Structure-Activity Relationship Guided Design of a Highly Sensitive Turn-on Fluorescent Probe for Imaging Inflammation-Associated-HOCl in Early Liver Fibrosis.

To explore the value of in-line phase-contrast imaging with computed tomography (ILPCI-CT) by synchrotron radiation (SR) for liver fibrosis. Liver fibrosis models were set up in 13 BALB/c mice by peritoneal injections of thioacetamide and evaluated by ILPCI-CT. Histological staging was used to categorize liver fibrosis into normal, mild fibrosis and advanced fibrosis groups. Microvessel density (MVD), the ratio of total vessel length to volume (L/V), the ratio of total number of branching points to liver volume (P/V) and the distribution of vessel diameter were assessed. The CT images showed slightly high-density shadows around the portal tracts in the fibrosis group. Three-dimensional reconstruction can detect vascular and nodular changes on the surface of fibrotic livers. The MVDs between the three groups were significantly different (P = 0.024). L/V was significantly different between the three groups (P = 0.014). There was a positive correlation between MVD and P/V. Fibrous material can be detected by ILPCI-CT even in the early stage of fibrosis. MVD, L/V, P/V and the distribution of vessel diameter were consistent with fibrosis-related angiogenesis progress. Three-dimensional reconstruction is a promising method to visualize morphological changes of the fibrotic liver. • ILPCI-CT can detect fibrous material even in the early stage of liver fibrosis. • MVD, L/V, P/V, and the distribution of vascular diameter reflect pathological angiogenesis. • 3D reconstruction could be a promising approach for detecting liver fibrosis.

BackgroundLiver fibrosis is caused by chemicals or viral infection. The progression of liver fibrosis results in hepatocellular carcinogenesis in later stages. Recent studies have revealed the importance of DNA hypermethylation in the progression of liver fibrosis to hepatocellular carcinoma (HCC). However, the importance of DNA methylation in the early-stage liver fibrosis remains unclear.MethodsTo address this issue, we used a pathological mouse model of early-stage liver fibrosis that was induced by treatment with carbon tetrachloride (CCl4) for 2 weeks and performed a genome-wide analysis of DNA methylation status. This global analysis of DNA methylation was performed using a combination of methyl-binding protein (MBP)-based high throughput sequencing (MBP-seq) and bioinformatic tools, IPA and Oncomine. To confirm functional aspect of MBP-seq data, we complementary used biochemical methods, such as bisulfite modification and in-vitro-methylation assays.ResultsThe genome-wide analysis revealed that DNA methylation status was reduced throughout the genome because of CCl4 treatment in the early-stage liver fibrosis. Bioinformatic and biochemical analyses revealed that a gene associated with fibrosis, secreted phosphoprotein 1 (Spp1), which induces inflammation, was hypomethylated and its expression was up-regulated. These results suggest that DNA hypomethylation of the genes responsible for fibrosis may precede the onset of liver fibrosis. Moreover, Spp1 is also known to enhance tumor development. Using the web-based database, we revealed that Spp1 expression is increased in HCC.ConclusionsOur study suggests that hypomethylation is crucial for the onset of and in the progression of liver fibrosis to HCC. The elucidation of this change in methylation status from the onset of fibrosis and subsequent progression to HCC may lead to a new clinical diagnosis.

Angiotensin II type-2 receptor attenuates liver fibrosis progression by suppressing IRE1α-XBP1 pathway.

Nicotinamide riboside, an NAD+ precursor, attenuates the development of liver fibrosis in a diet-induced mouse model of liver fibrosis

To explore the protective effect of knock-down the expression of B lymphocyte induced maturation protein 1 (Blimp1) gene on early liver injury in carbon tetrachloride (CCl4)-induced mouse model of liver fibrosis. C57BL/6 mice were intraderitoneal injected with 5% CCl4 olive oil solution to create mouse model of hepatic fibrosis. The expression of Blimp1 gene in the mice was reduced by intraderitoneal injection of short hairpin RNA (shRNA) adeno-associated virus (AAV). The mice were randomly divided into 3 groups: blank test group (n=10), CCl4+AAV-shRNA-NC group (n=10) and CCl4+AAV-shRNA-Blimp1 group (n=10). After 27 days of preparation of the CCl4 mouse model, animal materials were carried out. Western blot and real-time PCR were used to detect the levels of Blimp1, α-smooth muscle actin (α-SMA), collagen type Ⅰ alpha 1 (COL1A1), collagen type Ⅲ alpha 1 (COL3A1), and their mRNA expression levels of liver tissue in each group. The serum of each group was separated to measure aspartate transaminase (AST) and alanine transaminase (ALT) by automatic biochemical analyzer. The pathological changes of liver tissue and the degree of liver fibrosis in the mice were detected by pathological staining including hematoxylin-eosin staining, Masson, and Sirius red. The expression levels of Blimp1 protein in the liver of CCl4+AAV-shRNA-NC group (2.036±0.244, t=3.690, P=0.002) were significantly increased than that of the blank test group. In the CCl4+AAV-shRNA-Blimp1 group, the expression of Blimp1 protein decreased to the basal level (0.783±0.249, t=6.223, P=0.003). Compared with the serum levels of ALT [(1 957.8±633.6) U/L] and AST [(1 808.8±260.1) U/L] in the CCl4+AAV-shRNA-NC group, the serum levels of ALT [(894.0±360.1) U/L, t=3.998, P=0.003] and AST [(820.0±100.6) U/L, t=6.141, P=0.004] in the CCl4+AAV-shRNA-Blimp1 group were significantly decreased. The pathological results of the CCl4+AAV-shRNA-Blimp1 group showed that compared with the CCl4+AAV-shRNA-NC group, the infiltration of inflammatory cells in the liver tissue was reduced and the degree of fibrosis was alleviated. The level of α-SMA (0.676±0.064, t=7.930, P=0.001), COL1A1 (1.426±0.143, t=6.364, P=0.003) and COL3A1 (1.124±0.198, t=3.440, P=0.026) of liver in the CCl4+AAV-shRNA-Blimp1 group were significantly decreased than that of CCl4+AAV-shRNA-NC group, and the mRNA expression levels were altered as well as their protein levels. Blimp1 plays an important role in CCl4-induced liver fibrosis in mice, and knock-down the expression of Blimp1 gene is beneficial to protect early liver injury in mice.

To explore whether Yes-associated protein (YAP) affects occurrence and progression of liver fibrosis by regulating epithelial-mesenchymal transition (EMT). In a 8-week-old C57BL/6 mouse model of CCl4-induced liver fibrosis, the effect of verteporfin (a YAP inhibitor) intervention was assessed with HE staining and by detecting liver biochemistry and expressions of YAP and EMT-related genes using immunohistochemistry and Western blotting. Transcriptome and proteomic sequencing and informatics analysis were used to investigate the main downstream pathways of YAP in liver fibrosis. Serum levels of YAP, N-cadherin, vimentin and Twist were examined in 60 healthy individuals, 60 patients with chronic hepatitis B (CHB), and 60 patients with HBV-related liver cirrhosis. In another 24 C57BL/6 mice, the effects of Twist inhibitor alone or in combination with harmine (a YAP activator) on CCl4-induced liver fibrosis were evaluated by histopathological examination and Western blotting. The mouse models of liver fibrosis showed obvious structural damages of the liver lobes with formation of pseudolobules, and verteporfin treatment significantly improved these pathologies and lowered plasma ALT and AST levels of the mice. Transcriptome and proteomic sequencing and informatics analysis suggested that N-cadherin and Twist were differentially expressed in liver fibrosis in close correlation with YAP. Inhibition of YAP obviously downregulated hepatic N-cadherin and Twist protein expressions in the mice with liver fibrosis. In patients with CHB and liver cirrhosis, serum levels of YAP elevated obviously with the severity of liver fibrosis and were significantly correlated with N-cadherin, vimentin and Twist levels. In mice with liver fibrosis, inhibiting Twist effectively improved liver inflammation and fibrosis, while the combined treatment with YAP activator worsened hepatic collagen fiber deposition and increased hepatic YAP and α-SMA expressions. EMT is an important pathogenic mechanism of liver fibrosis, and inhibiting YAP can alleviate liver fibrosis by reducing EMT.

Nicotinamide Riboside, an NAD + Precursor, Reduces Hepatic Stellate Cell Activation and Attenuates Liver Fibrosis in a Diet-induced Mouse Model of Liver Fibrosis (OR24-06-19)

Background: The application of mesenchymal stem cell (MSC) therapy in liver fibrosis treatment has been increasingly investigated in recent years. MSCs obtained from a variety of sources (e.g. bone marrow, umbilical cord blood and adipose tissue) have been studied and have achieved remarkable results. In this study, we compared the effects of adipose-derived mesenchymal stem cells (AD-MSC) transplantation with bone marrow-derived mesenchymal stem cell (BM-MSC) transplantation in a mouse model of liver fibrosis, induced by carbon tetrachloride (CCl4).
 Methods: Eight-week old mice were treated with CCl4 for 11 weeks to induce liver fibrosis then 5x105 cells were transplanted into mice via the tail vein.
 Results: After 21 days of transplantation, the results showed that the stem cell treated groups ameliorated better than the placebo group. MSC treated groups showed reduced AST and ALT levels, down-regulated expression of extracellular matrix (ECM) genes, and improved liver histopathology. Both sources of MSCs (bone marrow and adipose tissue) were effective in the mouse model of liver fibrosis.
 Conclusion: Our results also indicated that AD-MSC transplantation in mice accelerated liver regeneration better than BM-MSC transplantation.

The emerging incidence of pathogenic liver conditions is turning into a major concern for global health. Induction of pyroptosis in hepatocytes instigates cellular disintegration, which in turn liberates substantial quantities of pro-inflammatory intracellular substances, thereby accelerating the advancement of liver fibrosis. Consequently, directing therapeutic efforts towards inhibiting pyroptosis could potentially serve as an innovative approach in managing inflammation related chronic hepatic disorders. GSDMD-NTki/wt mice and Alb-creki/wt mice were generated using CRISPR/Cas9 technology. After crossing the two strains together, we induced conditional cell death by doxycycline to construct a mouse model of liver fibrosis. We analyzed differentially expressed genes by RNA sequencing and explored their biological functions. The efficacy of obeticholic acid (OCA) in the treatment of liver fibrosis was assessed. Doxycycline-treated GSDMD-NTki/wt × Alb-creki/wt mice showed severe liver damage, vacuolation of hepatocytes, increased collagen fibers, and accumulation of lipid droplets. The expression of liver fibrosis related genes was greatly increased in the doxycycline-treated mouse liver compared with untreated mouse liver. RNA-sequencing showed that upregulated differentially expressed genes were involved in inflammatory responses, cell activation, and metabolic processes. Treatment with OCA alleviated the liver fibrosis, with reduced ALT and AST levels seen in the GSDMD-NTki/wt × Alb-creki/wt mice. We successfully constructed a novel mouse model for liver fibrosis. This GSDMD-NT-induced fibrosis may be mediated by abnormal lipid metabolism. Our results demonstrated that we successfully constructed a mouse model of liver fibrosis, and GSDMD-NT induced fibrosis by mediating lipid metabolism.

To date, there are very few effective drugs for liver fibrosis treatment; therefore, it is urgent to develop novel therapeutic targets and approaches. In the present research, we sought to study the protective effect of sweroside contained in Lonicera japonica or blue honeysuckle berries in a mouse model of liver fibrosis and investigate the underlying mechanism. The mouse model of liver fibrosis in was induced by intraperitoneal injections of 10% CCl4 for 6weeks (three times/week). At the beginning of the fourth week, sweroside was intragastrically administered once a day and at the end of the treatment, biochemical and histological studies were investigated. The expression of FXR, miR-29a and the downstream targets were analyzed as well. Moreover, the effect of sweroside on cell proliferation was observed in human hepatic stellate cells (HSCs) (LX-2), along with using the siRNA for FXR and miR-29a inhibitor to investigate the underpinning of the anti-fibrotic effect of sweroside. Sweroside successfully protected the liver fibrosis in CCl4-induced mouse model, accompanied by miR-29a induction. Furthermore, sweroside also induced miR-29a in HSCs, resulting in the inhibition of COL1 and TIMP1. Our data also showed that either silencing miR-29a or knockdown of FXR in LX-2 cell abolished the inhibition of COL1 and TIMP1 as well as the inhibition of cell proliferation by sweroside treatment. In conclusion, sweroside exerted its anti-fibrotic effect in vivo and in vitro by up-regulation of miR-29a and repression of COL1 and TIMP1, which was at least in part through FXR.

The liver has the amazing capacity to repair itself after injury; however, the same processes that are involved in liver regeneration after acute injury can cause serious consequences during chronic liver injury. In an effort to repair damage, activated hepatic stellate cells trigger a cascade of events that lead to deposition and accumulation of extracellular matrix components causing the progressive replacement of the liver parenchyma by scar tissue, thus resulting in fibrosis. Although fibrosis occurs as a result of many chronic liver diseases, the molecular mechanisms involved depend on the underlying etiology. Since studying liver fibrosis in human subjects is complicated by many factors, mouse models of liver fibrosis that mimic the human conditions fill this void. This review summarizes the general mouse models of liver fibrosis and mouse models that mimic specific human disease conditions that result in liver fibrosis. Additionally, recent progress that has been made in understanding the molecular mechanisms involved in the fibrogenic processes of each of the human disease conditions is highlighted.

Hepatic fibrosis is a progressive disease, which is reversible in the early stages. The current monitoring methods have notable limitations that pose a challenge to early detection. In this study, we evaluated the utility of [18F]AlF-ND-bisFAPI positron emission tomography imaging of fibroblast activation protein (FAP) to monitor the progression of liver fibrosis. Two mouse models of liver fibrosis were established by bile duct ligation and carbon tetrachloride administration, respectively. Positron emission tomography imaging was performed with the FAP-specific radiotracer [18F]AlF-ND-bisFAPI for the evaluation of rat HSCs and mouse models of fibrosis and combined with histopathology, immunohistochemical staining, and immunoblotting to elucidate the relationships among radioactivity uptake, FAP levels, and liver fibrosis progression. Furthermore, [18F]AlF-ND-bisFAPI autoradiography was performed to assess tracer binding in liver sections from patients with varying degrees of liver fibrosis. Cell experiments demonstrated that [18F]AlF-ND-bisFAPI uptake was specific in activated HSCs. Compared with control mice, [18F]AlF-ND-bisFAPI uptake in livers increased in the early stages of fibrosis and increased significantly further with disease progression. Immunohistochemistry and western blot analyses demonstrated that FAP expression increased with fibrosis severity. In accordance with the findings in animal models, ex vivo autoradiography on human fibrotic liver sections showed that radioactivity increased as fibrosis progressed from mild to severe. [18F]AlF-ND-bisFAPI positron emission tomography imaging is a promising noninvasive method for monitoring the progression of liver fibrosis.

High mobility group proteins 1 and 2 (HMGB1 and HMGB2) are 80% conserved in amino acid sequence. The function of HMGB1 in inflammation and fibrosis has been extensively characterized. However, an unaddressed central question is the role of HMGB2 on liver fibrosis. In this study, we provided convincing evidence that the HMGB2 expression was significantly upregulated in human liver fibrosis and cirrhosis, as well as in several mouse liver fibrosis models. The carbon tetrachloride (CCl4) induced liver fibrosis mouse model was used. AAV8-Hmgb2 was utilized to overexpress Hmgb2 in the liver, while Hmgb2-/- mice were used for loss of function experiments. The HMGB2 inhibitor inflachromene and liposome-shHMGB2 (lipo-shHMGB2) were employed for therapeutic intervention. The serum HMGB2 levels were also markedly elevated in patients with liver fibrosis and cirrhosis. Deletion of Hmgb2 in Hmgb2-/- mice or inhibition of HMGB2 in mice using a small molecule ICM slowed the progression of CCl4-induced liver fibrosis despite constant HMGB1 expression. In contrast, AAV8-mediated overexpression of Hmgb2 enchanced CCl4-incuded liver fibrosis. Primary hepatic stellate cells (HSCs) isolated from Hmgb2-/- mice showed significantly impaired transdifferentiation and diminished activation of α-SMA, despite a modest induction of HMGB1 protein. RNA-seq analysis revealed the induction of top 45 CCl4-activated genes in multiple signaling pathways including integrin signaling and inflammation. The activation of these genes by CCl4 were abolished in Hmgb2-/- mice or in ICM-treated mice. These included C-X3-C motif chemokine receptor 1 (Cx3cr1) associated with inflammation, cyclin B (Ccnb) associated with cell cycle, DNA topoisomerase 2-alpha (Top2a) associated with intracellular component, and fibrillin (Fbn) and fibromodulin (Fmod) associated with extracellular matrix. We conclude that HMGB2 is indispensable for stellate cell activation. Therefore, HMGB2 may serve as a potential therapeutic target to prevent HSC activation during chronic liver injury. The blood HMGB2 level may also serve as a potential diagnostic marker to detect early stage of liver fibrosis and cirrhosis in humans.

Liver injury causes nodule and scar tissue formation and diffuse fibrosis, which are characteristic of liver cirrhosis. Since there are currently no efficacious therapies to prevent fibrosis, the development of animal models of liver fibrosis is necessary to facilitate further in vivo studies of this pathology. In this study, a mouse model of liver fibrosis was generated using Swiss mice and carbon tetrachloride (CCl4) treatment. Induction of liver fibrosis was analyzed using 0.8, 1.0, or 1.2 mL/kg CCl4 to determine the effective dose. In this study, we aimed to develop a standardized hepatic fibrosis mouse model by using CCl4 induction to facilitate further studies in this field. In Swiss mice, we evaluated the dose of CCl4 and the criteria of fibrosis, such as serum markers, fibrosis marker genes, and histopathology. Mice were administered CCl4 three times per week for 8 consecutive weeks. Body weights, survival rates, levels of serum markers (aspartate aminotransferase/alanine aminotransferase [AST/ALT]) and fibrosis markers (fibronectin, procollagen, nt5e, transforming growth factor-beta [TGF-β], and integrin), and histopathology (using hematoxylin and eosin [H&E] staining) were analyzed to determine the optimal dose of CCl4 for induction of liver fibrosis. Results showed that 1.0 mL/kg CCl4 was the most efficient dose for the establishment of a liver fibrosis mouse model. In a standardized liver fibrosis model, mice were treated with 1.0 mL/kg CCl4 three times per week for 11 consecutive weeks, and levels of serum markers (AST, ALT, bilirubin, and albumin), expression of fibrosis marker genes (using quantitative reverse transcription polymerase chain reaction [RT-PCR]), histopathology (using Hematoxylin and eosin staining), and connective tissue formation (using Massive trichrome staining) were analyzed. The outcomes showed that serum markers and the levels of fibrosis marker genes were significantly increased in the standardized liver fibrosis model. Additionally, we observed sharp increases in fibronectin and procollagen expression (1222.40 ± 4.20 and 241.35 ± 1.18, respectively), and the development of cirrhosis (fibrosis stage 3–5/6) in liver tissues of the standardized mouse model of hepatic fibrosis.

We identified Mac-2 (galectin-3) binding protein (Mac-2bp) as a novel diagnostic and liver fibrosis predicting biomarker for nonalcoholic steatohepatitis in humans. In mouse models, there are no serum biomarkers predicting liver disease severity. In this study, we developed a mouse Mac-2bp enzyme-linked immunosorbent assay (ELISA) system and determined its efficacy for predicting the severity of liver disease in mouse models, especially in non-alcoholic fatty liver disease (NAFLD) models. We established several rat monoclonal antibodies against mouse Mac-2bp, selected two clones for the ELISA, and checked the accuracy and reproducibility of the ELISA, especially for NAFLD models and liver fibrosis models. We also investigated the relationships between serum levels and hepatic gene expression of Mac-2bp in mouse models. Our ELISA system had high accuracy and reproducibility (R2 = 0.999). The intra-assay and inter-assay results for the coefficient of variation were 2.0-3.7% and 1.7-6.9%, respectively. The levels of bilirubin, hemoglobin, and chyle did not affect the Mac-2bp serum levels detected by our ELISA kit. In the mouse models, serum Mac-2bp levels increased with liver disease progression (F0/F1/F2/F3, 239.1 ± 36.7/259.1 ± 43.0/457.5 ± 162.0/643.7 ± 116.0ng/mL; P <0.0001), and were significantly correlated with hepatic gene expression of Mac-2bp (R = 0.42, P <0.0001). Our mouse Mac-2bp ELISA system effectively predicts severity of NAFLD and liver fibrosis in mouse models.