Injection Of Carbon Tetrachloride Research Articles

Preparation, physicochemical characterization, and bioactivity evaluation of berberine-entrapped albumin nanoparticles

Berberine (BBR) is an isoquinoline alkaloid with several clinical therapeutic applications. Its low water solubility, absorption, and cellular bioavailability diminish BBR's therapeutic efficacy. In this study, BBR was encapsulated into bovine serum albumin nanoparticles (BSA NPs) core to reduce BBR limitations and enhance its clinical therapeutic properties. Several physicochemical characterization tools, such as Dynamic Light Scattering and Ultraviolet–Visible spectroscopic measurements, field emission transmission electron microscopy surface morphology, Fourier transforms infrared spectroscopy, thermal stability analysis, and releasing studies, were used to evaluate the BBR-BSA NPs. Compared to BBR, BBR-BSA nanoparticles demonstrated superior free radical scavenging and antioxidant capacities, anti-hemolytic and anticoagulant efficacies, and antimicrobial activities, as demonstrated by the findings of the in vitro studies. Furthermore, a stressed pancreatic rat model was induced using a high-fat, high-sucrose diet plus carbon tetrachloride injection. The in vivo results revealed that BBR-BSA NPs substantially restored peripheral glucose metabolism and insulin sensitivity. Oral administration of BBR-BSA NPs also improved pancreatic β-cells homeostasis, upregulated pancreatic antioxidant mechanisms, inhibited oxidants generation, and attenuated oxidative injury in the stressed pancreatic tissues. In conclusion, our in vitro and in vivo results confirmed that BBR-BSA NPs demonstrated more potent antioxidant properties and restored pancreatic homeostasis compared to BBR.

Mechanism of Allicin for Improvement of Hepatic Fibrosis by Regulating Autophagy

Abstract: Objectives: To explore the effects of allicin on the expression of mitochondrial autophagyrelated proteins in liver tissues of hepatic fibrosis (HF) rats. Materials and Methods: 45 SD rats were randomly divided into Normal, Model, Low, Middle and High groups. The HF rat model was replicated intraperitoneal injection of carbon tetrachloride and ethanol gavage for 4 weeks. Detection of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in rat serum; HE and Masson staining to observe pathological changes; Evaluating LC 3B protein expression by Immunofluorescence; Western Blotting and qRT-PCR methods were used to detect PINK1, Parkin, Beclin 1, and LC3 gene and protein expression levels in rat liver tissues. Results: Compared with the Normal group, the degree of HF in the model group was obvious, the volume fraction of collagen increased significantly (p<0.001), the LC 3B protein expression significantly upregulation (p<0.001), and the rat serum ALT and AST increased significantly (p<0.001, respectively). Up-regulation of PINK1, Parkin, Beclin 1, and LC 3B mRNA and protein expression and LC 3II/LC 3I ratio in liver tissues (p<0.001, respectively); Compared with the model group, Allicin treated groups had reduced HF degree, collagen volume fraction decreased significantly (p<0.05, respectively), LC 3B protein was significantly reduced (p<0.05, respectively), and liver tissue PINK1, Parkin, Beclin 1, and LC3B mRNA and protein expressions, and LC 3II/LC 3I ratio were down-regulated (p<0.05, respectively). Conclusion:Allicin could improve hepatic fibrosis. The mechanism might be related to down-regulating the expression of PINK1, Parkin, Beclin 1, and LC 3 gene and proteins and interfering with the level of mitochondrial autophagy Keywords: Allicin, HF, PINK1, Parkin, Autophagy.

MiR-345–5p curbs hepatic stellate cell activation and liver fibrosis progression by suppressing hypoxia-inducible factor-1alpha expression

Hepatic fibrosis, as a common stage of multiple liver diseases, currently has no effective drug treatment. Emerging evidence shows that miRNAs participate in the progression of liver fibrosis. However, the potential role of miRNAs in hepatic fibrosis is not yet fully understood. Herein, we first confirmed that miR-345–5p expression was significantly decreased in activated hepatic stellate cells (HSCs) and fibrotic livers. Functional analysis showed that overexpression of miR-345–5p in human LX-2 cells suppressed the expression of profibrotic markers and cellular proliferation in vitro. Using a dual-luciferase assay, we demonstrated that miR-345–5p regulates HSC activation by targeting the 3′UTR of HIF-1α mRNA. In addition, overexpression of miR-345–5p in vivo alleviated murine liver fibrosis induced by carbon tetrachloride (CCl4) injection, high-fat diet (HFD) feeding and bile duct ligation (BDL). Furthermore, overexpression of miR-345–5p downregulated the expression of HIF-1α and fibrosis markers in livers from different fibrosis models. Collectively, we conclude that miR-345–5p mediates the activation of HSCs by targeting HIF-1α, which subsequently modulates TGFβ/Smad2/Smad3 signaling. Thus, miR-345–5p may become a novel therapeutic target for the treatment of liver fibrosis.

XBP1-mediated activation of the STING signalling pathway in macrophages contributes to liver fibrosis progression

Background & AimsXBP1 modulates the macrophage proinflammatory response, but its function in macrophage stimulator of interferon genes (STING) activation and liver fibrosis is unknown. X-box binding protein 1 (XBP1) has been shown to promote macrophage nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) activation in steatohepatitis. Herein, we aimed to explore the underlying mechanism of XBP1 in the regulation of STING signalling and the subsequent NLRP3 activation during liver fibrosis.MethodsXBP1 expression was measured in the human fibrotic liver tissue samples. Liver fibrosis was induced in myeloid-specific Xbp1-, STING-, and Nlrp3-deficient mice by carbon tetrachloride injection, bile duct ligation, or a methionine/choline-deficient diet.ResultsAlthough increased XBP1 expression was observed in the fibrotic liver macrophages of mice and clinical patients, myeloid-specific Xbp1 deficiency or pharmacological inhibition of XBP1 protected the liver against fibrosis. Furthermore, it inhibited macrophage NLPR3 activation in a STING/IRF3-dependent manner. Oxidative mitochondrial injury facilitated cytosolic leakage of macrophage self-mtDNA and cGAS/STING/NLRP3 signalling activation to promote liver fibrosis. Mechanistically, RNA sequencing analysis indicated a decreased mtDNA expression and an increased BCL2/adenovirus E1B interacting protein 3 (BNIP3)-mediated mitophagy activation in Xbp1-deficient macrophages. Chromatin immunoprecipitation (ChIP) assays further suggested that spliced XBP1 bound directly to the Bnip3 promoter and inhibited the transcription of Bnip3 in macrophages. Xbp1 deficiency decreased the mtDNA cytosolic release and STING/NLRP3 activation by promoting BNIP3-mediated mitophagy activation in macrophages, which was abrogated by Bnip3 knockdown. Moreover, macrophage XBP1/STING signalling contributed to the activation of hepatic stellate cells.ConclusionsOur findings demonstrate that XBP1 controls macrophage cGAS/STING/NLRP3 activation by regulating macrophage self-mtDNA cytosolic leakage via BNIP3-mediated mitophagy modulation, thus providing a novel target against liver fibrosis.Lay summaryLiver fibrosis is a typical progressive process of chronic liver disease, driven by inflammatory and immune responses, and is characterised by an excess of extracellular matrix in the liver. Currently, there is no effective therapeutic strategy for the treatment of liver fibrosis, resulting in high mortality worldwide. In this study, we found that myeloid-specific Xbp1 deficiency protected the liver against fibrosis in mice, while XBP1 inhibition ameliorated liver fibrosis in mice. This study concluded that targeting XBP1 signalling in macrophages may provide a novel strategy for protecting the liver against fibrosis.

Endogenous Follistatin-like 1 guarantees the immunomodulatory properties of mesenchymal stem cells during liver fibrotic therapy

BackgroundMesenchymal stem cell (MSC) therapy has been shown to be a promising option for liver fibrosis treatment. However, critical factors affecting the efficacy of MSC therapy for liver fibrosis remain unknown. Follistatin-like 1 (FSTL1), a TGF-β-induced matricellular protein, is documented as an intrinsic regulator of proliferation and differentiation in MSCs. In the present study, we characterized the potential role of FSTL1 in MSC-based anti-fibrotic therapy and further elucidated the mechanisms underlying its action.MethodsHuman umbilical cord-derived MSCs were characterized by flow cytometry. FSTL1low MSCs were achieved by FSTL1 siRNA. Migration capacity was evaluated by wound-healing and transwell assay. A murine liver fibrotic model was created by carbon tetrachloride (CCl4) injection, while control MSCs or FSTL1low MSC were transplanted via intravenous injection 12 weeks post CCl4 injection. Histopathology, liver function, fibrosis degree, and inflammation were analysed thereafter. Inflammatory cell infiltration was evaluated by flow cytometry after hepatic nonparenchymal cell isolation. An MSC-macrophage co-culture system was constructed to further confirm the role of FSTL1 in the immunosuppressive capacity of MSCs. RNA sequencing was used to screen target genes of FSTL1.ResultsFSTL1low MSCs had comparable gene expression for surface markers to wildtype but limited differentiation and migration capacity. FSTL1low MSCs failed to alleviate CCl4-induced hepatic fibrosis in a mouse model. Our data indicated that FSTL1 is essential for the immunosuppressive action of MSCs on inflammatory macrophages during liver fibrotic therapy. FSTL1 silencing attenuated this capacity by inhibiting the downstream JAK/STAT1/IDO pathway.ConclusionsOur data suggest that FSTL1 facilitates the immunosuppression of MSCs on macrophages and that guarantee the anti-fibrotic effect of MSCs in liver fibrosis.

MSCs cell fates in murine acute liver injury and chronic liver fibrosis induced by carbon tetrachloride.

Mesenchymal stem cells (MSCs) therapy has shown potential benefits in multiple diseases. However, their clinic performance is not as satisfactory as expected. This study aimed to provide an alternative explanation by comparing MSCs' fates in different liver diseases. The distribution and therapeutic effects of hMSCs were investigated in acute liver injury (ALI) and chronic liver fibrosis (CLF) mice models, respectively. The two models were induced by single or repeated injection of carbon tetrachloride (CCl4) separately. The increase of hMSCs exposure in the liver (AUCliver 0-72 h) were more significant in ALI than in CLF (177.1% vs. 96.2%). In the ALI model, the hMSCs exposures in the lung (AUClung 0-72 h) increased by nearly 50% while decreased by 60.7% in CLF. The efficacy satellite study indicated that hMSCs could significantly ameliorate liver injury in ALI, but its effects in CLF were limited. In the ALI, suppressed Natural Killer (NK) cell activities were observed, while NK cell activities were increased in CLF. The depletion of NK cells could increase hMSCs exposure in mice. For mice MSC (mMSCs), their cell fates in ALI were very similar to hMSCs in ALI: mMSCs' exposure in the liver and lung increased in ALI. In conclusion, our study revealed the distinct cell pharmacokinetic patterns of MSCs in ALI and CLF mice, which might be at least partially attributed to the different NK cell activities in the two liver diseases. This finding provided a novel insight into the varied MSCs' therapeutic efficacy in the clinic. Significance Statement Currently, there is little knowledge about the PK behavior of cell products like MSCs. This study was the first time investigating the influence of liver diseases on cell fates and efficacies of MSCs and the underneath rationale. The exposure was distinct between two representative liver disease models, which directly linked with the therapeutic performance that MSCs achieved. The difference could be attributed to the NK cells-mediated MSCs clearance.

Comprehensive Analysis of the Differentially Expressed Transcriptome with ceRNA Networks in a Mouse Model of Liver Cirrhosis

Background: Hepatic cirrhosis is the consequence of various chronic liver disease for which there is no curative treatment. In this study, based on RNA sequencing (RNA-seq) and subsequent bioinformatic analysis, we aim to explore the biological function of non-coding RNAs (ncRNAs) in hepatic cirrhosis. Methods: The hepatic cirrhosis models were induced by the intraperitoneal injection of carbon tetrachloride (CCl4). The transcriptome profile was aquired by RNA-seq, of which result was verified by quantitative real-time PCR (qRT-PCR). The competing endogenous RNA (ceRNA) networks were visualized by Cytoscape software. The enrichment analyses of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were conducted. Results: The differentially expressed transcript of liver cirrhosis is consist of 2369 mRNAs, 374 lncRNAs, 91 circRNAs and 242 miRNAs (|log2(fold change)|≥1 and P&lt;0.05). The RNA-seq results were highly consistent with qRT-PCR validation of DEGs (four upregulated and four down-regulated, including ENSMUSG00000047517, ENSMUST00000217449, novel-circ-001366, miR-383-5p, ENSMUSG00000078683, ENSMUST00000148206, novel-circ-002669 and miR-216a-5p). Based on ceRNA theory, a circRNA-lncRNA co-regulated ceRNA network was established. Enrichment analysis revealed the potential key regulatory process during the liver cirrhosis progression. Conclusion: In conclusion, the present study comprehensively analyzed differentially expressed transcripts in CCl4-induced liver cirrhosis. Our findings explored the gene signatures for liver cirrhosis’ diagnosis and precise treatment.

Sorafenib Attenuates Fibrotic Hepatic Injury Through Mediating Lysine Crotonylation.

BackgroundLiver fibrosis is an independent contributor of chronic liver diseases, and regressing liver fibrosis is considered a potential therapeutic target for chronic liver diseases. We aimed to explore the effects and mechanism of sorafenib in liver fibrosis.MethodsMale Sprague Dawley (SD) rats were subjected to subcutaneous injection of carbon tetrachloride (CCl4) for 8 weeks to induce liver fibrosis and then treated with sorafenib. The degree of liver fibrosis was analyzed by hematoxylin–eosin (H&E) staining, Masson staining, and Picrosirius red (PSR) staining. Serum biochemical indexes were detected by fully automatic biochemical analyzer or enzyme-linked immunosorbent assay (ELISA). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of pro-fibrotic genes. Immunohistochemical staining and Western blotting were carried out to evaluate the levels of lysine crotonylation.ResultsLiver index was reduced with oral sorafenib in CCl4-induced rats. Serum liver function (alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL)) and fibrosis indicators (type III procollagen (PC-III), hyaluronic acid (HA), and laminin (LN)) were attenuated with sorafenib treatment. Sorafenib improved the hepatic structure and fibrotic progression. The expression of fibrosis-related genes was remarkely reduced with sorafenib treatment. Meanwhile, sorafenib inhibited α-SMA and collagen I cumulation induced by CCl4 injection. Besides, protein lysine crotonylation especially the crotonylated H2BK12 (H2BK12cr) and crotonylated H3K18 (H3K18cr) were reversed by sorafenib, which were decreased in response to CCl4 treatment. Spearman correlation analysis shown lysine crotonylation expression was negatively correlated with serum fibrotic indicators. Conversely, crotonylation-regulated enzymes, which negatively regulate protein crotonylation, were increased in response to CCl4 treatment, while sorafenib reduced their expression.ConclusionSorafenib exerts significant anti-fibrotic effects through mediating crotonylation-regulated enzymes and protein crotonylation in fibrotic rats.

The combination of experimental periodontitis and oral microbiota from periodontitis patients aggravates liver fibrosis in mice.

Periodontitis (PD) is the sixth most prevalent disease around the world and is involved in the development and progression of multiple systemic diseases. Previous studies have reported that PD may aggravate liver injuries. The objective of this study was to investigate whether and how PD affects liver fibrosis. Ligature-induced PD (LIP) was induced in male C57/B6J mice, and sub-gingival plaques (PL) from patients with PD were applied to mouse teeth. Liver fibrosis was induced by carbon tetrachloride (CCl4 ) injection. The mice were randomly divided into six groups: Oil, Oil+LIP, Oil+LIP+PL, CCl4 , CCl4 +LIP, and CCl4 +LIP+PL. Alveolar bone resorption was evaluated by methylene blue staining. Hepatic function was analysed by serum alanine aminotransferase and hepatic hydroxyproline. Picrosirius red and α-smooth muscle actin (SMA) staining were used to evaluate the fibrotic area. RNA sequencing and quantitative RT-PCR were used to measure gene expression. Western blotting was used to measure protein levels. Flow cytometry was used to analyse the accumulation of immune cells. Mouse microbiota were analysed using 16S rRNA gene sequencing. Mice in the CCl4 +LIP+PL group displayed higher serum alanine aminotransferase and hepatic hydroxyproline as well as more Picrosirius red-positive and α-SMA-positive areas in liver samples than those of the CCl4 group, suggesting that PD (LIP+PL) aggravated CCl4 -induced hepatic dysfunction and liver fibrosis. Consistently, the expression of fibro-genic genes and the protein levels of transforming growth factor β were much higher in the CCl4 +LIP+PL group than in the CCl4 group. Flow cytometry revealed that PD increased the accumulation of immune cells, including Kupffer cells, B cells, and Th17 cells, in the liver of mice with CCl4 treatment. PD also increased the expression of inflammatory genes and activated pro-inflammatory nuclear factor-kappa B pathway in the livers of CCl4 -injected mice. Moreover, PD altered both oral and liver microbiota in CCl4 -injected mice. PD aggravates CCl4 -induced hepatic dysfunction and fibrosis in mice, likely through the increase of inflammation and alteration of microbiota in the liver.

MiR-146b-5p activation of hepatic stellate cells contributes to the progression of fibrosis by directly targeting HIPK1.

The present study aimed to explore the biological functions of microRNA (miR)-146b-5p and homeodomain interacting protein kinase 1 (HIPK1) in the progression of hepatic fibrosis (HF) and to identify the underlying mechanism. A rat HF model was established by administering a subcutaneous injection of carbon tetrachloride (CCl4). Relative levels of miR-146b-5p and HIPK1 in fibrotic rat liver tissues and the rat hepatic stellate cell (HSC) line HSC-T6 were measured by quantitative reverse transcription PCR, western blotting and immunohistochemistry. Following activation of HSC-T6 cells by lipopolysaccharide (LPS) induction, cell viability was examined by MTT assay. Transfection of miR-146b-5p mimic or inhibitor into HSC-T6 cells was performed, with the aim to identify the influence of miR-146b-5p on HSC-T6 cell behavior. The targeting relationship between miR-146b-5p and HIPK1 was predicted by TargetScan 7.2 and StarBase 3.0 and it was later verified by a dual-luciferase reporter assay. Through lentivirus transfection, the biological function of HIPK1 in regulating the progression of HF and the underlying mechanism were investigated. The results showed that miR-146b-5p was upregulated in liver tissues of rats with HF and activated HSC-T6 cells, while HIPK1 was downregulated in liver tissues of rats with HF and activated HSC-T6 cells. miR-146b-5p was able to upregulate the activation markers of LPS-induced HSC-T6 cells, upregulate COL1A1 and TGF-β, increase cell viability and contribute to fibrosis progression. HIPK1 was validated as the direct target of miR-146b-5p and its overexpression could effectively reduce the effect of miR-146b-5p in contribution to the progression of HF. In conclusion, miR-146b-5p was significantly upregulated during the progression of HF. By targeting and downregulating HIPK1, miR-146b-5p could significantly activate HSCs, upregulate COL1A1 and TGF-β and contribute to fibrosis progression. miR-146b-5p is a potential biomarker and therapeutic target for HF.

Deep learning for assessing liver fibrosis based on acoustic nonlinearity maps: an in vivo study of rabbits

This study aimed to assess liver fibrosis in rabbits by deep learning models based on acoustic nonlinearity maps. Injection of carbon tetrachloride was used to induce liver fibrosis. Acoustic nonlinearity maps, which were built by data of echo signals, were used as input data for deep learning model. Convolutional neural network (CNN), CNN combined with support vector machine (SVM), CNN combined with random forest and CNN combined with logistic regression were used as deep learning model. Nested 10-fold cross-validation was used to search hyperparameters and evaluate performance of models. Histologic examination of liver specimens of the rabbits was performed to evaluate the fibrosis stage. Receiver operator characteristic curve and area under curve (AUC) were used for estimating the probability of the correct prediction of liver fibrosis stages. A total of 600 acoustic nonlinearity maps were used. Model of CNN combined with SVM demonstrated the best diagnostic performance compared with all other methods for diagnosis of significant fibrosis (≥F2, AUC = 0.82), advanced fibrosis (≥F3, AUC = 0.88) and cirrhosis (F4, AUC = 0.90). Model of CNN showed the second highest AUCs. The deep learning model based on acoustic nonlinearity maps demonstrated potential for evaluation of liver fibrosis.

Intravenous iron injection does not impair liver function in a rat model of cirrhosis

There is a lack of convincing data concerning the safety of iron therapy in patients with advanced liver cirrhosis (LC). This study investigated the hepatic effects of ferric carboxymaltose, an intravenous iron supplement, in a rat model of cirrhosis. In total, 45 Sprague-Dawley rats were allocated into three groups: normal rats (control group, n=15), cirrhotic rats receiving intravenous normal saline (LC group, n=15), and cirrhotic rats receiving 20 mg/kg intravenous ferric carboxymaltose (LC-iron group, n=15). LC was induced by twice-weekly intraperitoneal injection of carbon tetrachloride. Biochemical parameters were compared at 0, 2, 14, and 28 days. Additionally, liver tissue samples were extracted from five rats in each group at 2, 14, and 28 days for evaluation of the degrees of hepatic fibrosis and iron deposition. Inflammatory and oxidative stress markers were also compared among groups. Serum alanine transferase levels did not significantly differ between the LC and LC-iron groups at 0 (443±110 vs. 444±117 IU/L, P>0.99), 2 (453±117 vs. 479±136 IU/L, P=0.84), 14 (1,535±1,058 vs. 1,578±711 IU/L, P=0.55), or 28 days (2,067±641 vs. 2,533±914 IU/L, P=0.15). The degree of hepatic fibrosis was comparable between the groups, although hepatic iron accumulation was greater in the LC-iron group than in the LC group. The levels of inflammatory and oxidative stress markers were significantly lower in the LC-iron group than in the LC group. In our rat model of cirrhosis, the administration of intravenous iron appears safe. However, further preclinical and clinical studies are warranted to confirm the safety and efficacy of intravenous iron in patients with LC or end-stage liver disease.

Rat bone marrow mesenchymal stem cells (BMSCs) inhibit liver fibrosis by activating GSK3\u03b2 and inhibiting the Wnt3a/\u03b2-catenin pathway

BackgroundBone marrow mesenchymal stem cells (BMSCs) can effectively alleviate liver fibrosis, which is a pathological injury caused by various chronic liver diseases. This study aimed to investigate the antifibrotic effects of BMSCs and elucidate the underlying mechanism by which BMSCs affect liver fibrosis in vitro and in vivo.MethodsAfter the rat liver fibrosis model was induced by continuous injection of carbon tetrachloride (CCl4), BMSCs were administered for 4 weeks, and histopathological analysis and liver function tests were performed. T6 hepatic stellate cells (HSC-T6 cells) were stimulated by TGF-β1, and the activation and proliferation of cells were analyzed by CCK-8 assays, flow cytometry, real-time PCR, western blotting and enzyme-linked immunosorbent assay (ELISA).ResultsOur data demonstrated that BMSCs effectively reduced the accumulation of collagen, enhanced liver functionality and ameliorated liver fibrosis in vivo. BMSCs increased the sub-G1 population in HSC-T6 cells. In addition, coculture with BMSCs reduced the expression of α-SMA, collagen I, cyclin-D1, and c-Myc in HSC-T6 cells and activated the phosphorylation of GSK3β. The GSK3β inhibitor SB216763 reversed the effect of BMSCs. The Wnt/β-catenin signalling pathway was involved in BMSC-mediated inhibition of HSC-T6 cell activation.ConclusionsOur data suggested that BMSCs exerted antifibrotic effects by activating the expression of GSK3β and inhibiting the Wnt3a/β-catenin signalling pathway.

MicroRNA-122 inhibits hepatic stellate cell proliferation and activation in vitro and represses carbon tetrachloride-induced liver cirrhosis in mice

ObjectiveThis study aimed to determine the roles of microRNA (miR)-122 in the activation of hepatic stellate cells (HSCs) and liver cirrhosis. MethodsRat primary HSCs were incubated with transforming growth factor-beta (TGF-β), during which miR-122 and EphB2 expression was measured. miR-122 mimic and/or pcDNA3.1 EphB2 was transfected into TGF-β-induced HSCs. A mouse model of liver cirrhosis was established via an intraperitoneal injection of carbon tetrachloride (CCl4), followed by the injection of miR-122 agomir. Levels of serum alanine transaminase (ALT) and aspartate aminotransferase (AST) were measured. Fibronectin (FN), alpha smooth muscle actin (α-SMA), Collagen I, miR-122, and EphB2 expression was evaluated in liver tissues and HSCs. Cell proliferation was measured using CCK-8 assay. Interactions between miR-122 and EphB2 were assessed using dual luciferase reporter assay. ResultsmiR-122 (0.15-fold) was downregulated and EphB2 (mRNA: 5.06-fold; protein: 2.35-fold) was upregulated after TGF-β induction of HSCs. Overexpressed miR-122 decreased proliferation and EphB2 (mRNA: 0.46-fold; protein: 0.62-fold), FN (mRNA: 0.45-fold; protein: 0.64-fold), α-SMA (mRNA: 0.48-fold; protein: 0.51-fold), and Collagen I (mRNA: 0.44-fold; protein: 0.51-fold) expression in HSCs, which was abrogated by EphB2 upregulation. miR-122 expression was reduced by 0.21-fold and serum ALT and AST levels were enhanced in mice following 8-week CCl4 induction along with increased expression of FN, α-SMA, and Collagen I in liver tissues, which was blocked by miR-122 overexpression. Moreover, EphB2 was a target gene of miR-122. ConclusionmiR-122 curtails HSC proliferation and activation by targeting EphB2 and suppresses liver cirrhosis in mice.

FSTL1 promotes liver fibrosis by reprogramming macrophage function through modulating the intracellular function of PKM2

ObjectiveFollistatin-like protein 1 (FSTL1) is widely recognised as a secreted glycoprotein, but its role in modulating macrophage-related inflammation during liver fibrosis has not been documented. Herein, we aimed to characterise...

Shugan Huoxue Huayu Fang attenuates carbon tetrachloride-induced hepatic fibrosis in rats by inhibiting transforming growth factor-β1/Smad signaling.

To investigate the potential mechanism by which Shugan Huoxue Huayu Fang (SGHXHYF) ameliorates liver fibrosis. Liver fibrosis was induced in rats by intraperitoneal injection of carbon tetrachloride (CCl4) in peanut oil solution (40%, 3 mL/kg body weight) twice a week for 8 weeks. A normal control group received the same volume of peanut oil alone. During weeks 5-8, the CCl4-injected rat groups were administered saline (vehicle control), colchicine (0.1 mg/mL, 1 mg/kg, positive control), or SGHXHYF (0.1 mg/mL; 0.3, 0.6 and 1.2 mg/kg) once daily by oral gavage. Rats were sacrificed 24 h after the last treatment. Blood samples were collected for measurement of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin (ALB), collagen Ⅰ and collagen Ⅲ levels. Liver samples were analyzed by histopathological staining, Masson's staining of extracellular matrix proteins, and immune-ohistochemical staining of αsmooth muscle actin (α-SMA). TGF-β1/Smad protein and mRNA levels were analyzed by Western blot and quantitative reverse transcription-polymerase chain reaction analysis, respectively. In vitro experiments were also performed using rat hepatic stellate cells (HSCs). Compared with the control animals, CCl4-exposed rats exhibited elevated serum levels of ALT, AST, ALP, collagen I, and collagen III; reduced serum levels of ALB; and increased collagen deposition and αSMA expression in liver sections, reflecting liver fibrosis. CCl4 also increased expression of TGF-β1 and the activated (phosphorylated) forms of Smad2 and Smad3 but reduced expression of the negative regulator Smad7 in the liver. Notably, concomitant administration of SGHXHYF to CCl4-exposed rats was found to significantly reverse or abolish the pro-fibrotic effects of CCl4 in the liver and reduced serum transferase levels. Analysis of HSCs in vitro confirmed that, mechanistically, SGHXHYF inhibited activation of the TGF-β1/Smad signaling pathway by downregulating phosphorylated Smad2 and Smad3 and upregulating Smad7 levels. SGHXHYF ameliorated CCl4-induced liver fibrosis by inhibiting the TGF-β1/Smad signaling pathway. These findings suggest that SGHXHYF may have clinical utility for the treatment or prevention of liver fibrosis.

A fast and accurate mouse model for inducing non-alcoholic steatohepatitis.

This study aimed to perform a head-to-head comparison of changes during NASH progression throughout 6-11 weeks of an experiment to supply a faster nutritional model in mimicking NASH to decrease the duration and cost of in vivo studies. New therapies are urgently needed because of the growing prevalence of non-alcoholic steatohepatitis (NASH) and the lack of an effective treatment approach. Currently, dietary interventions are the most efficient options. This study compared features of NASH in a murine model using protocol that combined special nutritional regimes based on the combination of 21.1% fat, 41% sucrose, and 1.25% cholesterol with weekly intraperitoneal injections of carbon tetrachloride (CCl4). Male C57BL/6J mice received either special compositions + CCl4 (NASH group) or standard chow diet (healthy control group) for 11 weeks. Liver histopathology based on hematoxylin and eosin (H&E) and Masson's Trichrome (TC) staining and biochemical analyses were used to assess disease progression. In C57BL/6J mice administered a high fat, high cholesterol, high sucrose diet and CCl4 for 8 weeks, steatohepatitis with pronounced hepatocyte ballooning, inflammation, steatosis, and fibrosis was observed. According to the NAFLD activity scoring system, the maximum NAS score was manifested after 8-9 weeks (NAS score: 6.75). Following this protocol also led to a significant increase in AST and ALT, total cholesterol, and total triglyceride serum levels in the NASH group. Following the special nutritional regime based on high fat, cholesterol, and sucrose in combination with CCL4 injections resulted in a NASH model using C57BL/6J mice in a shorter time compared to similar studies. The obtained histopathological NASH features can be advantageous for preclinical drug testing.

Exosome-mediated delivery of RBP-J decoy oligodeoxynucleotides ameliorates hepatic fibrosis in mice.

Rationale: Macrophages play multi-dimensional roles in hepatic fibrosis. Studies have implicated Notch signaling mediated by the transcription factor RBP-J in macrophage activation and plasticity. Additionally, we have previously shown that myeloid-specific disruption of RBP-J can ameliorate hepatic fibrosis in mice. Accordingly, we next asked whether blocking Notch signaling in macrophages could serve as a therapeutic strategy to treat hepatic fibrosis. In this study, we used a combination of transcription factor decoy oligodeoxynucleotides (ODNs) and exosomes to test this possibility.Methods: Hairpin-type decoy oligodeoxynucleotides (ODNs) were designed for the transcription factor RBP-J. The effects of RBP-J decoy ODNs on Notch signaling were evaluated by western blot, quantitative RT-PCR, luciferase reporter assays, and electrophoretic mobility shift assays. ODNs were loaded into HEK293T-derived exosomes by electroporation. A hepatic fibrosis mouse model was established by the intraperitoneal injection of carbon tetrachloride or bile duct ligation. Mice with hepatic fibrosis were administered exosomes loaded with RBP-J decoy ODNs via tail vein injection. The in vivo distribution of exosomes was analyzed by fluorescence labeling and imaging. Liver histology was examined using hematoxylin and eosin, Sirius red, and Masson staining, as well as immunohistochemical staining for Col1α1 and αSMA.Results: We found that RBP-J decoy ODNs could be efficiently loaded into exosomes and inhibit the activation of Notch signaling. Furthermore, exosomes administered via the tail vein were found to be primarily taken up by hepatic macrophages in mice with liver fibrosis. Importantly, RBP-J decoy ODNs delivered by exosomes could efficiently inhibit Notch signaling in macrophages and ameliorate hepatic fibrosis in mice.Conclusions: Combined, our data showed that the infusion of exosomes loaded with RBP-J decoy ODNs represents a promising therapeutic strategy for the treatment of hepatic fibrosis.

Dual therapy with zinc acetate and rifaximin prevents from ethanol-induced liver fibrosis by maintaining intestinal barrier integrity.

BACKGROUNDHepatic overload of gut-derived lipopolysaccharide dictates the progression of alcoholic liver disease (ALD) by inducing oxidative stress and activating Kupffer cells and hepatic stellate cells through toll-like receptor 4 signaling. Therefore, targeting the maintenance of intestinal barrier integrity has attracted attention for the treatment of ALD. Zinc acetate and rifaximin, which is a nonabsorbable antibiotic, had been clinically used for patients with cirrhosis, particularly those with hepatic encephalopathy, and had been known to improve intestinal barrier dysfunction. However, only few studies focused on their efficacies in preventing the ALD-related fibrosis development. AIMTo investigate the effects of a combined zinc acetate with rifaximin on liver fibrosis in a mouse ALD model.METHODSTo induce ALD-related liver fibrosis, female C57BL/6J mice were fed a 2.5% (v/v) ethanol-containing Lieber-DeCarli liquid diet and received intraperitoneal carbon tetrachloride (CCl4) injection twice weekly (1 mL/kg) for 8 wk. Zinc acetate (100 mg/L) and/or rifaximin (100 mg/L) were orally administered during experimental period. Hepatic steatosis, inflammation and fibrosis as well as intestinal barrier function were evaluated by histological and molecular analyses. Moreover, the direct effects of both agents on Caco-2 barrier function were assessed by in vitro assays.RESULTSIn the ethanol plus CCl4-treated mice, combination of zinc acetate and rifaximin attenuated oxidative lipid peroxidation with downregulation of Nox2 and Nox4. This combination significantly inhibited the Kupffer cells expansion and the proinflammatory response with blunted hepatic exposure of lipopolysaccharide and the toll-like receptor 4/nuclear factor kB pathway. Consequently, liver fibrosis and hepatic stellate cells activation were efficiently suppressed with downregulation of Mmp-2, -9, -13, and Timp1. Both agents improved the atrophic changes and permeability in the ileum, with restoration of tight junction proteins (TJPs) by decreasing the expressions of tumor necrosis factor α and myosin light chain kinase. In the in vitro assay, both agents directly reinforced ethanol or lipopolysaccharide-stimulated paracellular permeability and upregulated TJPs in Caco-2 cells.CONCLUSIONDual therapy with zinc acetate and rifaximin may serve as a strategy to prevent ALD-related fibrosis by maintaining intestinal barrier integrity.

COX-2/sEH Dual Inhibitor PTUPB Alleviates CCl 4 -Induced Liver Fibrosis and Portal Hypertension.

Background: 4-(5-phenyl-3-{3-[3-(4-trifluoromethylphenyl)-ureido]-propyl}-pyrazol-1-yl) -benzenesulfonamide (PTUPB), a dual cyclooxygenase-2 (COX-2)/soluble epoxide hydrolase (sEH) inhibitor, was found to alleviate renal, pulmonary fibrosis and liver injury. However, few is known about the effect of PTUPB on liver cirrhosis. In this study, we aimed to explore the role of PTUPB in liver cirrhosis and portal hypertension (PHT).Method: Rat liver cirrhosis model was established via subcutaneous injection of carbon tetrachloride (CCl4) for 16 weeks. The experimental group received oral administration of PTUPB (10 mg/kg) for 4 weeks. We subsequently analyzed portal pressure (PP), liver fibrosis, inflammation, angiogenesis, and intra- or extrahepatic vascular remodeling. Additionally, network pharmacology was used to investigate the possible mechanisms of PTUPB in live fibrosis.Results: CCl4 exposure induced liver fibrosis, inflammation, angiogenesis, vascular remodeling and PHT, and PTUPB alleviated these changes. PTUPB decreased PP from 17.50 ± 4.65 to 6.37 ± 1.40 mmHg, reduced collagen deposition and profibrotic factor. PTUPB alleviated the inflammation and bile duct proliferation, as indicated by decrease in serum interleukin-6 (IL-6), liver cytokeratin 19 (CK-19), transaminase, and macrophage infiltration. PTUPB also restored vessel wall thickness of superior mesenteric arteries (SMA) and inhibited intra- or extrahepatic angiogenesis and vascular remodeling via vascular endothelial growth factor (VEGF), von Willebrand factor (vWF), etc. Moreover, PTUPB induced sinusoidal vasodilation by upregulating endothelial nitric oxide synthase (eNOS) and GTP-cyclohydrolase 1 (GCH1). In enrichment analysis, PTUPB engaged in multiple biological functions related to cirrhosis, including blood pressure, tissue remodeling, immunological inflammation, macrophage activation, and fibroblast proliferation. Additionally, PTUPB suppressed hepatic expression of sEH, COX-2, and transforming growth factor-β (TGF-β).Conclusion: 4-(5-phenyl-3-{3-[3-(4-trifluoromethylphenyl)-ureido]-propyl}-pyrazol-1-yl)- benzenesulfonamide ameliorated liver fibrosis and PHT by inhibiting fibrotic deposition, inflammation, angiogenesis, sinusoidal, and SMA remodeling. The molecular mechanism may be mediated via the downregulation of the sEH/COX-2/TGF-β.