivate of itaconate, mitigated hepatic damage by improving liver function, reducing histopathological damage, and decreasing the death of hepatocytes. Additionally, OI decreased myeloperoxidase and thiobarbituric acid reactive substances (TBARS) levels in the ALI model. OI also inhibited the inflammatory response by reducing pro-inflammatory cytokine secretion (IL-6, TNF-α, IL-1β, and MCP-1) and infiltration of macrophages and neutrophils in the ALI model. However, administration of ML385, a specified Nrf2 inhibitor, eliminated the protective properties of OI in the CCl4-induced liver injury model by increasing hepatic damage and oxidative stress. Furthermore, OI increased the expression and nuclear translocation of Nrf2 and elevated the expression of heme oxygenase-1 and NAD(P)H quinone oxidoreductase 1, while knockdown of Nrf2 eliminated these effects in murine hepatocyte NCTC 1469 under CCl4 treatment. Moreover, we found that OI reduced serum High-mobility group box 1 (HMGB1) levels in CCl4-treated mice. Finally, OI inhibited nuclear translocation of factor-kappa B (NF-𝜅B) and inflammatory cytokine production in murine macrophages. In conclusion, these results indicated that OI ameliorated CCl4-induced ALI by mitigating oxidative stress and the inflammatory response. The possible mechanism was associated with the elevation of Nrf2 nuclear translocation and inhibition of HMGB1 mediated the nuclear translocation of NF-𝜅B.

Antioxidant activities of anastatin A & B derivatives and compound 38c's protective effect in a mouse model of CCl4-induced acute liver injury.

Anastatins A and B, two flavonoid compounds isolated from desert plant Anastatica hierochuntica, have protective activities for primary rat hepatocytes. Anastatins A and B, and their derivatives, were synthesized by our group previously. In this study, the antioxidant activity and cytotoxicity of these compounds were studied using chemical assessment methods, cell proliferation inhibition experiments, and cell oxidative damage models. The best compound, 38c, was used to study the hepatoprotection activity and mechanism by using a CCl4-induced liver injury model in mice. The results show that most of these flavonoid compounds have good antioxidant activity and low cytotoxicity in vitro. Among them, the most potent compound was 38c, which exhibited a protective effect on CCl4-induced hepatic injury by suppressing the amount of CYP2E1. These findings indicate that anastatin flavonoid derivatives have potential therapeutic utility against oxidative hepatic injury.

A high-throughput targeted metabolomics method for the quantification of 104 non-polar metabolites in cholesterol, eicosanoid, and phospholipid metabolism: application in the study of a CCl4-induced liver injury mouse model.

Much evidence suggested that cholesterol, eicosanoid and phospholipid metabolism plays crucial roles in inflammation, atherosclerosis, carcinogenesis, etc. Therefore, fast and accurate quantification of the metabolites in these metabolic pathways is necessary for discovering the molecular mechanisms and biomarkers of related diseases. In this assay, ultra-high performance liquid chromatography combined with triple quadrupole mass spectrometry platform (UPLC-QqQ-MS) based protocols were developed to simultaneously quantify a total of 104 key metabolites including 32 phospholipids (PLs), 44 eicosanoids (EAs), 28 oxysterols and bile acids (BAs), within 15 minutes. Validation results showed that this method is stable, sensitive and accurate for analyzing different matrix samples. Next, this method was used to characterize the metabolic phenotype of a CCl4-induced liver injury model. The results showed that polyunsaturated fatty acids (PUFA) and PUFA acyl-phospholipids (PFA-PLs) were down-regulated and the levels of saturated fatty acyl-phospholipids (SFA-PLs) and EAs were up-regulated in both the liver tissue and plasma of the CCl4-injury group. BAs were up-regulated in plasma, but down-regulated in the liver tissue of the CCl4-injury group. Immunohistochemistry assay demonstrated that the expression levels of cytosolic phospholipase A2 (cPLA2), phosphorylated cytosolic phospholipase A2 (p-cPLA2), secreted phospholipase A2-IIA (sPLA2-IIA) and lysophosphatidylcholine acyltransferase 1 (LPCAT1) were all up-regulated. According to our results, we drew a diagram of the CCl4-induced acute liver injury molecular mechanism. Moreover, we found that the area under the receiver operating characteristic curve (AUC) of 7α-hydroxycholesterol and 7β-hydroxycholesterol was 1.0, which indicates that the two metabolites have significant potential for the diagnosis of acute liver injury. The outstanding performance of this analytical method proves its further usefulness for mechanism studies and biomarker screening of related diseases.

Clove (Syzygium aromaticum) and honey extracts significantly reduce inflammatory cytokines and liver function enzymes in experimental rats fed on carbon tetrachloride (CCl4)

Liver is the key organ (detoxifier) that gets injured by reactive oxygen species (ROS) which deplete the body's stores of anti-oxidants. ROS is present in all chronic liver diseases leading to oxidative stress. Consumption of foods high in anti-oxidants replenishes and supplies the body with important anti-oxidants which break down ROS lowering inflammatory responses and preventing liver damage. In this work, clove and honey, which are known to be rich in anti-oxidants extracts were evaluated for their potential to breakdown ROS, Reactive nitrogen species (RNS) and lower oxidative stress in rats. Purified extracts were tested in vitro for ROS scavenging by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and in vivo using CCl4-induced liver injury model. A total of 50 experimental rats were assigned to 5 groups each consisting of 10 animals and treated as follows; 1. CCl4 group: fed on CCl4 in olive oil 2. honey group: fed CCl4 then honey extract, 3. clove group: CCl4 then clove extract, 4. double negative control group: rats fed on standard diet and 5 Negative control: fed olive oil, At 10 weeks, liver histopathology was done and sera collected for liver and kidney function tests. CCl4 -only treated rats showed fibroblastic cells proliferation and inflammatory cells infiltration which was absent in the control animals. Clove extract did not exhibit fibroblastic cells proliferation at all. Both clove and honey significantly reduced cytokines (TGF-β, TNF-α and EGF) and liver function enzymes'(ALT, AST and GGT) activity (58.5,56; 119,121 and 6.1; 6.3) compared to the CCl4 group (97; 191 and 10.1). Clove highly inhibited DPPH (77.2%) compared to honey (6.5%) and conferred better liver protection against CCl4. These findings indicate that clove and honey contain compounds that have anti-oxidant activity with the ability to reverse liver fibrosis. Frequent consumption of these foods may lower liver damage and prevent development of some liver diseases. Public health education on nutritional benefits of these foods is strongly encouraged.

Oxidative stress and inflammatory responses in the liver of swamp eel (Monopterus albus) exposed to carbon tetrachloride

“Liver and gall syndrome” disease often caused dramatic economic losses in swamp eel (Monopterus albus) which is one of the most economically important freshwater aquaculture fish in China. Carbon tetrachloride (CCl4)-induced liver injury model has been evidenced as a useful method to screen the hepatoprotective agents to study and prevent this disease in fish. Therefore, in order to provide a better research platform for prevention and treatment liver diseases in swamp eel, we evaluated the hepatotoxicity effects of CCl4 on swamp eel based on the acute toxicity and biochemical responses of glutamate pyruvate transaminase (GPT), glutamate oxalate transaminase (GOT), lactate dehydrogenase (LDH), catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MDA), as well as mRNA expression levels of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO). The 96 h LD50 value of CCl4 in swamp eel was 2.54 g/kg. Sublethal levels of CCl4 induced time-depended liver injury in swamp eel, evidenced by the significant increases of serum GPT, GOT and LDH levels at the first 48 h injection, but restoration after 96 h injection. CCl4 led to oxidative stress, supported by the enhanced of MDA contents, and decreased of SOD and CAT activities. Moreover, CCl4 caused hepatic inflammatory reaction, evidenced by the significant changes of the transcription of COX and 5-LO genes in liver tissues. Overall, our results suggested that lipid peroxidation, antioxidant enzymes and COX and 5-LO pathways are important mechanisms involved in CCl4-induced liver injury, and could be used as the effective biomarkers of CCl4-induced liver injury model to screen the hepatoprotective agents in swamp eel.

Eosin fluorescence: A diagnostic tool for quantification of liver injury

Hepatitis is one of the most common life threatening diseases. The diagnosis is mainly based on biochemical analysis such as liver function test. However, histopathological evaluation of liver serves far better for more accurate final diagnosis. The goal of our study was to evaluate the eosin fluorescence pattern in CCl4-induced liver injury model compared with normal and different treatment groups. For this purpose, liver tissues were stained with H/E and examined under bright field microscope but the fluorescence microscopy of H/E stained slides provided an interesting fluorescence pattern and was quite helpful in identifying different structures. Interesting fluorescence patterns were obtained with FITC, Texas Red and Dual channel filter cubes that were quite helpful in identifying different morphological features of the liver. During the course of hepatic injury, liver cells undergo necrosis, apoptosis and overall cellular microenvironment is altered due to the modification of proteins and other intracellular molecules. Intensified eosin fluorescence was observed around the central vein of injured liver compared to normal indicating enhanced binding of eosin to the more exposed amino acid residues. To conclude, eosin fluorescence pattern varies with the health status of a tissue and can be used further for the diagnosis and quantification of severity of various liver diseases.

Mice with Platelet/Megakaryocyte-Specific Deletion of TGF-β1 Are Partially Protected from Liver Fibrosis in a CCl4-Induced Liver Injury Model

Platelets contain a high concentration of transforming growth factor β1 (TGFβ1). Platelet TGFβ1 has been shown to contribute to plasma TGFβ1 levels and resultant cardiac fibrosis and systolic dysfunction in a murine pressure overload model. TGFβ1 has also been reported to be involved in the development of liver fibrosis, but the cellular source of TGFβ1 was not known. In this study, we tested whether platelet TGFβ1 contributes to liver fibrosis by comparing three groups of mice: 1) mice with platelet-specific deletion of TGFβ1 (PF4Cre/Tgfb1flox/flox), 2) littermate control Tgfb1flox/flox, and 3) WT C57Bl/6 mice. Mice were injected with CCl4 (1ml/kgBW) with peanut oil (1:100) i.p. 2 times a week for 6 weeks. CCl4-induced liver injury was evaluated by in situ hybridization of two liver-specific genes, albumin and Cyp2e, which are expressed in periportal and pericentral areas of the liver, respectively. Liver fibrosis was quantified by slide stitching picrosirius red stained whole liver sections under polarized light. Total collagen content in liver homogenate was evaluated by hydroxyproline assay. Blood was collected before and after CCl4 injections by retrobulbar puncture method into tubes containing 0.1 vol of 3.8% Na-citrate containing10µM PGE1to minimize platelet activation in vitro. TGFβ1 levels in plasma were measured by ELISA.PF4Cre/Tgfb1flox/flox mice had > 90% lower levels of total TGFβ1 in their platelets and ~50% lower plasma TGFβ1 levels than WT or littermate control mice, (TGFβ1 levels in 109 platelets/ml were 73 ± 10 ng in WT and 4.0 ± 1.0 ng in PF4CreTgfb1flox/flox mice, p<0.0001; and in plasma 2.5 ± 0.7 ng/ml in WT and 1.3 ± 0.2 ng/ml in PF4CreTgfb1flox/flox; p<0.001). Plasma TGFβ1 levels increased 24 hours after CCl4 injection in WT (3 ng/ml before and 6 ng/ml after CCl4 injection), but not in PF4Cre/Tgfb1flox/flox mice (1.2 ng/ml before and 1.5 ng/ml after CCl4 injection). In situ hybridization showed high expression of Cyp2e gene primarily in the pericentral vein area where damage occurred within 3 days after the first CCl4 injection. However, albumin gene expression was normal in all non-damaged periportal areas. Liver fibrosis was ~ 20% less in PF4Cre/Tgfb1flox/flox mice (n=11) compared to WT (n=14), or littermate control mice as quantified by both picrosirius red staining and hydroxyproline assays (picrosirius staining area fraction was 2.6 ± 0.4 % in PF4Cre/Tgfb1flox/flox, 3.2 ± 0.6 % in WT, and 3 ± 0.4 % in littermate controls; p=0.004 between WT and PF4Cre and P=0.07 for littermate control vs. PF4Cre). There was a lower trend in total collagen content in PF4CreTgfb1flox/flox mice compared to combined controls mice (collegen in PF4Cre/Tgfb1flox/flox was 0.8 ± 0.1 ng/ml and 1.6 ± 1 ng/ml in controls (p=0.07). Staining platelets in frozen sections of liver after 1 day of CCl4 injection with anti-CD41 (eBioscience) showed platelet accumulation in the damaged area of the liver, so we created a transient thrombocytopenia in WT mice by injecting a single dose of anti-GP1bα (2.5 mg/kg) antibody (R300, Emfret), which caused an 80% decrease in platelet count within 24 hours, and we found a 20% reduction in fibrosis in the CCl4 treated thrombocytopenic mice compared to isotype-matched antibody-injected mice (p=0.02). Because myofibroblast accumulation is a hallmark of tissue fibrosis, we quantified this in the damaged area by counting aSMA and vimentin double-stained cells and found reduced myofibroblast accumulation in PF4Cre/Tgfb1flox/flox mice vs. WT or littermate control mice. Evidence of active TGFβ1 signaling was detected as transient loss of phosphoSmad2 signaling in damaged tissue after 1 day of CCl4 challenge, but intensity increased as the liver tissue began to repair after 3 days. PhosphoSmad2 signaling intensity was 20% less in PF4Cre/Tgfb1flox/flox mice compared to WT mice after 3 days. Liver function was evaluated by measuring plasma levels of albumin and globin and we found that albumin to globin ratio was decreased in WT mice, but not in PF4Cre/Tgfb1flox/flox mice after 3 days of CCl4 injection.We conclude that platelet-derived TGFβ1 contributes at least partially to the development of liver fibrosis and the deterioration of liver function in the CCl4-induced liver injury model. These data have important implications for understanding TGFβ1 biology in various tissue injuries and in assessing the role of TGFβ1 in murine models of human diseases. DisclosuresNo relevant conflicts of interest to declare.

Protective effect of Trillium tschonoskii saponin on CCl4-induced acute liver injury of rats through apoptosis inhibition

To explore hepatoprotective role and underlying mechanisms of Trillium tschonoskii Maxim (TTM), 36 rats were randomly divided into control, CCl4-induced liver injury model, and biphenyl dimethyl dicarboxylate (DDB) and low-, moderate-, and high-dose TTM treatment groups. After CCl4-induced model establishment, the rats from DDB and TTM groups were administrated with DDB at 0.2 g/kg per day and TTM at 0.1, 0.5, and 1.0 g/kg per day, while the rats from control and model groups were administrated with saline. After 5 days of treatments, all rats were sacrificed for determining serum ALT and AST levels and liver index, examining histopathological changes in liver through HE and TUNEL staining, and evaluating TNF-α and IL-6 mRNA expression by real-time PCR, and caspase-3, Bcl-2, and Bax expression by Western blot. Results indicated that CCl4 could induce acute liver injury and abnormal liver function in rats with obvious hepatomegaly, increased liver index, high ALT and AST levels, up-regulated TNF-α and IL-6, and overexpressed Bax and caspase-3. However, DDB and TTM could execute protective role in CCl4-induced liver injury in rats through reducing ALT and AST levels, rescuing hepatomegaly, down-regulating inflammatory factors and inhibiting hepatocyte apoptosis in a dose-dependent manner. Therefore, TTM has obvious protective role in CCl4-induced liver injury of rats through inhibiting hepatocyte apoptosis.

Liver Injury Assessment by Vetscan VS2 Analyzer and Most Frequently Used ALT/GTP Reagent.

Liver injury is estimated by serum alanine aminotransferases (ALT) levels in experimental animal models. Laboratories use various techniques to measure ALT levels including assay reagents and chemistry analyzers. VetScan VS2 (VS2) is widely used in veterinary clinics and research laboratories for highly reproducible, convenient and effective testing. Alternatively, ALT liquid reagent is used by laboratories to estimate liver injury in animal studies. The aim of this study was to perform comparative analyses of data obtained from these two assays in two different animal models. In this study, we used two different mouse models and compared the ALT levels measured using VetScan VS2 chemistry analyzer and ALT liquid reagent. Immunohistochemical analysis of hepatic tissue was also performed to document liver pathology. The first model is a high fat diet feeding model that results in a mild hepatic steatosis (fat accumulation in the liver) without elevation of ALT levels. For a severe liver injury model, we employed a hepatotoxin-induced liver injury model (carbon tetrachloride, CCl4), which leads to the development of hepatic fibrosis and very high ALT levels. VetScan VS2 and ALT reagent gave different values of ALT for all animal groups. However, linear regression analysis showed a significantly high association between ALT levels obtained by VS2 and ALT liquid reagent in a high-fat feeding model with no liver injury. For the CCl4 induced liver injury model, serum dilution (5 and 10 times) was performed to obtain accurate results with ALT reagent. ALT levels acquired from both techniques showed a close association. Interestingly, this correlation was closer when serum was diluted 5 fold. This study demonstrates that both methods give similar results when evaluating liver injury in animal studies. However, the serum dilution factor is critical for severe liver injury assessment when using ALT reagent and requires some optimization. In this regard, VetScan VS2 is easier to use and gives comparable results.

Synthesis and evaluation of asiatic acid derivatives as anti-fibrotic agents: Structure/activity studies

Fibrotic diseases are characterized by the over-accumulation of fibrous components in the extracellular matrix and the liver, which can lead to liver cirrhosis. Current treatment options cannot reverse or halt liver fibrosis, motivating a search for newer treatment options. Previously, we showed that asiaticoside, a bioactive triterpene glycoside from Centella asiatica, has anti-fibrotic properties. Here, the aglycone asiatic acid was chemically modified, and these derivatives were evaluated for their potential as anti-fibrotic agents. The data obtained from in vivo testing of these compounds in a rodent CCl4-induced liver injury model are discussed. The information obtained from these studies may be useful in the design of novel anti-fibrotic agents.

Tonsil-derived stromal cells reduces CCl4-induced liver fibrosis in mice via autophagy activation (P2180)

Abstract Liver transplantation is the treatment of choice for chronic liver failure due to fibrosis, however, it faces several difficulties, including donor shortage, surgery-related complications, immunological rejection, and high medical cost. Cell transplantation therapy would be the minimally invasive and alternative methods, potentially with fewer complications. As cell source, we isolated mesenchymal stromal cells from human palatine tonsils (T-MSCs) and injected into CCl4-induced liver injury model of the mice. We found that T-MSCs were differentiated into hepatocyte-like cells under 25 ng/ml insulin-like growth factor, 25 ng/ml hepatocyte growth factor, 10-7 M dexamethasone, and 10 ng/ml oncostatin M for three weeks in vitro. After injecting into CCl4-liver damaged mice, T-MSCs were differentiated into albumin positive cells but were not detected in the group of mice with normal liver. During this process, T-MSCs showed accumulation of autophagosome-bound LC3B, upregulation of proautophagic beclin-1, and ubiquitin positive in vivo. Our data imply that T-MSCs contributed to reduction of CCl4-induced liver fibrosis through autophagy activation pathway.

Evaluation of antioxidant activities of water-soluble polysaccharides from brown alga Hizikia fusiformis

Water-soluble polysaccharides were isolated from the brown alga Hizikia fusiformis and separated using hydrophobic chromatography on an ME-1 resin column. Three major polysaccharide fractions, FG, GM-1 and GM-2, were analyzed by high-performance liquid chromatography, Fourier-transform infrared and 13C nuclear magnetic resonance spectroscopy. These three fractions showed free radical scavenging activity against hydroxyl radical and 1,1-diphenyl-2-picrylhydrazyl radical in vitro. Among these three fractions, GM-2 had the best free radical scavenging activity. Thus, only GM-2 was used in the acute oxidative stress experiments in the CCl4-induced liver injury model. GM-2 also protected against oxidative injury induced by CCl4in vivo.

Potential Implications for Monitoring Serum Bile Acid Profiles in Circulation with Serum Proteome for Carbon Tetrachloride-Induced Liver Injury/Regeneration Model in Mice

Bile acids have recently emerged as versatile signaling molecules, and their signaling pathway is a promising target for the treatment of metabolic diseases. Here, we developed a highly sensitive and high-throughput quantification method for six taurine- and glycine-conjugated bile acids using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after solid-phase extraction (SPE-MALDI-TOF MS). In a carbon tetrachloride (CCl4)-induced liver injury/regeneration model in mice, serum bile acid profiles were monitored, and the same samples were separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), and protein spots that significantly changed in quantity in a serial time points were identified by MALDI-TOF MS. Serum taurocholic acid (TCA) concentration was significantly elevated earlier than the increase of serum aspartate aminotransferase (AST)/alanine aminotransferase (ALT) activity, a potentially sensitive marker for minimal hepatic damage. Furthermore, TCA peaked at 20 h after treatment when massive serum proteins appeared in circulation. It should be noted that direct MALDI-imaging mass spectrometry (IMS) has succeeded in showing a hepatic lobular distribution change of TCA, predominantly seen in zone 1 area whereas necrotic changes were dominant in zone 3 area. The in-depth analysis of bile acid profiles in circulation with hepatic lobular distribution is a strong basis to understand the serum proteome in CCl4-induced liver injury model.

Activation of natural killer cells inhibits liver regeneration in toxin-induced liver injury model in mice via a tumor necrosis factor-α-dependent mechanism

Liver lymphocytes are enriched in natural killer (NK) cells, and activation of NK cells by injection of polyinosinic-polycytidylic acid (poly I:C) inhibits liver regeneration in the partial hepatectomy model via production of IFN-gamma. However, the role of NK cells in liver regeneration in a model of carbon tetrachloride (CCl(4))-induced liver injury remains unknown. In this study, we investigated the effect of activation of NK cells induced by poly I:C on liver regeneration in the CCl(4) model. Administration of poly I:C suppressed liver regeneration in CCl(4)-treated mice. Depletion of NK cells but not Kupffer cells or T cells restored liver regeneration in poly I:C/CCl(4)-treated mice. Poly I:C and CCl(4) cotreatment synergistically induced accumulation of NK cells in the liver and NK cell production of IFN-gamma and tumor necrosis factor (TNF)-alpha. Serum levels of these two cytokines were also synergistically induced after poly I:C and CCl(4) treatment. Finally, blockage of TNF-alpha but not IFN-gamma restored liver regeneration in poly I:C/CCl(4)-treated mice. Taken together, these findings suggest that poly I:C treatment inhibits liver regeneration in the CCl(4)-induced liver injury model via induction of NK cell production of TNF-alpha.