Mouse Model Of Hepatic Fibrosis Research Articles

Experimental study on H2O2 activation of HSC-T6 and hepatic fibrosis in cholestatic mice by "Yajieshaba"

Ethnopharmacological relevanceYajieshaba (YJSB), approved by the Yunnan Provincial Food and Drug Administration in 2008, are known for their anti-inflammatory, antiviral, and pro-apoptotic properties, effectively treating Hepatic fibrosis (HF). However, its mechanism of action remains unclear. Aim of the studyThe objective of this investigation is to explore how YJSB influences the TGF-β1/Smad signaling pathway as a strategy for reducing HF. MethodsThe establishment of a HF model in mice involved ligation of the common bile duct, followed by administration of YJSB. Body and liver weights were measured, and the liver index calculated. Serum levels of ALT, AST, ALP, TBA, and TBIL were assessed using colorimetric methods. Additionally, liver homogenates were analyzed for PIIINP, Col-IV, LN, HA, and Hyp, as well as TGF-β1 activity, using ELISA. Histological analyses of liver sections, stained with H&E, Ag, and Masson's trichrome, were performed to examine inflammation and the accumulation of collagen and reticular fibers. These studies aimed to elucidate the pharmacodynamic effects of YJSB on HF in mice with bile duct obstruction. The target pathways of YJSB were preliminarily identified through immunofluorescence detection of TGF-β1, P-Smad2L, P-Smad2C, P-Smad3L, P-Smad3C, and Smad4 proteins. In vitro experiments included the induction of hepatic stellate cell (HSC-T6) activation by H2O2. A cell injury model was established for HSC-T6, and the CCK-8 assay was used to determine the optimal YJSB concentration and treatment duration. After pirfenidone (PFD) administration, which inhibits the TGF-β1/Smad pathway, the effects of YJSB on HSC-T6 cell proliferation were observed. ELISA assays quantified Col-III, α-SMA, and Col-I in cell lysates to assess YJSB's impact on collagen synthesis in HSC-T6 cells. Western blot analysis was performed to assess the protein levels within the TGF-β1/Smad signaling cascade. ResultsIn the HF mouse model, administration of YJSB notably augmented the body weight and reduced the liver index. Concurrently, there was an elevation in serum concentrations of ALP, AST, ALT, TBA, and TBIL. Similarly, in the liver homogenates of HF mice, increases were observed in the levels of HA, PIIINP, Col-IV, LN, Hyp, and TGF-β1. Histological assessments using H&E, Ag, and Masson stains indicated a substantial diminution in liver tissue damage. Through immunofluorescence analysis, it was discerned that YJSB modulated the expression of TGF-β1, P-Smad2L, P-Smad2C, and P-Smad3L downwards, while elevating P-Smad3C and Smad4 protein expressions. Additional investigations revealed a significant reduction in α-SMA, Col-I, and Col-III levels in cell culture fluids, suggesting a decrease in collagen synthesis and a protective role against cellular damage. Western blot analyses demonstrated that the TGF-β1/Smad pathway inhibitor, PFD, acted in synergy with YJSB, enhancing its regulatory effects on this pathway, decreasing levels of TGF-β1, P-Smad2L, P-Smad2C, P-Smad3L, and promoting the expression of P-Smad3C. ConclusionsYJSB demonstrates a pharmacodynamic effect against HF, enhancing liver functionality and effectively mitigating the damage associated with bile duct obstruction. The proposed action mechanism of YJSB involves modulation of the TGF-β1/Smad signaling pathway. Research indicates that YJSB might play a role in suppressing the movement, programmed cell death, and activation of HSC-T6, potentially decelerating the advancement of hepatic fibrosis.

Glycyrrhizic acid ameliorates hepatic fibrosis by inhibiting oxidative stress via AKR7A2

BackgroundHepatic fibrosis is a reversible pathological phenomenon caused by the abnormal proliferation of connective tissues in the liver for self-repair after persistent liver injury. Among these tissues, the activation status of hepatic stellate cells (HSCs) is crucial. Glycyrrhizic acid (GA) agents have been proven to have excellent anti-fibrosis effects, but their targets are unclear. PurposeTo investigate the anti-hepatic fibrosis effect of GA and its target in activated HSCs. MethodsA mouse model of hepatic fibrosis was prepared with 20 % carbon tetrachloride (CCl4) and GA was administered continuously for 4 weeks. Subsequently, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), type Ⅲ procollagen peptide (P III P), laminin (LN), hyaluronic acid (HA), and type Ⅳ collagen (Col Ⅳ) were measured. Liver tissues were subjected to hematoxylin and eosin (HE), Masson, and Sirius red staining and proteome sequencing analysis. Based on LX-2 cells, activity-based protein profiling (ABPP) was used to investigate the potential targets of GA, which was further validated by the cellular thermal shift assay (CETSA), immunofluorescence co-localization, molecular docking, small interfering RNA (siRNA) and western blot (WB) assays. ResultsIn vivo, GA significantly reduced serum ALT, AST, HA, P III P, Col IV, and LN levels. HE, Masson, and Sirius red staining showed that GA significantly ameliorated hepatic inflammatory response and collagen deposition in CCl4-treated mice. Proteome sequencing results showed that GA mainly regulated glutathione S-transferase family members involved in glutathione metabolism. In vitro, GA significantly inhibited LX-2 cell proliferation and reduced reactive oxygen species accumulation. ABPP suggested that aldo-keto reductase family 7 member A2 (AKR7A2) was the major binding protein of GA in LX-2 cells. CETSA, fluorescence co-localization, molecular docking, and surface plasmon resonance further validated GA binding to AKR7A2. The WB results showed that GA up-regulated AKR7A2 expression both in vitro and in vivo and was corroborated by siRNA experiments. ConclusionGA targeted AKR7A2 in LX-2 cells to defend against sustained oxidative stress injury, thereby inhibiting the proliferation of activated HSCs and reversing hepatic fibrosis.

Liver Fibrosis Amelioration by Macrophage-Biomimetic Polydopamine Nanoparticles via Synergistically Alleviating Inflammation and Scavenging ROS.

The progression of liver fibrosis is determined by the interaction of damaged hepatocytes, active hepatic stellate cells, and macrophages, contributing to the development of oxidative stress and inflammatory environments within the liver. Unfortunately, the current pharmacological treatment for liver fibrosis is limited by its inability to regulate inflammation and oxidative stress concurrently. In this study, we developed a cell membrane biomaterial for the treatment of liver fibrosis, which we designated as PM. PM is a biomimetic nanomaterial constructed by encapsulating polydopamine (PDA) with a macrophage membrane (MM). It is hypothesized that PM nanoparticles (NPs) can successfully target the site of inflammation, simultaneously inhibit inflammation, and scavenge reactive oxygen species (ROS). In vitro experiments demonstrated that PM NPs exhibited strong antioxidant properties and the ability to neutralize pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β). Moreover, the capacity of PM NPs to safeguard cells from oxidative stress and their anti-inflammatory efficacy in an inflammatory model were validated in subsequent cellular experiments. Additionally, PM NPs exhibited a high biocompatibility. In a mouse model of hepatic fibrosis, PM NPs were observed to aggregate efficiently in the fibrotic liver, displaying excellent antioxidant and anti-inflammatory properties. Notably, PM NPs exhibited superior targeting, anti-inflammatory, and ROS scavenging abilities in inflamed tissues compared to MM, PDA, or erythrocyte membrane-encapsulated PDA. Under the synergistic effect of anti-inflammation and antioxidant, PM NPs produced significant therapeutic effects on liver fibrosis in mice. In conclusion, the synergistic alleviation of inflammation and ROS scavenging by this specially designed nanomaterial, PM NPs, provides valuable insights for the treatment of liver fibrosis and other inflammatory- or oxidative stress-related diseases.

Ba-Qi-Rougan formula alleviates hepatic fibrosis by suppressing hepatic stellate cell activation via the MSMP/CCR2/PI3K pathway

Ethnopharmacological relevanceThe Ba-Qi-Rougan formula (BQRGF) is a traditional and effective compound prescription from Traditional Chinese Medicine (TCM) utilized in treating hepatic fibrosis (HF). Aim of the studyWe aimed to evaluate the therapeutic efficacy of BQRGF on HF and explore the underlying mechanisms of action. Materials and methodsUPLC-Q-TOF-MS technology was employed to identify the material basis of BQRGF. Mice with carbon tetrachloride (CCl4)-induced HF received BQRGF at three doses (3.87, 7.74, and 15.48 g/kg per day). We examined serum and liver biochemical indicators and liver histology to assess the therapeutic impact. Primary mouse cells were isolated and utilized for experimental analysis. MSMP expression levels were examined in vitro and in vivo experimental models, including human and mouse tissue. Furthermore, lentivirus and small interfering RNA (siRNA) transfections were employed to manipulate microseminoprotein (MSMP) expression in LO2 cells (human normal liver cells). These manipulated LO2 cells were then co-cultured with LX2 human hepatic stellate cells (HSCs). Through the modulation of MSMP expression in co-cultured cells, administering recombinant MSMP (rMSMP) with or without BQRGF-medicated serum, and using specific pathway inhibitors or agonists in LX2 cells, we elucidated the underlying mechanisms. ResultsA total of 48 compounds were identified from BQRGF, with 12 compounds being absorbed into the bloodstream and 9 compounds being absorbed into the liver. Four weeks of BQRGF treatment in the HF mouse model led to significant improvements in biochemical and molecular assays and histopathology, particularly in the medium and high-dose groups. These improvements included a reduction in the level of liver injury and fibrosis-related factors. MSMP levels were elevated in human and mouse fibrotic liver tissues, and this increase was mitigated in HF mice treated with BQRGF. Moreover, primary cells and co-culture studies revealed that BQRGF reduced MSMP expression, decreased the expression of the hepatic stellate cell (HSC) activation markers, and suppressed critical phosphorylated protein levels in the CCR2/PI3K/AKT pathway. These findings were further validated using CCR2/PI3K/AKT signaling inhibitors and agonists in MSMP-activated LX2 cells. ConclusionsCollectively, our results suggest that BQRGF combats HF by diminishing MSMP levels and inhibiting MSMP-induced HSC activation through the CCR2/PI3K/AKT pathway.

Sodium alginate combined with oxymatrine ameliorates CCl4-induced chemical hepatic fibrosis in mice

Hepatic fibrosis (HF) is a challenging clinical problem. Both sodium alginate (SA) and oxymatrine (OM) can be used to treat HF; however, the influence of viscosity on the therapeutic efficacy of sodium alginate is currently unknown. This study used a CCl4-induced HF mouse model to screen the specifications and doses of SA and investigate its therapeutic effects on HF in combination with OM. Sodium alginate of different viscosities ameliorated HF in mice, with 232 mPa·s SA delivered at a dose of 100 mg/kg showing remarkable therapeutic effect, characterized by reduced aspartate transaminase/alanine transaminase levels, reduced expression of α-SMA, collagen I, and other related genes, and increased abundance of beneficial intestinal probiotics such as Lactococcus and Blautia. The combination treatment further improved other related indices and increased the abundance of Phascolarctobacterium and Oscillospiraceae. These results suggest that the oral administration of SA may improve HF via the “gut-liver axis” based on the gut microbiota and has potential clinical applications.

Knockdown of TXNDC5 alleviates CCL4-induced hepatic fibrosis in mice by enhancing endoplasmic reticulum stress

BackgroundHepatic fibrosis is a common pathological process in many chronic liver diseases. TXNDC5 has been shown to be involved in the progression of renal and pulmonary fibrosis. However, the role of TXNDC5 in hepatic fibrosis is unknown. The purpose of this study is to explore the role and mechanism of TXNDC5 in hepatic fibrosis. MethodsWe used TGF-β1 to activate LX-2 cells and reduced TXNDC5 expression by short hairpin RNA. Cell viability was assessed by CCK-8 assay. Cell apoptosis was analyzed by flow cytometry or Tunel assay. The fibrosis-related proteins and endoplasmic reticulum stress (ERs)-related proteins were measured by western blot. ELISA was performed to detect COL1AL levels and MMP2/9 activities in cell medium. A mouse model of hepatic fibrosis was constructed by intraperitoneal injection of CCL4. HE and Masson staining were performed to assess fibrosis in mouse liver tissue. ResultsThe results show that TXNDC5 was up-regulated in activated LX-2 cells and CCL4-induced hepatic fibrosis mice. Knockdown of TXNDC5 inhibited the viability of activated LX-2 cells and the production of collagen COL1A1. Knockdown of TXNDC5 promoted apoptosis of activated LX-2 cells. Mechanically, inhibition of TXNDC5 enhanced ERs, and the ERs inhibitor 4-Phenylbutyric acid (4-PBA) reversed the effect of TXNDC5 on activated LX-2 cells. More importantly, knockdown of TXNDC5 alleviated CCl4-induced hepatic fibrosis in mice. ConclusionsKnockdown of TXNDC5 may reduce hepatic fibrosis by regulating ERs, and targeting TXNDC5 seems to be a candidate treatment for hepatic fibrosis.

Axitinib attenuates the progression of liver fibrosis by restoring mitochondrial function.

Liver fibrosis can progress to cirrhosis and hepatocellular carcinoma, which may eventually lead to liver failure and even death. No direct anti-fibrosis drugs are available at present. Axitinib is a new generation of potent multitarget tyrosine kinase receptor inhibitors, but its role in liver fibrosis remains unclear. In this study, a CCl4-induced hepatic fibrosis mouse model and a TGF-β1-induced hepatic stellate cell model were used to explore the effect and mechanism of axitinib on hepatic fibrosis. Results confirmed that axitinib could alleviate the pathological damage of liver tissue induced by CCl4 and inhibit the production of glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase. It also inhibited collagen and hydroxyproline deposition and the protein expression of Col-1 and α-SMA in CCl4-induced liver fibrosis. In addition, axitinib inhibited the expression of CTGF and α-SMA in TGF-β1-induced hepatic stellate cells. Further studies showed that axitinib inhibited mitochondrial damage and reduced oxidative stress and NLRP3 maturation. The use of rotenone and antimycin A confirmed that axitinib could restore the activity of mitochondrial complexes I and III, thereby inhibiting the maturation of NLRP3. In summary, axitinib inhibits the activation of HSCs by enhancing the activity of mitochondrial complexes I and III, thereby alleviating the progression of liver fibrosis. This study reveals the strong potential of axitinib in the treatment of liver fibrosis.

The effect of hepatic stellate cell derived-IL-11 on hepatocyte injury in hepatic fibrosis

AimsThis study aimed to elucidate the role of Interleukin-11 (IL-11) in hepatic fibrosis (HF) and its potential as a therapeutic target for HF treatment. Materials and methodsWe investigated IL-11 expression in patients with varying degrees of liver injury through ELISA and immunohistochemistry. A CCl4-induced HF mouse model was constructed to study IL-11 expression and cell apoptosis using Western blotting (WB) and other techniques. The expression of IL-11 was silenced using rAAV8 in the mouse model. In vitro stimulation of hepatic stellate cells (LX-2) with TGF-β1, and of LO-2 cells with exogenous IL-11, were performed. Cell supernatants of TGF-β1-stimulated LX-2 were used to culture LO-2 cells, with apoptosis monitored via flow cytometry and WB. Key findingsIncreased IL-11 levels were observed in patients and the HF mouse model, with silencing reducing IL-11 expression. In vitro experiments revealed increased endogenous IL-11 in TGF-β1-stimulated LX-2 cells and an increase in apoptotic index, IL11RA, and gp130 in IL-11-stimulated LO-2 cells. Cell apoptosis was reduced in the siRNA/IL11, siRNA/IL11RA, and anti-IL11 groups. WB and immunohistochemistry results showed upregulated p-JNK, p-ERK, and p-P53 expressions in the CCl4-induced HF mouse model and IL-11-treated LO-2 cells. SignificanceOur findings suggest IL-11 enhances LX-2 cell activation and proliferation, and promotes LO-2 cell apoptosis through JNK/ERK signaling pathways. This suggests that targeting IL-11 secretion may serve as a potential therapeutic strategy for HF, providing a foundation for its clinical application in HF treatment.

Aqueous extract of Amydrium sinense (Engl.) H. Li alleviates hepatic fibrosis by suppressing hepatic stellate cell activation through inhibiting Stat3 signaling.

Background: The present study aimed to investigate the protective effect of the water extract of Amydrium sinense (Engl.) H. Li (ASWE) against hepatic fibrosis (HF) and clarify the underlying mechanism. Methods: The chemical components of ASWE were analysed by a Q-Orbitrap high-resolution mass spectrometer. In our study, an in vivo hepatic fibrosis mouse model was established via an intraperitoneal injection of olive oil containing 20% CCl4. In vitro experiments were conducted using a hepatic stellate cell line (HSC-T6) and RAW 264.7 cell line. A CCK-8 assay was performed to assess the cell viability of HSC-T6 and RAW264.7 cells treated with ASWE. Immunofluorescence staining was used to examine the intracellular localization of signal transducer and activator of transcription 3 (Stat3). Stat3 was overexpressed to analyse the role of Stat3 in the effect of ASWE on HF. Results: Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that candidate targets of ASWE, associated with protective effects against hepatic fibrosis, were related to inflammation response. ASWE ameliorated CCl4-induced liver pathological damage and reduced the liver index and alanine transaminase (ALT) and aspartate transaminase (AST) levels. ASWE also decreased the serum levels of collagen Ⅰ (Col Ⅰ) and hydroxyproline (Hyp) in CCl4-treated mice. In addition, the expression of fibrosis markers, including α-SMA protein and Acta2, Col1a1, and Col3a1 mRNA, was downregulated by ASWE treatment in vivo. The expression of these fibrosis markers was also decreased by treatment with ASWE in HSC-T6 cells. Moreover, ASWE decreased the expression of inflammatory markers, including the Tnf-α, Il6 and Il1β, in RAW264.7 cells. ASWE decreased the phosphorylation of Stat3 and total Stat3 expression and reduced the mRNA expression of the Stat3 gene in vivo and in vitro. ASWE also inhibited the nuclear shuttling of Stat3. Overexpression of Stat3 weakened the therapeutic effect of ASWE and accelerated the progression of HF. Conclusion: The results show that ASWE protects against CCl4-induced liver injury by suppressing fibrosis, inflammation, HSC activation and the Stat3 signaling pathway, which might lead to a new approach for preventing HF.

Eupatilin Ameliorates Hepatic Fibrosis and Hepatic Stellate Cell Activation by Suppressing β-catenin/PAI-1 Pathway.

The activation of hepatic stellate cells (HSCs) has proved to be pivotal in hepatic fibrosis. Therefore, the suppression of HSC activation is an effective anti-fibrotic strategy. Although studies have indicated that eupatilin, a bioactive flavone found in Artemisia argyi, has anti-fibrotic properties, the effect of eupatilin on hepatic fibrosis is currently unclear. In this study, we used the human hepatic stellate cell line LX-2 and the classical CCl4-induced hepatic fibrosis mouse model for in vitro and vivo experiments. We found that eupatilin significantly repressed the levels of the fibrotic markers COL1α1 and α-SMA, as well as other collagens in LX-2 cells. Meanwhile, eupatilin markedly inhibited LX-2 cell proliferation, as verified by the reduced cell viability and down-regulation of c-Myc, cyclinB1, cyclinD1, and CDK6. Additionally, eupatilin decreased the level of PAI-1 in a dose-dependent manner, and knockdown of PAI-1 using PAI-1-specific shRNA significantly suppressed the levels of COL1α1, α-SMA, and the epithelial-mesenchymal transition (EMT) marker N-cadherin in LX-2 cells. Western blotting indicated that eupatilin reduced the protein level of β-catenin and its nuclear translocation, while the transcript level of β-catenin was not affected in LX-2 cells. Furthermore, analysis of histopathological changes in the liver and markers of liver function and fibrosis revealed that hepatic fibrosis in CCl4-treated mice was markedly alleviated by eupatilin. In conclusion, eupatilin ameliorates hepatic fibrosis and hepatic stellate cell activation by suppressing the β-catenin/PAI-1 pathway.

Anti-inflammatory and antifibrotic effects of CBP/β-catenin inhibitor for hepatocytes: small molecular inhibitor, OP-724 possibly improves liver function.

Wnt/β-catenin signals are associated with several functions, including organ fibrosis. A synthetic small molecule, OP-724 (prodrug of C-82), an inhibitor of cyclic AMP response element-binding protein (CREB)-binding protein (CBP)/β-catenin, has demonstrated antifibrotic activity in mouse models of hepatic fibrosis. OP-724 is mediated by profibrotic and antifibrotic cells, such as hepatic stellate cells, macrophages, and neutrophils. In this study, the direct effects of C-82 on hepatocytes in hepatic inflammation were investigated. Immortalized human hepatocytes were pretreated with inflammatory cytokines. Moreover, the alteration of mRNA and protein expressions of cytokines and chemokines associated with hepatic inflammation and fibrosis, and of mitochondria-related molecules after C-82 treatment were analyzed in this study. The mRNA expression of several proinflammatory and profibrotic chemokines was upregulated by the stimulation of these inflammatory cytokines. In addition, this increase was prevented by C-82. In particular, the protein secretion of CCL2, CCL5, CXCL1, CXCL9, and CXCL10 was noticeably upregulated by TNFα and prevented by additional C-82. Moreover, C-82 increased the VEGF-A and FGF-2 proteins, categorized as anti-inflammatory and antifibrotic molecules, respectively. It also increased the expression of mitochondrial components and mitochondrial membrane potential. In conclusion, C-82 inhibits hepatocyte-mediated proinflammation and fibrogenesis. It also directly activates the mitochondrial function, thus improving liver dysfunction.

Exploration of potential mechanism of Rougan formula against hepatic fibrosis by network analysis and experimental assessment

Ethnopharmacological relevanceRougan Formula (RG) has long been clinically applied to treat hepatic fibrosis in patients with different chronic liver diseases. However, the core active substances and the potential pharmacological mechanisms of RG remain unclear. Aim of the studyThe purpose of this study is to explore bioactive components, key targets, and potential mechanisms of RG by performing network pharmacological analyses and experimental model validation. Materials and methodsAll chemical components in RG extract were identified using ultraperformance liquid chromatography-quadrupole/time-of-flight tandem mass technology. The candidate components and drug targets of RG, as well as disease-related genes, were extracted from TCMSP and GeneCards databases. The potential pathways related to genes were predicted by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. The core bioactive components, key targets, and signaling pathways were ultimately obtained by analyzing protein-protein interaction (PPI) and component-target-pathway (C-T-P) networks. Subsequently, the efficacy and underlying mechanisms of RG on hepatic fibrosis were experimentally validated in transforming growth factor-beta 1 (TGF-β1)-induced hepatic stellate cell activation model and CCL4-induced hepatic fibrosis mouse model. ResultsA total of 52 components in RG extract were obtained, and 22 of them were selected as the core bioactive components. Five hundred and thirty-nine overlapped targets were determined by matching drug targets with disease-related targets. The results of PPI and C-T-P network analyses revealed 100 key targets and 19 signaling pathways associated with RG efficacy. In vitro and in vivo studies further verified that RG exerted a significant anti-hepatic fibrotic effect by suppressing the activation of hepatic stellate cells by downregulating the TGF-β1/Smads signaling pathway. ConclusionsThese results may provide some evidence for further clinical research and development of RG formula as an effective and safe drug for hepatic fibrosis treatment.

Yao medicine Amydrium hainanense suppresses hepatic fibrosis by repressing hepatic stellate cell activation via STAT3 signaling.

Ethnopharmacological relevance: Hepatic fibrosis (HF) occurs in response to chronic liver injury and may easily develop into irreversible liver cirrhosis or even liver cancer. Amydrium hainanense water extract (AHWE) is a water-soluble component extracted from the Yao medicine Amydrium hainanense (H.Li, Y.Shiao & S.L.Tseng) H.Li, which is commonly used for treating inflammatory diseases in folk. Previous evidence suggested that AHWE significantly inhibited hepatic stellate cell activation. However, little is known regarding the therapeutic effect of AHWE in HF and its underlying action mechanism. Objective: Investigation of the therapeutic effect of AHWE in HF and its underlying mechanism. Methods: The therapeutic effect of AHWE was tested in vivo using an HF mouse model via an intraperitoneal injection of carbon tetrachloride (CCl4). Histological evaluation of liver injury and fibrosis were tested by H&E staining and Masson's trichrome staining. Serum levels of ALT, AST, collagen type I (Col I), and hydroxyproline (HYP) were measured. The mRNA expression of liver fibrotic and inflammatory genes were tested, and the protein levels of alpha smooth muscle actin (α-SMA) and signal transducers and activators of transcription 3 (STAT3) were analyzed. The in vitro experiments were conducted using HSC-T6 and RAW264.7 cell lines. Results: Treatment with AHWE significantly reversed histopathological liver damage and liver function abnormalities in CCl4 mouse model. Also, the serum levels of ALT, AST, Col I, and HYP in CCl4-induced HF mice were improved in AHWE treatment. Further, AHWE showed a remarkable inhibitory effect on the expression of fibrosis markers (Acta2, Col1a1, and Col3a1) and inflammatory factors (Stat3, Tnfa, Il6, and Il1b) induced by CCl4. The results of in vitro experiments were consistent with those obtained in vivo. In addition, it is shown that STAT3 signaling was involved in the anti-fibrotic effects of AHWE as evidenced by STAT3 overexpression. Conclusion: The present study proposed a novel ethnomedicine for HF and suggested the underlying role of STAT3 signaling pathway regulation in this anti-fibrotic effect of the proposed medicine. These findings would serve as solid scientific evidence in support of the development of AHWE as a novel alternative or complementary therapy for HF prevention and treatment.

Kinin B1 receptor blockade attenuates hepatic fibrosis and portal hypertension in chronic liver diseases in mice

Background and aimsKinin B1 receptors (B1Rs) are implicated in the pathogenesis of fibrosis. This study examined the anti-fibrotic effects of B1R blockade with BI 113823 in two established mouse models of hepatic fibrosis induced by intraperitoneal carbon tetrachloride (CCl4) injection or bile duct ligation (BDL). The mechanisms underlying the protection afforded by B1R inhibition were examined using human peripheral blood cells and LX2 human hepatic stellate cells (HSCs).MethodsFibrotic liver diseases were induced in mice by intraperitoneal carbon tetrachloride (CCl4) injection for 6 weeks, and by bile duct ligation (BDL) for 3 weeks, respectively. Mice received daily treatment of vehicle or BI 113823 (B1R antagonist) from onset of the experiment until the end of the study.ResultsB1Rs were strongly induced in fibrotic mouse liver. BI 113823 significantly attenuated liver fibrosis and portal hypertension (PH), and improved survival in both CCl4 and BDL mice. BI 113823 significantly reduced the expression of fibrotic proteins α-SMA, collagens 1, 3, 4, and profibrotic growth factors PDGF, TGFβ, CTGF, VEGF, proliferating cell nuclear antigen; and reduced hepatic Akt phosphorylation in CCl4- and BDL-induced liver fibrosis. BI 113823 also reduced expression of Cytokines IL-1, IL-6; chemokines MCP-1, MCP-3 and infiltration of inflammatory cells; and inhibited human monocyte and neutrophil activation, transmigration, TNF-α & MPO production in vitro. BI 113823 inhibited TGF-β and B1R agonist-stimulated human-HSC activation, contraction, proliferation, migration and fibrosis protein expression, and inhibited activation of PI3K/Akt signalling pathway.ConclusionsB1Rs merits consideration as a novel therapeutic target for chronic liver fibrosis and PH.

Myrrhone inhibits the progression of hepatic fibrosis by regulating the abnormal activation of hepatic stellate cells.

We focus on exploring the antihepatic fibrosis effect of Myrrhone (Myr), a compound extracted from myrrh, and its effective target. Mouse hepatic stellate cells (HSCs) were cultured in vitro and activated by transforming growth factor-β induction. After Myr intervention, cell viability was assessed by theCell Counting Kit-8 assay. The α-smooth muscle actin(α-SMA) and Collagen I levels were measured by immunofluorescence, and the expressions of tumor necrosis factor-α, interleukin-6, andmatrix metalloproteinase-9were examined by enzyme-linked immunosorbent assay, and the p-Smad3 protein level in HSCs was determined by Western Blot. Small molecule-protein docking and pull-down experiments were conducted to validate the binding capacity between Nard and Smad3. In animal experiments, a mouse model of hepatic fibrosis was established with carbon tetrachloride. Myr was administered by gavage daily to determine the serum alanine aminotransferase and aspartate transaminase levels. The severity of hepatic fibrosis was evaluated by Masson staining, the α-SMA and Collagen I expressions were measured by immunohistochemistry, and the histopathological changes were examined by Sirius red and hematoxylin and eosin staining. Myr suppressed the abnormal activation of HSCs, inhibited the cell viability, downregulated the α-SMA and Collagen I, and inhibited the p-Smad3 expression. After silencing Smad3, the effect of Myr was inhibited. Molecular docking and pull-down experiments revealed the presence of a targeted binding relationship between Myr and Smad3. In mouse experiments, Myr could inhibit hepatic fibrosis. This study discovers that Myr can affect the phosphorylation of Smad3, and inhibit the activation of HSCs and the progression of hepatic fibrosis.

Improved anti-fibrotic effects by combined treatments of simvastatin and NS-398 in experimental liver fibrosis models.

Background/AimsEfficient anti-fibrotic therapies are required for the treatment of liver cirrhosis. Hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) and cyclooxygenase-2 (COX-2) inhibitors have been reported to have anti-fibrotic effects. Here, we investigated whether combined treatment with a statin and a COX-2 inhibitor has synergistic anti-fibrotic effects.MethodsThe effects of treatment strategies incorporating both simvastatin and a COX-2 inhibitor, NS-398, were investigated using an immortalized human hepatic stellate cell line (LX-2) and a hepatic fibrosis mouse model developed using thioacetamide (TAA) in drinking water. Cellular proliferation was investigated via 5-bromo-2-deoxyuridine uptake. Pro- and anti-apoptotic factors were investigated through Western blotting and real-time polymerase chain reaction analysis.ResultsThe evaluation of the anti-proliferative effects on LX-2 cells showed that the observed effects were more pronounced with combination therapy than with single-drug therapy. Moreover, hepatic fibrosis and collagen deposition decreased significantly in TAA-treated mice in response to the combined treatment strategy. The mechanisms underlying the anti-fibrotic effects of the combination therapy were investigated. The effects of the combination therapy were correlated with increased expression levels of extracellular signal-regulated kinase 1/2 signaling molecules, upregulation of the Bax/Bcl-2 signaling pathway, inhibition of the transforming growth factor-β signaling pathway, and inhibition of tissue inhibitor of matrix metalloproteinases 1 and 2.ConclusionsThe combination of simvastatin and NS-398 resulted in a synergistic anti-fibrotic effect through multiple pathways. These findings offer a theoretical insight into the possible clinical application of this strategy for the treatment of advanced liver diseases with hepatic fibrosis.

Trimetazidine inhibits liver fibrosis and hepatic stellate cell proliferation and blocks transforming growth factor-β (TGFβ)/Smad signaling in vitro and in vivo

ABSTRACT Trimetazidine (TMZ) has been used extensively to treat coronary artery disease and to reduce fibrosis. Liver fibrosis is a reversible process. However, the impacts of TMZ on liver fibrosis triggered by CCl4 and on hepatic stellate cells in liver fibrosis remain to be elaborated. In the current study, the liver fibrosis models were constructed by using CCl4-induced mice and TGF-β-induced hepatic stellate cells. The involvement of TMZ in liver fibrosis was subsequently investigated. In the CCl4-induced hepatic fibrosis mouse model, it was shown that the expression levels of alanine aminotransferase and aspartate aminotransferase were reduced after TMZ treatment; the expression levels of the extracellular matrix proteins colla1 and α-SMA were down-regulated; furthermore, the expression levels of TGFβ/Smad signaling proteins were inhibited. In TGF-β-induced hepatic stellate cells, compared to the TGF-β-induced group, cell proliferation and migration were inhibited after TMZ treatment; meanwhile, extracellular matrix protein and TGFβ/Smad signaling protein expression levels followed the same trend as in the hepatic fibrosis model. In conclusion, TMZ could block the TGFβ/Smad signaling in liver fibrosis model, with inhibiting liver fibrosis and hepatic stellate cell proliferation. This may broaden the application sphere of TMZ in liver fibrosis therapy.

Exosomal microRNA-618 derived from mesenchymal stem cells attenuate the progression of hepatic fibrosis by targeting Smad4

ABSTRACT Hepatic fibrosis (HF) is a pathological phenomenon that occurs during the process of long-term damage and repair in the liver. This condition will lead to the development of cirrhosis and even liver cancer if untreated. Previous evidence has shown that exosomes derived from mesenchymal stem cells (MSCs), carrying microRNAs (miRs), can affect the pathogenesis of HF. Therefore, the present study aimed to identify novel exosomal miRs derived from MSCs that play a critical role in the progression of HF. Next, the expression data of differentially expressed miRs (DEMs) of patients with liver cirrhosis and healthy controls were obtained from the Gene Expression Omnibus dataset. DEMs were analyzed using Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Moreover, to further confirm the function of exosomal miR-618 derived from MSCs on the pathogenesis of HF in vivo, a mouse model of HF was established. The results of the present study suggested that a close associated existed between DEMs and HF. Based on the results of the bioinformatics analysis, miR-618 was one of the main downregulated miRs involved in cirrhosis. In addition, miR-618 could be transferred from MSCs to LX-2 cells via exosomes; exosomal miR-618 derived from MSCs inhibited the viability and migration of LX-2 cells that were treated with TGF-β. Furthermore, exosomal miR-618 derived from MSCs attenuated the progression of HF via targeting Smad4. These findings indicated that treatment of exosomal miR-618 derived from MSCs might serve as a new strategy for HF.

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.

Dendropanoxide, a Triterpenoid from Dendropanax morbifera, Ameliorates Hepatic Fibrosis by Inhibiting Activation of Hepatic Stellate Cells through Autophagy Inhibition.

Hepatic fibrosis results from chronic liver damage and is characterized by excessive accumulation of extracellular matrix (ECM). In this study, we showed that dendropanoxide (DPX), isolated from Dendropanax morbifera, had anti-fibrotic effects on hepatic fibrosis by inhibiting hepatic stellate cell (HSC) activation. DPX suppressed mRNA and protein expression of α-SMA, fibronectin, and collagen in activated HSCs. Moreover, DPX (40 mg/kg) treatment significantly lowered levels of liver injury markers (aspartate aminotransferase and alanine transaminase), expression of fibrotic markers, and deposition of ECM in a carbon tetrachloride-induced mouse model. Anti-fibrotic effects of DPX were comparable to those of silymarin in a hepatic fibrosis mouse model. As a possible mechanism of anti-fibrotic effects, we showed that DPX inhibited autophagosome formation (LC3B-II) and degradation of p62, which have important roles in HSC activation. These findings suggest that DPX inhibits HSC activation by inhibiting autophagy and can be utilized in hepatic fibrosis therapy.