Mouse Model Of Liver Fibrosis Research Articles

Shear-Wave Elastography Using Commercially Available Ultrasound in a Mouse Model of Chronic Liver Disease

Elastography is currently used clinically to diagnose the degree of liver stiffness. We sought to develop a shear-wave elastography (SWE) measurement method using ultrasound in mice and to compare its results with those of other noninvasive tests for liver fibrosis. We divided male mice into three groups (normal (G1), liver fibrosis (G2), and fatty liver (G3)). We measured mouse liver SWE values and compared them with T1rho and T2 values from magnetic resonance imaging results. We also compared the SWE values with the expression levels of a serum liver fibrosis biomarker (Mac-2-binding protein (M2BP)) and hepatic genes. SWE values significantly increased over time in G2 but did not change in G3. T1rho values in G2 and G3 were significantly increased compared with those in G1. T2 values in G2 did not increase compared with those in group 1. T2 values in G3 significantly increased compared with those in groups 1 and 2. In G2, SWE values significantly and positively correlated with T1rho values. SWE values significantly correlated with serum M2BP levels in G2 but did not correlate with inflammatory gene expression. We could measure SWE values to assess the degree of liver fibrosis in mouse models of liver disease.

Demethylzeylasteral attenuates hepatic stellate cell activation and liver fibrosis by inhibiting AGAP2 mediated signaling

BackgroundLiver fibrosis is a common cause of chronic liver disease. If left untreated, it can ultimately develop into liver cirrhosis or hepatocellular carcinoma. However, a direct antifibrotic therapy is currently unavailable. A re-examination of existing chemicals might be a potential strategy for finding more lead compounds against liver fibrosis. Demethylzeylasteral (T-96), a naturally occurring bioactive compound found in Tripterygium wilfordii Hook. f. (TwHf) possesses multiple pharmacological properties. However, its antifibrotic potential has not yet been fully evaluated. PurposeThis study aimed to investigate the antifibrotic properties of T-96 and its underlying molecular mechanisms. MethodsThe antifibrotic properties of T-96 were investigated in three types of hepatic stellate cells (HSCs) and in a CCl4-induced liver fibrosis mouse model. The effect of T-96 on the proliferation, migration, and activation of HSCs was detected using CCK-8 and scratch/wound healing assays. Hepatic inflammation and fibrosis were evaluated by H&E, Masson's trichrome stain, and Sirius Red staining. The expression of inflammatory and fibrogenic genes was detected by quantitative real-time PCR (qRT-PCR) and western blotting. RNA sequencing (RNA-seq) was performed to explore the potential molecular mechanisms mediating the antifibrotic effect of T-96, which was verified by dual-luciferase reporter assay, qRT-PCR, western blotting, immunofluorescence, and immunoprecipitation analysis. ResultsThe T-96 treatment significantly suppressed the proliferation, migration, and activation of HSCs in vitro. The administration of T-96 attenuated hepatic injury, inflammation, and fibrosis progression in mice with CCl4-induced liver fibrosis. In addition, the RNA-seq of fibrotic liver tissues and subsequent functional verification indicated that the key mechanisms of the antifibrotic effect of T-96 were mediated by suppressing the expression of AGAP2 (Arf GAP with GTPase-like domain, ankyrin repeat and PH domain 2), inhibiting the subsequent phosphorylation of focal adhesion kinase (FAK) and protein kinase B (AKT), and finally reducing the expression of fibrosis-related genes. ConclusionOur results provide the first insight that T-96 exerts potent antifibrotic effects both in vitro and in vivo by inhibiting the AGAP2 mediated FAK/AKT signaling axis, and that T-96 may serve as a potential therapeutic candidate for the treatment of liver fibrosis.

Study of the negative regulation of transforming growth factor beta type II receptor to inhibit the occurrence and development of liver fibrosis with miR-217

Objective: To observe the effect of miR-217 on angiotensin II (AngII)-induced hepatic stellate cells (HSCs) activation, and carbon tetrachloride (CCl4)-induced overexpression in mice, so as to clarify miR-217 role in liver fibrosis. Methods: HSCs were stimulated with AngⅡ and the changes condition in the expression level of miR-217 were detected. HSCs were divided into control group, AngII-treated group and AngⅡ+miR-217-treated group. The expression levels of alpha-smooth muscle actin, fibroblast-specific protein 1 and collagen Ⅰ (Collagen Ⅰ) in each group were detected. The target gene of mir-217 was screened and verified by Targetscan and Dual luciferase gene reporter assay. Real-time quantitative PCR and Western blot were used to detect the effect of miR-217 on the expression level of transforming growth factor beta type Ⅱ receptor (TGFBR2). A CCl4-induced mouse liver fibrosis model was constructed. Masson staining and Sirius red staining were used to detect the effect of miR-217 overexpression on the progression of liver fibrosis in CCl4 mice. Data of two groups were compared using t-test. Data of multiple groups were statistically analyzed with one-way ANOVA. Results: The expression level of miR-217 was downregulated by AngⅡ-stimulated HSC cells. The expression levels of α-smooth muscle actin, fibroblast-specific protein 1 and Collagen Ⅰ induced by AngⅡ was inhibited by miR-217 mimics transfection. The 3'-UTR of TGFBR2 had specifically bind miR-217. The mRNA and protein expression levels of TGFBR2 was inhibited with miR-217 mimics transfection in HSCs. The overexpression of miR-217 had inhibited the expression levels of Collagen Ⅰ and Ⅲ in CCl4 mice and alleviated the progression of liver fibrosis . Conclusion: miR-217 regulates liver fibrosis by targeting TGFBR2, inhibits AngII-induced HSC activation, and slows down the process of liver fibrosis in CCl4 mice, suggesting that miR-217 may have an inhibitory effect on liver fibrosis.

Wogonoside attenuates liver fibrosis by triggering hepatic stellate cell ferroptosis through SOCS1/P53/SLC7A11 pathway.

Wogonoside (WG) is a flavonoid chemical component extracted from Scutellaria baicalensis, which exerts therapeutic effects on liver diseases. Ferroptosis, a novel form of programmed cell death, regulates diverse physiological/pathological processes. In this study, we attempted to investigate a novel mechanism by which WG mitigates liver fibrosis by inducing ferroptosis in hepatic stellate cells (HSCs). A CCl4 -induced mouse liver fibrosis model and a rat HSC line were employed for in vivo and in vitro experiments, both treated with WG. Firstly, the levels of the fibrotic markers α-smooth muscle actin (α-SMA) and α1(I)collagen (COL1α1) were effectively decreased by WG in CCl4 -induced mice and HSC-T6 cells. Additionally, mitochondrial condensation and mitochondrial ridge breakage were observed in WG-treated HSC-T6 cells. Furthermore, ferroptotic events including depletion of SLC7A11, GPX4 and GSH, and accumulation of iron, ROS and MDA were discovered in WG-treated HSC-T6 cells. Intriguingly, these ferroptotic events did not appear in hepatocytes or macrophages. WG-elicited HSC ferroptosis and ECM reduction were dramatically abrogated by ferrostatin-1 (Fer-1), a ferroptosis inhibitor. Importantly, our results confirm that SOCS1/P53/SLC7A11 is a signaling pathway which promotes WG attenuation of liver fibrosis. On the contrary, WG mitigated liver fibrosis and inducted HSC-T6 cell ferroptosis were hindered by SOCS1 siRNA and pifithrin-α (PFT-α). These findings demonstrate that SOCS1/P53/SLC7A11-mediated HSC ferroptosis is associated with WG alleviating liver fibrosis, which provides a new clue for the treatment of liver fibrosis.

FRI157 - Direct anti-inflammatory and anti-fibrotic effects of a novel long-acting Glucagon/GIP/GLP-1 triple agonist, HM15211, in thioacetamide-induced mouse model of liver injury and fibrosis

FRI157 - Direct anti-inflammatory and anti-fibrotic effects of a novel long-acting Glucagon/GIP/GLP-1 triple agonist, HM15211, in thioacetamide-induced mouse model of liver injury and fibrosis

735-P: Direct Anti-Inflammatory and Antifibrotic Effects of a Novel Long-Acting Glucagon/GIP/GLP-1 Triple Agonist, HM15211, in Thioacetamide-Induced Mouse Model of Liver Injury and Fibrosis

HM152 is a novel long-acting triple agonist consisting of Glucagon/GIP/GLP-1 triple agonist conjugated to human IgG FC fragment via short PEG linker. Previously, therapeutic benefits of HM152 were demonstrated in diet-induced animal models of NASH and fibrosis. Here, direct anti-inflammatory and -fibrotic effects of HM152 were further evaluated in TAA (thioacetamide) -induced liver injury and fibrosis mouse, and investigate underlying mechanism. To induce liver injury and fibrosis, gradually increased dose of TAA was injected to mouse for 12 weeks, and HM152 was administered during last weeks. Interestingly, HM152 treatment significantly reduced hepatic hydroxyproline (-51% vs. Veh, p<0.01) , Sirius red positive area (-65% vs. Veh, p<0.001) , and fibrosis score (0.7 for HM152 vs. 3.0 for Veh, p<0.001) . Considering baseline fibrosis score at week 2 (1.0) , HM152 could confer both potential reversal effect on pre-existing fibrosis and prevention effect on fibrogenesis. Consistently, expression of hepatic marker genes for fibrosis (i.e. collagen-1α1) and inflammation (i.e. F4/80, TNF-α) were significantly reduced in HM152 group. Furthermore, multiplex cytokine analysis revealed that HM152 treatment was associated with robust reduction across pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6. Significant reduction in blood level of liver enzymes was also confirmed. Mechanistically, PMA/LPS-induced pro-inflammatory cytokine secretion of THP-1 cell was significantly attenuated by HM15211. TGF-β-induced collagen production was also reduced in LX2 cell. In conclusion, HM152 markedly improved liver inflammation and fibrosis in TAA mice, and related mechanism was elaborated by in vitro studies. Thus, HM152 could be a novel therapeutic option for fibrosis due to NASH. Human study is ongoing to assess the clinical relevance of these findings. Disclosure J.Kim: None. S.Lee: n/a. I.Choi: None. J.Lee: None. Y.Kim: None. S.Lee: None. H.Kwon: Employee; Hanmi Pharm. Co., Ltd. E.Park: Employee; Hanmi Pharm. Co., Ltd. J.Choi: Employee; Hanmi Pharm. Co., Ltd. S.Bae: None. D.Kim: None.

Upregulation of GLT25D1 in Hepatic Stellate Cells Promotes Liver Fibrosis via the TGF-β1/SMAD3 Pathway In Vivo and In vitro.

Collagen β(1-O) galactosyltransferase 25 domain 1 (GLT25D1) is associated with collagen production and glycosylation, and its knockout in mice results in embryonic death. However, its role in liver fibrosis remains elusive, particularly in hepatic stellate cells (HSCs), the primary collagen-producing cells associated with liver fibrogenesis. Herein, we aimed to elucidate the role of GLT25D1 in HSCs. Bile duct ligation (BDL)-induced mouse liver fibrosis models, primary mouse HSCs (mHSCs), and transforming growth factor beta 1 (TGF-β1)-stimulated LX-2 human hepatic stellate cells were used in in vivo and in vitro studies. Stable LX-2 cell lines with either GLT25D1 overexpression or knockdown were established using lentiviral transfection. RNA-seq was performed to investigate the genomic differences. HPLC-MS/MS were used to identify glycosylation sites. Scanning electronic microscopy (SEM) and second-harmonic generation/two-photon excited fluorescence (SHG/TPEF) were used to image collagen fibril morphology. GLT25D1 expression was upregulated in nonparenchymal cells in human cirrhotic liver tissues. Meanwhile, its knockdown attenuated collagen deposition in BDL-induced mouse liver fibrosis and inhibited mHSC activation. GLT25D1 was overexpressed in activated versus quiescence LX-2 cells and regulated in vitro LX-2 cell activation, including proliferation, contraction, and migration. GLT25D1 also significantly increased liver fibrogenic gene and protein expression. GLT25D1 upregulation promoted HSC activation and enhanced collagen expression through the TGF-β1/SMAD signaling pathway. Mass spectrometry showed that GLT25D1 regulated the glycosylation of collagen in HSCs, affecting the diameter of collagen fibers. Collectively, the upregulation of GLT25D1 in HSCs promoted the progression of liver fibrosis by affecting HSCs activation and collagen stability.

A new mouse model of radiation-induced liver disease reveals mitochondrial dysfunction as an underlying fibrotic stimulus

Background & AimsHigh-dose irradiation is an essential tool to help control the growth of hepatic tumors, but it can cause radiation-induced liver disease (RILD). This life-threatening complication manifests itself months following radiation therapy and is characterized by fibrosis of the pericentral sinusoids. In this study, we aimed to establish a mouse model of RILD to investigate the underlying mechanism of radiation-induced liver fibrosis.MethodsUsing a small animal image-guided radiation therapy platform, an irradiation scheme delivering 50 Gy as a single dose to a focal point in mouse livers was designed. Tissues were analyzed 1 and 6 days, and 6 and 20 weeks post-irradiation. Irradiated livers were assessed by histology, immunohistochemistry, imaging mass cytometry and RNA sequencing. Mitochondrial function was assessed using high-resolution respirometry.ResultsAt 6 and 20 weeks post-irradiation, pericentral fibrosis was visible in highly irradiated areas together with immune cell infiltration and extravasation of red blood cells. RNA sequencing analysis showed gene signatures associated with acute DNA damage, p53 activation, senescence and its associated secretory phenotype and fibrosis. Moreover, gene profiles of mitochondrial damage and an increase in mitochondrial DNA heteroplasmy were detected. Respirometry measurements of hepatocytes in vitro confirmed irradiation-induced mitochondrial dysfunction. Finally, the highly irradiated fibrotic areas showed markers of reactive oxygen species such as decreased glutathione and increased lipid peroxides and a senescence-like phenotype.ConclusionsBased on our mouse model of RILD, we propose that irradiation-induced mitochondrial DNA instability contributes to the development of fibrosis via the generation of excessive reactive oxygen species, p53 pathway activation and a senescence-like phenotype.Lay summaryIrradiation is an efficient cancer therapy, however, its applicability to the liver is limited by life-threatening radiation-induced hepatic fibrosis. We have developed a new mouse model of radiation-induced liver fibrosis, that recapitulates the human disease. Our model highlights the role of mitochondrial DNA instability in the development of irradiation-induced liver fibrosis. This new model and subsequent findings will help increase our understanding of the hepatic reaction to irradiation and to find strategies that protect the liver, enabling the expanded use of radiotherapy to treat hepatic tumors.

Quantitative Magnetization EXchange MRI Measurement of Liver Fibrosis Model in Rodents.

Quantitative MRI can elucidate the complex microstructural changes in liver disease. The Magnetization EXchange (MEX) method estimates macromolecular fraction, such as collagen, and can potentially aid in this task. MEX sequence, and its derived quantitative macromolecular fraction, should correlate with collagen deposition in rodents liver fibrosis model. Prospective. Sixteen adults Sprague-Dawley rats and 13 adults C57BL/6 strain mice given carbon tetrachloride (CCl4 ) twice weekly for 6 or 8 weeks. A 7 T scanner. MEX sequence (selective suppression and magnetization exchange), spin-echo and gradient-echo scans. Macromolecular fraction (F) and T1 were extracted for each voxel and for livers' regions of interest, additional to calculating the percentage of F > 0.1 pixels in F maps (high-F). Histology included staining with hematoxylin and eosin, picrosirius red and Masson trichrome, and inflammation scoring. Quantitative collagen percentage calculated using automatic spectral-segmentation of the staining. Comparing CCl4 -treated groups and controls using Welch's t-test and paired t-test between different time points. Pearson's correlation used between ROI MEX parameters or high-F fraction, and quantitative histology. F or T1 , and inflammation scores were tested with one-sided t-test. P < 0.05 was deemed significant. Rats: F values were significantly different after 6 weeks of treatment (0.10 ± 0.02) compared to controls (0.080 ± 0.003). After 8 weeks, F significantly increased (0.11 ± 0.02) in treated animals, while controls are not significant (0.0814 ± 0.0008, P=0.079). F correlated with quantitative histology (R=0.87), and T1 was significantly different between inflammation scores (1: 1332 ± 224 msec, 2: 2007 ± 464 msec). Mice: F was significantly higher (0.062 ± 0.006) in treatment group compared to controls (0.042 ± 0.006). F and high-F fraction correlated with quantitative histology (R=0.88; R=0.84). T1 was significantly different between inflammation scores (1:1366 ± 99 msec; 2:1648 ± 45 msec). MEX extracted parameters are sensitive to collagen deposition and inflammation and are correlated with histology results of mouse and rat liver fibrosis model. 1 TECHNICAL EFFICACY: Stage 3.

Resveratrol ameliorates liver fibrosis induced by nonpathogenic Staphylococcus in BALB/c mice through inhibiting its growth

BackgroundThe altered gut microbiota is implicated in the pathogenesis of liver fibrosis. Resveratrol is a candidate for the treatment of liver fibrosis, which could ameliorate the dysregulation of gut microbiota in mice. This study aimed to clarify the role and mechanism of resveratrol in gut microbiota during liver fibrosis.MethodsA mouse model of liver fibrosis induced by CCl4 was conducted to assess the effect of resveratrol on liver fibrosis. The changes of gut microbiota in liver fibrotic mice after resveratrol intervention were assessed using 16S ribosomal RNA sequencing. The mechanism of the gut microbiota dysregulation in liver fibrosis was investigated by Sirius red staining, immunohistochemical assay, bacterial translocation (BT), EUB338 fluorescence in situ hybridization, immunofluorescence, trans-epithelial electrical resistance analysis and paracellular permeability analysis.ResultsResveratrol relieved CCl4-induced liver fibrosis. Besides, resveratrol restrained the gut microbiota Staphylococcus_lentus and Staphylococcus_xylosus in the liver fibrotic mice, and the Staphylococcus_xylosus and Staphylococcus_lentus facilitated the occurrence of BT and the cultures of them enhanced the permeability of intestine. The in vivo assay corroborated that the excessive Staphylococcus_xylosus and Staphylococcus_lentus canceled the protecting effect of resveratrol on liver fibrosis, and Staphylococcus_xylosus or Staphylococcus_lentus alone had a limited impact on the liver injury of normal mice.ConclusionResveratrol ameliorated liver fibrosis by restraining the growth of Staphylococcus_xylosus and Staphylococcus_lentus.

Sorafenib-loaded silica-containing redox nanoparticles for oral anti-liver fibrosis therapy.

Liver fibrosis is a chronic disease resulting from repetitive or prolonged liver injury with limited treatment options. Sorafenib has been reported to be a potential antifibrotic agent; however, its therapeutic effect is restricted because of its low bioavailability and severe adverse effects in the gastrointestinal (GI) tract. In this study, we developed sorafenib-loaded silica-containing redox nanoparticles (sora@siRNP) as an oral nanomedicine to treat liver fibrosis. The designed siRNP were prepared by self-assembly of amphiphilic block copolymers, which possess antioxidant nitroxide radicals as a side chain of the hydrophobic segment and porous silica particles in the nanoparticle core. The silica moieties in the core formed a crosslink between the self-assembling block copolymers to afford stable drug absorption, which could be useful in harsh GI conditions after oral drug administration. Based on in vitro evaluation, sora@siRNP exerted antiproliferative and antifibrotic effects against hepatic stellate cells (HSCs) and low toxicity against normal endothelial cells. A pharmacokinetic study showed that siRNP significantly improved the bioavailability and distribution of sorafenib in the liver. In an in vivo study using a mouse model of CCl4-induced liver fibrosis, oral administration of sora@siRNP significantly suppressed the fibrotic area in comparison to free sorafenib administration. In mice with CCl4-induced fibrosis, free sorafenib administration did not suppress the expression of α-smooth muscle actin; however, mice treated with sora@siRNP showed significantly suppressed expression of α-smooth muscle actin, indicating the inhibition of HSC activation, which was confirmed by in vitro experiments. Moreover, oral administration of free sorafenib induced severe intestinal damage and increased leakage into the gut, which can be attributed to the generation of reactive oxygen species (ROS). Our antioxidant nanocarriers, siRNP, reduced the adverse effects of local ROS scavenging in the GI tract. Our results suggest that sora@siRNP could serve as a promising oral nanomedicine for liver fibrosis.

HMSCs-derived exosome circCDK13 inhibits liver fibrosis by regulating the expression of MFGE8 through miR-17-5p/KAT2B.

To investigate the effects of human bone marrow mesenchymal stem cells (hMSCs)-derived exosome circCDK13 on liver fibrosis and its mechanism. Exosomes derived from hMSCs were extracted and identified by flow cytometry and osteogenic and adipogenic induction, and the expressions of marker proteins on the surface of exosomes were detected by western blot. Cell proliferation was measured by CCK8 assay, the expression of active markers of HSCs by immunofluorescence, and the expressions of fibrosis-related factors by western blot. A mouse model of liver fibrosis was established by intraperitoneal injection of thioacetamide (TAA). Fibrosis was detected by HE staining, Masson staining, and Sirius red staining. Western blot was utilized to test the expressions of PI3K/AKT and NF-κB pathway related proteins, dual-luciferase reporter assay and RIP assay to validate the binding between circCDK13 and miR-17-5p as well as between miR-17-5p and KAT2B, and ChIP to validate the effect of KAT2B on H3 acetylation and MFGE8 transcription. hMSCs-derived exosomes inhibited liver fibrosis mainly through circCDK13. Dual-luciferase reporter assay and RIP assay demonstrated the binding between circCDK13 and miR-17-5p as well as between miR-17-5p and KAT2B. Further experimental results indicated that circCDK13 mediated liver fibrosis by regulating the miR-17-5p/KAT2B axis, and KAT2B promoted MFGE8 transcription by H3 acetylation. Exo-circCDK13 inhibited PI3K/AKT and NF-κB signaling pathways activation through regulating the miR-17-5p/KAT2B axis. hMSCs-derived exosome circCDK13 inhibited liver fibrosis by regulating the expression of MFGE8 through miR-17-5p/KAT2B axis.

Liver fibrosis inhibits lethal injury through D-galactosamine/lipopolysaccharide-induced necroptosis

Objective: To explore the new mechanism of liver fibrosis through D-galactosamine/lipopolysaccharide (D-GalN/LPS)-induced necroptosis as an entry point to inhibit lethal injury. Methods: The carbon tetrachloride (CCl4)-induced mouse model of liver fibrosis was established. At 6 weeks of fibrosis, the mice were challenged with a lethal dose of D-GalN/LPS, and the normal mice treated with the same treatment were used as the control. The experiment was divided into four groups: control group (Control), acute injury group (D-GalN/LPS), liver fibrosis group (Fib), and liver fibrosis + acute challenge group (Fib + D-GalN/LPS). Quantitative PCR and immunofluorescence were used to analyze the expression of necroptosis key signal molecules RIPK1, RIPK3, MLKL and/or P-MLKL in each group. Normal mice were treated with inhibitors targeting key signaling molecules of necroptosis, and then given an acute challenge. The inhibitory effect of D-GalN/LPS-induced-necroptosis on acute liver injury was evaluated according to the changes in transaminase levels and liver histology. Liver fibrosis spontaneous ablation model was established, and then acute challenge was given. Necroptosis key signal molecules expression was analyzed in liver tissue of mice in each group and compared by immunohistochemistry. The differences between groups were compared with t-test or analysis of variance. Results: Quantitative PCR and immunofluorescence assays result showed that D-GalN/LPS-induced significant upregulation of RIPK1, RIPK3, MLKL and/or P-MLKL. Necroptosis key signal molecules inhibition had significantly reduced D-GalN/LPS-induced liver injury, as manifested by markedly reduced serum ALT and AST levels with improvement in liver histology. Necroptosis signaling molecules expression was significantly inhibited in fibrotic livers even under acute challenge conditions. Additionally, liver fibrosis with gradual attenuation of fibrotic ablation had inhibited D-GalN/LPS-induced necroptosis. Conclusion: Liver fibrosis may protect mice from acute lethal challenge injury by inhibiting D-GalN/LPS-induced necroptosis.

Vitamin A - modified Betulin polymer micelles with hepatic targeting capability for hepatic fibrosis protection

Targeting hepatic stellate cells (HSCs) can improve the therapeutic efficacy of medicines used to treat hepatic fibrosis. The present work aimed to study the feasibility of homing devices with vitamin A(VA) chemically attached for delivering betulin(Bt)specifically to HSCs. The manufacture and characterisation of VA modified poly (ethylene glycol) -poly (lactide-co-glycolide) block copolymer micelles loaded with Bt (Bt/ VAPPMs) and their potential therapeutic benefits in vitro and in vivo are described in this paper. Bt/VAPPMs were made in a nearly spherical core-shell configuration with diameters under 200nm.In vitro release study showed that Bt/VAPPMs exhibited steady and continuous release for over 168 hours. Bt/VAPPMs had good biocompatibility at the cellular level, according to the safety evaluation, and elicited no inflammatory response in mice. More importantly, as uptake behavior studied in cells and bioimaging experiments in vivo, Bt/VAPPMs exhibited an instinctive liver- targeting capability to focus on activated HSCs. Efficacy tests revealed that administering Bt/VAPPMs effectively inhibits collagen I expression in LX-2 cells in vitro, and this effect was also seen in a mouse model of liver fibrosis. Overall, results demonstrated that Bt/VAPPMs is a promising drug delivery system that possesses specific HSCs targeting ability for treating hepatic fibrosis.

EP3 enhances adhesion and cytotoxicity of NK cells toward hepatic stellate cells in a murine liver fibrosis model

Natural killer (NK) cells exhibit antifibrotic properties in liver fibrosis (LF) by suppressing activated hepatic stellate cell (HSC) populations. Prostaglandin E2 (PGE2) plays a dual role in innate and adaptive immunity. Here, we found that E-prostanoid 3 receptor (EP3) was markedly downregulated in NK cells from liver fibrosis mice and patients with liver cirrhosis. NK cell-specific deletion of EP3 aggravated hepatic fibrogenesis in mouse models of LF. Loss of EP3 selectively reduced the cytotoxicity of the CD27+CD11b+ double positive (DP) NK subset against activated HSCs. Mechanistically, deletion of EP3 impaired the adhesion and cytotoxicity of DP NK cells toward HSCs through modulation of Itga4-VCAM1 binding. EP3 upregulated Itga4 expression in NK cells through promoting Spic nuclear translocation via PKC-mediated phosphorylation of Spic at T191. Activation of EP3 by sulprostone alleviated CCL4-induced liver fibrosis in mice. Thus, EP3 is required for adhesion and cytotoxicity of NK cells toward HSCs and may serve as a therapeutic target for the management of LF.

Guizhi Fuling pill attenuates liver fibrosis in vitro and in vivo via inhibiting TGF-β1/Smad2/3 and activating IFN-γ/Smad7 signaling pathways

ABSTRACT Liver fibrosis resulting from chronic liver injuries (CLI) is a common health problem globally. Guizhi Fuling pill (GZFL), a modern preparation from traditional Chinese medicine, exhibited anti-dysmenorrhea, anti-inflammatory, and immune-regulative effects. However, the effect of GZFL on liver fibrosis remains unknown. In this research, LX-2 cells were stimulated with acetaldehyde for mimicking liver fibrosis progression in vitro. In addition, carbon tetrachloride (CCl4)-induced mouse model of liver fibrosis was established as well. The data revealed GZFL obviously suppressed the proliferation and triggered the apoptosis of acetaldehyde-stimulated LX-2 cells. In addition, GZFL prevented acetaldehyde-induced activation of LX-2 cells via downregulation of TGF-β1, p-Smad2, p-Smad3, CUGBP1, and upregulation of p-STAT1 and Smad7. Meanwhile, GZFL significantly alleviated CCl4‑induced liver fibrosis, as evidenced by the decrease of ALT and AST levels. Moreover, GZFL downregulated the expressions of TGF-β1, p-Smad2, p-Smad3, and CUGBP1 in CCl4-treated mice. Furthermore, GZFL remarkably elevated the levels of IFN-γ, p-STAT1, and Smad7 in CCl4-treated mice. To sum up, GZFL was able to inhibit liver fibrosis in vitro and in vivo through suppressing TGF-β1/Smad2/3-CUGBP1 signaling and activating IFN-γ/STAT1/Smad7 signaling. Thus, GZFL might have a potential to act as a therapeutic agent for anti-fibrotic therapy.

Mitigation of liver fibrosis via hepatic stellate cells mitochondrial apoptosis induced by metformin

Liver fibrosis, a disease characterized by the excessive accumulation of extracellular matrix originating from activated hepatic stellate cells (HSCs), is a common pathological response to chronic liver injury resulting from a variety of insults. However, drugs that effectively block the activation of HSCs have still not been adequately investigated. This study demonstrates that metformin decreased the number of activated-HSCs through induction of apoptosis, but did not impact numbers of hepatocytes. Metformin upregulated BAX activation with facilitation of BIM, BAD and PUMA; downregulated Bcl-2 and Bcl-xl, but did not affect Mcl-1. Additionally, metformin induced cytochrome c release from mitochondria into the cytoplasm, directly triggering caspase-9-mediated mitochondrial apoptosis. The decline in mitochondrial membrane potential (ΔΨm) and deposition of superoxide in mitochondria accelerated the destruction of the integrity of mitochondrial membrane. Moreover, we verified the therapeutic effect of metformin in our mouse model of liver fibrosis associated with nonalcoholic steatohepatitis (NASH) in which hepatic function, NASH lesions and fibrosis were improved by metformin. In conclusion, this study indicated that metformin has significant therapeutic value in NASH-derived liver fibrosis by inducing apoptosis in HSCs, but does not affect the proliferation of hepatocytes.

NUAK1 promotes organ fibrosis via YAP and TGF-β/SMAD signaling.

Fibrosis is a central pathway that drives progression of multiple chronic diseases, yet few safe and effective clinical antifibrotic therapies exist. In most fibrotic disorders, transforming growth factor-β (TGF-β)-driven scarring is an important pathologic feature and a key contributor to disease progression. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are two closely related transcription cofactors that are important for coordinating fibrogenesis after organ injury, but how they are activated in response to tissue injury has, so far, remained unclear. Here, we describe NUAK family kinase 1 (NUAK1) as a TGF-β-inducible profibrotic kinase that is up-regulated in multiple fibrotic organs in mice and humans. Mechanistically, we show that TGF-β induces a rapid increase in NUAK1 in fibroblasts. NUAK1, in turn, can promote profibrotic YAP and TGF-β/SMAD signaling, ultimately leading to organ scarring. Moreover, activated YAP and TAZ can induce further NUAK1 expression, creating a profibrotic positive feedback loop that enables persistent fibrosis. Using mouse models of kidney, lung, and liver fibrosis, we demonstrate that this fibrogenic signaling loop can be interrupted via fibroblast-specific loss of NUAK1 expression, leading to marked attenuation of fibrosis. Pharmacologic NUAK1 inhibition also reduced scarring, either when initiated immediately after injury or when initiated after fibrosis was already established. Together, our data suggest that NUAK1 plays a critical, previously unrecognized role in fibrogenesis and represents an attractive target for strategies that aim to slow fibrotic disease progression.

Fibroblast activation protein activated antifibrotic peptide delivery attenuates fibrosis in mouse models of liver fibrosis

In liver fibrosis, activated hepatic stellate cells are known to overexpress fibroblast activation protein. Here we report a targeted antifibrotic peptide-delivery system in which fibroblast activation protein, which is overexpressed in fibrotic regions of the liver, liberates the antifibrotic peptide melittin by cleaving a fibroblast activation protein-specific site in the peptide. The promelittin peptide is linked to pegylated and maleimide-functionalized liposomes, resulting in promelittin-modified liposomes. The promelittin-modified liposomes were effective in reducing the viability of activated hepatic stellate cells but not that of control cells. In three types of liver fibrosis mouse models, intravenously administered promelittin-modified liposomes significantly reduces fibrotic regions. In addition, in the bile duct ligation mouse model promelittin-modified liposome-treatment increases overall survival. Although this peptide-delivery concept was tested for liver fibrosis, it can potentially be adapted to other fibrotic diseases.

Investigating the effect of gender on liver fibrosis and associated sarcopenia

Gender influences the incidence of liver fibrosis, which has a 2:1 male-to-female ratio and different outcomes in the two genders, including sarcopenia that affects 40-60% of patients with advanced liver fibrosis. Thus, we assessed the influence of gender in the pathophysiological mechanisms of liver fibrosis, regeneration and associated muscle dysfunction. A mouse model of liver fibrosis was obtained by injecting increasing doses of carbon tetrachloride (CCl4, from 0.17 to 0.72 mL/g body weight) for a maximum of 12 weeks, followed by a 8 week-washout period.