CCl4-induced Liver Fibrosis Research Articles

Aloin Attenuates Oxidative Stress, Inflammation, and CCl4-Induced Liver Fibrosis in Mice: Possible Role of TGF-β/Smad Signaling.

Liver fibrosis refers to the excessive buildup of extracellular matrix (ECM) components in liver tissue. It is considered a pathological response to liver damage for which there is no effective treatment. Aloin, an anthraquinone compound isolated from the aloe plant, has shown good pharmacological effects in the treatment of gastric cancer, ulcerative colitis, myocardial hypertrophy, traumatic brain injury, and other diseases; however, its specific impact on liver fibrosis remains unclear. To address this gap, we conducted a study to explore the mechanisms underlying the potential antifibrotic effect of aloin. We constructed a mouse liver fibrosis model using carbon tetrachloride (CCl4) dissolved in olive oil as a modeling drug. Additionally, a cellular model was developed by using transforming growth factor β1 (TGF-β1) as a stimulus applied to hepatic stellate cells. After aloin intervention, serum alanine aminotransferase, hepatic hydroxyproline, and serum aspartate aminotransferase were reduced in mice after aloin intervention compared to CCl4-mediated liver injury without aloin intervention. Aloin relieved the oxidative stress caused by CCl4 via reducing hepatic malondialdehyde in liver tissue and increasing the level of superoxide dismutase. Aloin treatment decreased interleukin (IL)-1β, IL-6, and tumor necrosis factor-α and increased the expression of IL-10, which inhibited the inflammatory response in liver injury. In addition, aloin inhibited the activation of hepatic stellate cells and reduced the level of α-smooth muscle actin (α-SMA) and collagen type I. In cell and animal experiments, aloin attenuated liver fibrosis, acting through the TGF-β/Smad2/3 signaling pathway, and mitigated CCl4- and TGF-β1-induced inflammation. Thus, the findings of this study provided theoretical data support and a new possible treatment strategy for liver fibrosis.

Hepatocellular Brg1 promotes CCl4-induced liver inflammation, ECM accumulation and fibrosis in mice.

Hepatic fibrosis is a progressive pathological process involving the exhaustion of hepatocellular regenerative capacity and ultimately leading to the development of cirrhosis and even hepatocellular carcinoma. Brg1, the core subunit of the SWI/SNF chromatin-remodeling complex, was recently identified as important for liver regeneration. This study investigates the role of Brg1 in hepatic fibrosis development. Hepatocyte-specific Brg1 knockout mice were generated and injected with carbon tetrachloride (CCl4) for 4, 6, 8, and 12 weeks to induce liver fibrosis. Afterwards, liver fibrosis and liver damage were assessed. Brg1 expression was significantly increased in the fibrotic liver tissue of wild-type mice, as compared to that of untreated wild-type mice. The livers of the Brg1 knockout animals showed reduced liver inflammation, extracellular matrix accumulation, and liver fibrosis. TNF-α and NF-κB-mediated inflammatory response was reduced in Brg1 knockout animals. Brg1 promotes the progression of liver fibrosis in mice and may therefore be used as a potential therapeutic target for treating patients with liver fibrosis due to chronic injury.

Ginsenoside Rb1 induces hepatic stellate cell ferroptosis to alleviate liver fibrosis via the BECN1/SLC7A11 axis

Liver fibrosis is primarily driven by the activation of hepatic stellate cells (HSCs), a process associated with ferroptosis. Ginsenoside Rb1 (GRb1), a major active component extracted from Panax ginseng, inhibits HSC activation. However, the potential role of GRb1 in mediating HSC ferroptosis remains unclear. This study examined the effect of GRb1 on liver fibrosis both in vivo and in vitro, using CCl4-induced liver fibrosis mouse model and primary HSCs, LX-2 cells. The findings revealed that GRb1 effectively inactivated HSCs in vitro, reducing alpha-smooth muscle actin (α-SMA) and type I collagen (Col1A1) levels. Moreover, GRb1 significantly alleviated CCl4-induced liver fibrosis in vivo. From a mechanistic standpoint, the ferroptosis pathway appeared to be central to the antifibrotic effects of GRb1. Specifically, GRb1 promoted HSC ferroptosis both in vivo and in vitro, characterized by increased glutathione depletion, malondialdehyde production, iron overload, and accumulation of reactive oxygen species (ROS). Intriguingly, GRb1 increased Beclin 1 (BECN1) levels and decreased the System Xc-key subunit SLC7A11. Further experiments showed that BECN1 silencing inhibited GRb1-induced effects on HSC ferroptosis and mitigated the reduction of SLC7A11 caused by GRb1. Moreover, BECN1 could directly interact with SLC7A11, initiating HSC ferroptosis. In conclusion, the suppression of BECN1 counteracted the effects of GRb1 on HSC inactivation both in vivo and in vitro. Overall, this study highlights the novel role of GRb1 in inducing HSC ferroptosis and promoting HSC inactivation, at least partly through its modulation of BECN1 and SLC7A11.

Corylin Attenuates CCl4-Induced Liver Fibrosis in Mice by Regulating the GAS6/AXL Signaling Pathway in Hepatic Stellate Cells.

Liver fibrosis is reversible when treated in its early stages and when liver inflammatory factors are inhibited. Limited studies have investigated the therapeutic effects of corylin, a flavonoid extracted from Psoralea corylifolia L. (Fabaceae), on liver fibrosis. Therefore, we evaluated the anti-inflammatory activity of corylin and investigated its efficacy and mechanism of action in ameliorating liver fibrosis. Corylin significantly inhibited inflammatory responses by inhibiting the activation of mitogen-activated protein kinase signaling pathways and the expression of interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha in human THP-1 and mouse RAW264.7 macrophages. Furthermore, corylin inhibited the expression of growth arrest-specific gene 6 in human hepatic stellate cells (HSCs) and the activation of the downstream phosphoinositide 3-kinase/protein kinase B pathway. This inhibited the activation of HSCs and the expression of extracellular matrix proteins, including α-smooth muscle actin and type I collagen. Additionally, corylin induced caspase 9 and caspase 3 activation, which promoted apoptosis in HSCs. Moreover, in vivo experiments confirmed the regulatory effects of corylin on these proteins, and corylin alleviated the symptoms of carbon tetrachloride-induced liver fibrosis in mice. These findings revealed that corylin has anti-inflammatory activity and inhibits HSC activation; thus, it presents as a potential adjuvant in the treatment of liver fibrosis.

Comparative study between the effect of mesenchymal stem cells microvesicles versus ozone on induced liver injury in adult male albino rats (Histological & Immunohistochemical study)

Liver disease accounts for approximately 2 million deaths er year worldwide. Liver fibrisis results from chronic injury to the liver. If not effectively treated in time, liver fibrosis may transform into liver cirrhosis. MVs are recognized as potential biomarkers and important theraputic tools for a wide sectrum of diseases. Medical ozone has the ability to protect the body against pathological conditions caused by oxidative stress. The influence of ozone and MVs on CCL4 induced liver fibrosis was investigated in this study. Forty-eight adult male albino rats were divided into four equal groups. I control, II CCL4 group, III ozone and IV microvesicles groups. Liver fibrosis was induced in group II, III & IV using 12 SC injections (0.5 ml/kg body weight) of CCL4 dissolved in olive oil twice ber week for weeks. Blood samples were obtained to estimate serum ALT & AST. Liver tissues were processed for measurment of GSH & SOD, light and electron microscopic examination. H&E staine sections og group II showed dilated congested sinusoids and centralveins, mononuclear infiltrations, vacuolations and dark nuclei. Ultrastructurally, group II revealed irregular heterochromatic nuclei of hepatocytes, small scanty mitochondria & vacuolations. Morphometric & statistical analyses were performed. Group III showed some improvement, however, group IV showed more imrovement. The results indicates that MVs caused marked improvement than ozone against CCL4 induced liver damage via antioxidant & antiinflammatory properties.

Targeting GPR65 alleviates hepatic inflammation and fibrosis by suppressing the JNK and NF-κB pathways

BackgroundG-protein coupled receptors (GPCRs) are recognized as attractive targets for drug therapy. However, it remains poorly understood how GPCRs, except for a few chemokine receptors, regulate the progression of liver fibrosis. Here, we aimed to reveal the role of GPR65, a proton-sensing receptor, in liver fibrosis and to elucidate the underlying mechanism.MethodsThe expression level of GPR65 was evaluated in both human and mouse fibrotic livers. Furthermore, Gpr65-deficient mice were treated with either bile duct ligation (BDL) for 21 d or carbon tetrachloride (CCl4) for 8 weeks to investigate the role of GPR65 in liver fibrosis. A combination of experimental approaches, including Western blotting, quantitative real-time reverse transcription‑polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA), confocal microscopy and rescue studies, were used to explore the underlying mechanisms of GPR65’s action in liver fibrosis. Additionally, the therapeutic potential of GPR65 inhibitor in the development of liver fibrosis was investigated.ResultsWe found that hepatic macrophages (HMs)-enriched GPR65 was upregulated in both human and mouse fibrotic livers. Moreover, knockout of Gpr65 significantly alleviated BDL- and CCl4-induced liver inflammation, injury and fibrosis in vivo, and mouse bone marrow transplantation (BMT) experiments further demonstrated that the protective effect of Gpr65 knockout is primarily mediated by bone marrow-derived macrophages (BMMs). Additionally, in vitro data demonstrated that Gpr65 silencing and GPR65 antagonist inhibited, while GPR65 overexpression and application of GPR65 endogenous and exogenous agonists enhanced the expression and release of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and transforming growth factor-β (TGF-β), all of which subsequently promoted the activation of hepatic stellate cells (HSCs) and the damage of hepatocytes (HCs). Mechanistically, GPR65 overexpression, the acidic pH and GPR65 exogenous agonist induced up-regulation of TNF-α and IL-6 via the Gαq-Ca2+-JNK/NF-κB pathways, while promoted the expression of TGF-β through the Gαq-Ca2+-MLK3-MKK7-JNK pathway. Notably, pharmacological GPR65 inhibition retarded the development of inflammation, HCs injury and fibrosis in vivo.ConclusionsGPR65 is a major regulator that modulates the progression of liver fibrosis. Thus, targeting GPR65 could be an effective therapeutic strategy for the prevention of liver fibrosis.

Redox-sensitive high-mobility group box-1 isoforms contribute to liver fibrosis progression and resolution in mice

High-mobility group box-1 (HMGB1) significantly increases and undergoes post-translational modifications (PTMs) in response to liver injury. Since oxidative stress plays a major role in liver fibrosis and induces PTMs in proteins, we hypothesized that redox-sensitive HMGB1 isoforms contribute to liver fibrosis progression and resolution. We used ESI-LC-MS (electrospray ionization-liquid chromatography-mass spectrometry) to study PTMs of HMGB1 during fibrosis progression and resolution. Conditional knockout mice were used for functional analyses. We identified that disulfide ([O]) and sulfonated ([SO3]) HMGB1 increase during carbon tetrachloride-induced liver fibrosis progression, however, while [O] HMGB1 declines, [SO3] HMGB1 drops but remains, during fibrosis resolution. Conditional knockout of Hmgb1 revealed that production of [O] and [SO3] HMGB1 occurs mostly in hepatocytes. Co-injection of [O] HMGB1 worsens carbon tetrachloride-induced liver fibrosis more than co-injection of [H] HMGB1. Conversely, ablation of [O] Hmgb1 in hepatocytes reduces liver fibrosis. Moreover, ablation of the receptor for advanced-glycation end-products (Rage) reveals that the profibrogenic effect of [O] HMGB1 is mediated by RAGE signaling in hepatic stellate cells (HSCs). Notably, injection of [SO3] HMGB1 accelerates fibrosis resolution due to RAGE-dependent stimulation of HSC apoptosis. Importantly, gene signatures activated by redox-sensitive HMGB1 isoforms in mice, classify patients with fibrosis according to fibrosis and inflammation scores. Dynamic changes in hepatocyte-derived [O] and [SO3] HMGB1 signal through RAGE-dependent mechanisms on HSCs to drive their profibrogenic phenotype and fate, contributing to progression and resolution of liver fibrosis. Since oxidative stress plays a major role in liver fibrosis and induces post-translational modifications of proteins, we hypothesized that redox-sensitive HMGB1 isoforms contribute to liver fibrosis progression and resolution. This study is significant because a rise in [H] HMGB1 could flag 'patient at risk', the presence of [O] HMGB1 could suggest 'disease in progress or active scarring', while the appearance of [SO3] HMGB1 could point at 'resolution under way'. The latter could be used as a readout for response to pharmacological intervention with anti-fibrotic agents.

Triptolide attenuates CCL4-induced liver fibrosis by regulating the differentiation of CD4+ T cells in mice

Liver fibrosis is a major global health issue, and immune dysregulation is a main contributor. Triptolide is a natural immunosuppressive agent with demonstrated effectiveness in ameliorating liver fibrosis, but whether it exerts anti-liver fibrotic effects via immunoregulation remains obscure. In this study, first, by employing a CCL4-induced liver fibrosis mouse model, we demonstrated that triptolide could alleviate pathological damage to liver tissue and attenuate liver function damaged by CCL4. In addition, triptolide inhibited the expression of liver fibrotic markers such as hydroxyproline, collagen type IV, hyaluronidase, laminin, and procollagen type III, and the protein expression of α-SMA in CCL4-induced liver fibrosis. Second, with the help of network pharmacology, we predicted that triptolide’s anti-liver fibrotic effects might occur through the regulation of Th17, Th1, and Th2 cell differentiation, which indicated that triptolide might mitigate liver fibrosis via immunoregulation. Finally, multiplex immunoassays and flow cytometry were adopted to verify this prediction. The results suggested that triptolide could reverse the aberrant expression of inflammatory cytokines caused by CCL4 and regulate the differentiation of Th1, Th2, Th17, and Treg cells. In conclusion, triptolide could attenuate CCL4-induced liver fibrosis by regulating the differentiation of CD4+ T cells. The results obtained in this study extended the application of triptolide and introduced a new mechanism of triptolide’s anti-liver fibrotic effects.

Role of macrophage-to-myofibroblast transition in chronic liver injury and liver fibrosis

BackgroundChronic liver injury contributes to liver fibrosis, which is characterized by the excessive deposition of extracellular matrix (ECM) components. ECM is mainly composed of myofibroblasts. Recently, macrophage-to-myofibroblasts transition (MMT), has been identified as a novel origin for myofibroblasts. However, the potential functions of MMT in chronic liver injury and liver fibrosis remain unknown.MethodsTo clarify the transformation of fibrotic cells in hepatic fibrosis, liver specimens were collected from people at different stages in the progression of hepatic fibrosis and stained with immunofluorescence. Models of hepatic fibrosis such as the CCL4 model, HFD-induced NAFLD model, MCD-induced NAFLD model and ethanol-induced AFLD model were demonstrated and were stained with immunofluorescence.ResultsHere, we uncovered macrophages underwent MMT in clinical liver fibrosis tissue samples and multiple animal models of chronic liver injury. MMT cells were found in specimens from patients with liver fibrosis on the basis of co-expression of macrophage (CD68) and myofibroblast (a-SMA) markers. Moreover, macrophages could transform into myofibroblasts in CCL4-induced liver fibrosis model, high-fat diet (HFD) and methionine-choline-deficient diet (MCD)-induced nonalcoholic fatty liver diseases (NAFLD) model, and ethanol-induced alcoholic fatty liver diseases (AFLD) model. In addition, we highlighted that MMT cells mainly had a predominant M2 phenotype in both human and experimental chronic liver injury.ConclusionsTaken together, MMT acts a crucial role in chronic liver injury and liver fibrosis.

Inhibition of chloride intracellular channel protein 1 (CLIC1) ameliorates liver fibrosis phenotype by activating the Ca2+-dependent Nrf2 pathway

Persistent damage to liver cells leads to liver fibrosis, which is characterized by the accumulation of scar tissue in the liver, ultimately leading to cirrhosis and serious complications. Because it is difficult to reverse cirrhosis once it has progressed, the primary focus has been on preventing the progression of liver fibrosis. However, studies on therapeutic agents for liver fibrosis are still lacking. Here, we investigated that the natural dipeptide cyclic histidine-proline (CHP, also known as diketopiperazine) shows promising potential as a therapeutic agent in models of liver injury by inhibiting the progression of fibrosis through activation of the Nrf2 pathway. To elucidate the underlying biological mechanism of CHP, we used the Cellular Thermal Shift Assay (CETSA)-LC–MS/MS, a label-free compound-based target identification platform. Chloride intracellular channel protein 1 (CLIC1) was identified as a target whose thermal stability is increased by CHP treatment. We analyzed the direct interaction of CHP with CLIC1 which revealed a potential interaction between CHP and the E228 residue of CLIC1. Biological validation experiments showed that knockdown of CLIC1 mimicked the antioxidant effect of CHP. Further investigation using a mouse model of CCl4-induced liver fibrosis in wild-type and CLIC1 KO mice revealed the critical involvement of CLIC1 in mediating the effects of CHP. Taken together, our results provide evidence that CHP exerts its anti-fibrotic effects through specific binding to CLIC1. These insights into the mechanism of action of CHP may pave the way for the development of novel therapeutic strategies for fibrosis-related diseases.

HMGB2 is a potential diagnostic marker and therapeutic target for liver fibrosis and cirrhosis.

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

Obeticholic acid-loaded exosomes attenuate liver fibrosis through dual targeting of the FXR signaling pathway and ECM remodeling

End-stage of liver fibrosis as a precancerous state could lead to cirrhosis and hepatocellular carcinoma which liver transplantation is the only effective treatment. Previous studies have indicated that farnesoid X receptor (FXR) agonists, such as obeticholic acid (OCA) protect against hepatic injuries. However, free OCA administration results in side effects in clinical trials that could be alleviated by applying bio carriers such as MSC-derived exosomes (Exo) with the potential to mimic the biological regenerative effect of their parent cells, as proposed in this study. Loading OCA into the Exo was conducted via water bath sonication. Ex vivo bio distribution studies validated the Exo-loaded OCA more permanently accumulated in the liver. Using CCL4-induced liver fibrosis, we proposed whether Exo isolated from human Warton’s Jelly mesenchymal stem cells loaded with a minimal dosage of OCA can facilitate liver recovery. Notably, Exo-loaded OCA exerted additive anti-fibrotic efficacy on histopathological features in CCL4-induced fibrotic mice. Compared to baseline, Exo-mediated delivery OCA results in marked improvements in the fibrotic-related indicators as well as serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations. Accordingly, the synergistic impact of Exo-loaded OCA as a promising approach is associated with the inactivation of hepatic stellate cells (HSCs), extracellular matrix (ECM) remodeling, and Fxr-Cyp7a1 cascade on CCL4-induced liver fibrosis mice. In conclusion, our data confirmed the additive protective effects of Exo-loaded OCA in fibrotic mice, which suggests a valuable therapeutic strategy to combat liver fibrosis. Furthermore, the use of Exo for accurate drug delivery to the liver tissue can be inspiring.

Hepatoprotective Effect of Medicine Food Homology Flower Saffron against CCl4-Induced Liver Fibrosis in Mice via the Akt/HIF-1α/VEGF Signaling Pathway.

Liver fibrosis refers to a complex inflammatory response caused by multiple factors, which is a known cause of liver cirrhosis and even liver cancer. As a valuable medicine food homology herb, saffron has been widely used in the world. Saffron is commonly used in liver-related diseases and has rich therapeutic and health benefits. The therapeutic effect is satisfactory, but its mechanism is still unclear. In order to clarify these problems, we planned to determine the pharmacological effects and mechanisms of saffron extract in preventing and treating liver fibrosis through network pharmacology analysis combined with in vivo validation experiments. Through UPLC-Q-Exactive-MS analysis, a total of fifty-six nutrients and active ingredients were identified, and nine of them were screened to predict their therapeutic targets for liver fibrosis. Then, network pharmacology analysis was applied to identify 321 targets for saffron extract to alleviate liver fibrosis. Functional and pathway enrichment analysis showed that the putative targets of saffron for the treatment of hepatic fibrosis are mainly involved in the calcium signaling pathway, the HIF-1 signaling pathway, endocrine resistance, the PI3K/Akt signaling pathway, lipid and atherosclerosis, and the cAMP signaling pathway. Based on the CCl4-induced liver fibrosis mice model, we experimentally confirmed that saffron extract can alleviate the severity and pathological changes during the progression of liver fibrosis. RT-PCR and Western blotting analysis confirmed that saffron treatment can prevent the CCl4-induced upregulation of HIF-1α, VEGFA, AKT, and PI3K, suggesting that saffron may regulate AKT/HIF-1α/VEGF and alleviate liver fibrosis.

Vanillic acid alleviates liver fibrosis through inhibiting autophagy in hepatic stellate cells via the MIF/CD74 signaling pathway

MIF/CD74 signaling pathway and autophagy may be closely related to liver fibrosis. Vanillic acid (VA) is likely to have an anti-liver fibrosis effect, although related studies have not been reported. The aim of this study was to verify the role of hepatic stellate cells (HSCs) autophagy and the MIF/CD74 signaling pathway in the pathogenesis of liver fibrosis, and to investigate the effect of VA on liver fibrosis through in vivo and in vitro experiments. Our results showed that VA significantly attenuated CCl4-induced liver fibrosis. The alleviation of liver fibrosis with VA treatment was associated with a reduction of MIF, CD74, α-SMA, LC3B and Collagen 1. In addition, VA, MIF inhibitor (ISO-1) and autophagy inhibitor (3-MA) markedly inhibited the proliferation and migration of HSCs. This study indicates that VA could protect against HSCs activation, proliferation and migration by inhibiting the autophagy in HSCs via the MIF/CD74 signaling pathway so that alleviates liver fibrosis.

SAT005 Effect Of OSU-ERβ-12, A Carborane-based ERβ-selective Agonist, On Hepatic Fibrosis And NASH

Abstract Disclosure: P. Kumar: None. T.H. Helms: None. H. Radomska: None. W. Tyler: None. S.K. Kulp: None. C. Bennett: None. C.C. Coss: None. Background: Non-alcoholic fatty liver disease (NAFLD) is a major cause of chronic liver disease worldwide. NAFLD includes a range of conditions from simple steatosis to non-alcoholic steatohepatitis (NASH), advanced fibrosis, and cirrhosis that can ultimately lead to liver failure and/or hepatocellular carcinoma (HCC). Hepatic stellate cells (HSCs) are essential to this pathologic fibrogenesis. In response to liver injury, they deposit collagen, fibronectin, and release the potent pro-fibrotic cytokine transforming growth factor-β (TGFβ). Estrogens have been shown to suppress the TGF-β-induced expression but the molecular mechanism is not completely understood. Estrogens are protective against multiple aspects of NAFLD/NASH pathophysiology. Their therapeutic potential however is limited by undesirable side effects attributed to ERα activation. Selective modulation of ERβ can provide therapeutic benefits of estrogens with reduced ERα-associated toxicities and has shown promising results in several preclinical NASH models. We hypothesize that beneficial estrogen pharmacology in NAFLD-NASH is driven by ERβ activation and ERβ-selective modulators can provide anti-NASH benefit. Our objective was to investigate the role of ERβ signaling and the therapeutic potential of the novel carborane ERβ-selective agonist, OSU-ERβ-12 (ERβ-12), in fibrosis and NASH models. Methods. Efficacy of ERβ-12 was tested in the CCl4 model of hepatic fibrosis by administering CCl4 to mice over 4 weeks at which point intervention with estradiol, LY500307 (LY) and ERβ-12 started. Combined treatment and CCL4 continued for 2 weeks when the CCl4 ceased, and therapy continued for 2 more weeks until sacrifice. We assessed fibrosis via algorithmic quantification of picrosirius red stain. Anti-fibrotic activity of ERβ-12 was assessed in vitro in a human HCC cell (HepG2)/human HSC (LX-2) co-culture system treated with ERβ-12, TGFβ or the combination for 24 h. Expression of pro-fibrotic, TGFβ pathway-related markers was assessed by qRT-PCR and western blotting in liver tissue and cell lysates. Results. ERβ-12 significantly reduced CCl4-induced liver fibrosis in mice at both non-selective (100 mg/kg) and ERβ-selective (10 mg/kg) doses. ERα-related toxicity (reduced urogenital weight) was not observed at the ERβ-selective dose. Reduced fibrosis was associated with decreased mRNA expression of pro-fibrotic and TGFβ pathway genes in livers of ERβ-12 treated mice. ERβ mRNA expression in HepG2/LX-2 co-culture cells was elevated compared to individual cell types. ERβ agonists (ERβ-12, LY) reduced TGFβ-mediated expression of pro-fibrotic genes: MCP-1, TGFβ, COL1A1. This effect was reversed with the pan-ER antagonist fulvestrant. Conclusion. These data suggest that ERβ agonism inhibits TGFβ-mediated liver fibrosis and supports further investigation of ERβ-targeted therapeutic candidates. Presentation: Saturday, June 17, 2023

Hepatoprotective and antifibrotic activity of watercress extract in a model of CCl4-induced liver fibrosis in Wistar rats

Hepatoprotective and antifibrotic activity of watercress extract in a model of CCl4-induced liver fibrosis in Wistar rats

Fluorofenidone alleviates liver fibrosis by inhibiting hepatic stellate cell autophagy via the TGF-β1/Smad pathway: implications for liver cancer.

Liver fibrosis is a key stage in the progression of various chronic liver diseases to cirrhosis and liver cancer, but at present, there is no effective treatment. This study investigated the therapeutic effect of the new antifibrotic drug fluorofenidone (AKF-PD) on liver fibrosis and its related mechanism, providing implications for liver cancer. The effects of AKF-PD on hepatic stellate cell (HSC) autophagy and extracellular matrix (ECM) expression were assessed in a carbon tetrachloride (CCl4)-induced rat liver fibrosis model. In vitro, HSC-T6 cells were transfected with Smad2 and Smad3 overexpression plasmids and treated with AKF-PD. The viability and number of autophagosomes in HSC-T6 cells were examined. The protein expression levels of Beclin-1, LC3 and P62 were examined by Western blotting. The Cancer Genome Atlas (TCGA) database was used for comprehensively analyzing the prognostic values of SMAD2 and SMAD3 in liver cancer. The correlation between SMAD2, SMAD3, and autophagy-related scores in liver cancer was explored. The drug prediction of autophagy-related scores in liver cancer was explored. AKF-PD attenuated liver injury and ECM deposition in the CCl4-induced liver fibrosis model. In vitro, the viability and number of autophagosomes in HSCs were reduced significantly by AKF-PD treatment. Meanwhile, the protein expression of FN, α-SMA, collagen III, Beclin-1 and LC3 was increased, and P62 was reduced by the overexpression of Smad2 and Smad3; however, AKF-PD reversed these effects. SMAD2 and SMAD3 were hazardous factors in liver cancer. SMAD2 and SMAD3 correlated with autophagy-related scores in liver cancer. Autophagy-related scores could predict drug response in liver cancer. AKF-PD alleviates liver fibrosis by inhibiting HSC autophagy via the transforming growth factor (TGF)-β1/Smadpathway. Our study provided some implications about how liver fibrosis was connected with liver cancer by SMAD2/SMAD3 and autophagy.

Reversal of fibrosis and portal hypertension by Empagliflozin treatment of CCl4-induced liver fibrosis: Emphasis on gal-1/NRP-1/TGF-β and gal-1/NRP-1/VEGFR2 pathways

To date, liver fibrosis has no clinically approved treatment. Empagliflozin (EMPA), a highly selective sodium-glucose-cotransporter-2 (SGLT2) inhibitor, has shown ameliorative potential in liver diseases without revealing its full mechanisms. Neuropilin-1 (NRP-1) is a novel regulator of profibrogenic signaling pathways related to hepatic stellate cells (HSCs) and hepatic sinusoidal endothelial cells (HSECs) that modulates intrahepatic profibrogenic and angiogenic pathways. Herein, EMPA's antifibrotic potentials and effects on galactin-1 (Gal-1)/NRP-1 signaling pathways have been evaluated in an experimental liver fibrosis rat model by testing different EMPA dose regimens. EMPA treatment brought a dose-dependent decrease in Gal-1/NRP-1 hepatic expression. This was coupled with suppression of major HSCs pro-fibrotic pathways; transforming growth factor-β (TGF-β)/TGF-βRI/Smad2 and platelet-derived growth factor-beta (PDGF-β) with a diminution of hepatic Col 1A1 level. In addition, EMPA prompted a protuberant suppression of the angiogenic pathway; vascular endothelial growth factor (VEGF)/VEGF-receptor-2 (VEGFR-2)/SH2-Domain Containing Adaptor Protein-B (Shb), and reversal of altered portal hypertension (PHT) markers; endothelin-1 (ET-1) and endothelial nitric oxide synthase (eNOS). The amelioration of liver fibrosis was coupled with a remarkable improvement in liver aminotransferases and histologic hepatic fibrosis Ishak scores. The highest EMPA dose showed a good safety profile with minimal changes in renal function and glycemic control. Thus, the current study brought about novel findings for a potential liver fibrosis treatment modality via targeting NRP-1 signaling pathways by EMPA.

CARD9 deficiency aggravated nonalcoholic steatohepatitis in mice through increasing inflammatory response

Nonalcoholic steatohepatitis (NASH), a subtype of nonalcoholic fatty liver disease (NAFLD), is the leading cause of liver-related morbidity worldwide. Caspase recruitment domain family member 9 (CARD9), a myeloid cell-specific signaling protein, belongs to the CARD protein family. However, its role in NASH is unknown. Therefore, this study aimed to investigate the role of CARD9 in the development of NASH. NASH models were established using CARD9-knockout and wild-type mice. They were either fed a methionine/choline deficient (MCD) diet for 6 weeks or a high-fat high-cholesterol (HFHC) diet for 16 weeks. Liver fibrosis model was also developed using CCl4. CARD9 deficiency accelerated steatohepatitis development in MCD or HFHC diet-fed mice, accompanied by an upregulation of fibrosis, adipogenesis, and proinflammatory genes. CARD9 deficiency was found to exacerbate CCl4-induced liver fibrosis. In vitro studies demonstrated that CARD9 deficiency induced the expression of S100a8/a9 through Toll-like receptor in Kupffer cells treated with palmitate. This led to an increased expression of proinflammatory, fibrosis, and lipid metabolism-related genes in NASH progression. These results highlight the role of CARD9 in the development of NASH and provide new insights into the therapeutic strategies for NASH.

The application of a novel platform of multiparametric magnetic resonance imaging in a bioenvironmental toxic carbon tetrachloride-induced mouse model of liver fibrosis

The use of multiparametric magnetic resonance imaging (MRI) to distinguish complex histopathological changes in liver fibrosis has not yet been systematically established. The purpose of this study is to gauge the efficacy of a cutting-edge MRI platform for evaluating ecotoxicologically hazardous carbon tetrachloride (CCl4) induced liver fibrosis, while also scrutinizing the relationship between MRI and its histopathological features. Thirty-six mice were randomly divided into 6 groups, each with 6 mice. Control mice received an intraperitoneal injection of olive oil, while the experimental mice received different doses of intraperitoneal injection of CCl4. Both sets underwent this process twice per week over a duration of 5 weeks. MRI measurements encompassed T1WI, T2WI, T1 mapping, T2 mapping, T2* mapping. Liver fibrosis and inflammation were assessed and classified using Metavir and activity scoring systems. CCl4 successfully induced liver fibrosis in mice, showing an increasing extent of liver fibrosis and liver function damage with the increasing dosage of CCl4. Compared with the control group, T1, ΔT1, and T2 in the experimental group were considerably elevated (P < 0.05) than those in the control group. Spearman's correlation showed that the correlation of Native T1 and △T1 with fibrosis (r = 0.712, 0.678) was better than with inflammation (r = 0.688, 0.536). T2 correlation with inflammation (r = 0.803) was superior to fibrosis (r = 0.568). ROC analysis showed that the AUC of Native T1 was highest (0.906), followed by ΔT1 (0.852), while the AUC increased to 0.945 when all relevant MRI parameters were combined. T1 is the most potent MRI parameter for evaluating CCl4-induced liver fibrosis, followed by ΔT1. Meanwhile, T2 may not be suitable for evaluating liver fibrosis but is more suitable for evaluating liver inflammation.