CCl4-induced Liver Fibrosis Mouse Model Research Articles

Transcriptomic and proteomic investigation of the ameliorative effect of total polyphenolic glycoside extract on hepatic fibrosis in Lamiophlomis rotata Kudo via the AGE/RAGE pathway

Ethnopharmacological relevanceDuring the regression of liver fibrosis, a decrease in hepatic stellate cells (HSCs) can occur through apoptosis or inactivation of activated HSCs (aHSCs). A new approach for antifibrotic therapy involves transforming hepatic myofibroblasts into a quiescent-like state. Lamiophlomis rotata (Benth.) Kudo (L. rotata), an orally available Tibetan herb, has traditionally been used to treat skin disease, jaundice, and rheumatism. In our previous study, we found that the total polyphenolic glycoside extract of L. rotata (TPLR) promotes apoptosis in aHSCs for the treatment of hepatic fibrosis. However, whether TPLR induces aHSCs to become inactivated HSCs (iHSCs) is unclear, and the underlying mechanism remains largely unknown. PurposeThis study aimed to examine the impact of TPLR on the phenotypes of hepatic stellate cells (HSCs) during the regression of liver fibrosis and explore the potential mechanism of action. MethodsThe effect of TPLR on the phenotypes of hepatic stellate cells (HSCs) was assessed using immunofluorescence (IF) staining, reverse transcription-polymerase chain reaction (RT–PCR), and Western blotting. Transcriptomic and proteomic methods were employed to identify the main signaling pathways involved. Based on the omics results, the likely mechanism of TPLR on the phenotypes of aHSCs was confirmed through overexpression and knockdown experiments in TGF-β1-activated LX-2 cells. Using a CCl4-induced liver fibrosis mouse model, we evaluated the anti-hepatic fibrosis effect of TPLR and explored its potential mechanism based on omics findings. ResultsTPLR was found to induce the differentiation of aHSCs into iHSCs by significantly decreasing the protein expression of α-SMA and Desmin. Transcriptomic and proteomic analyses revealed that the AGE/RAGE signaling pathway plays a crucial role in the morphological transformation of HSCs following TPLR treatment. In vitro experiments using RAGE overexpression and knockdown demonstrated that the mechanism by which TPLR affects the phenotype of HSCs is closely associated with the RAGE/RAS/MAPK/NF-κB axis. In a model of liver fibrosis, TPLR obviously inhibited the generation of AGEs and alleviated liver tissue damage and fibrosis by downregulating RAGE and its downstream targets. ConclusionThe AGE/RAGE axis plays a pivotal role in the transformation of activated hepatic stellate cells (aHSCs) into inactivated hepatic stellate cells (iHSCs) following TPLR treatment, indicating the potential of TPLR as a therapeutic agent for the management of liver fibrosis.

Metformin exerts anti-liver fibrosis effect based on the regulation of gut microbiota homeostasis and multi-target synergy

Liver fibrosis can progress to cirrhosis if left untreated. Therefore, identifying effective antifibrotic drugs is crucial. This study aimed to investigate the role and potential mechanism of metformin in treating hepatic fibrosis based on the synergistic effect of multiple targets and the “intestine-liver axis” theory. A CCl4-induced liver fibrosis mouse model was established. We measured liver function, liver fibrosis indicators, oxidative stress and inflammation indices. Hematoxylin and eosin and Masson's trichrome staining were used to detect collagen deposition. The expression of apoptotic proteins, TGF-β/Smads and TIMP-1/MMPs was assessed. 16S rRNA and untargeted metabolomics (liquid chromatography-mass spectrometry) were used to assess mouse intestinal flora and metabolites, performing a comprehensive correlation analysis. Metformin improved the general status and liver function and decreased liver collagen deposition in CCl4-induced liver fibrotic mice. Compared with the control group, IL-6, TNF-α and COX-2 serum levels in the liver fibrosis group increased. Although not significantly different, the serum inflammatory marker levels in the metformin group were lower than those in the model group. Metformin decreased serum MDA and increased serum SOD activity, which increased and decreased, respectively, in the model group. Furthermore, metformin inhibited liver cell apoptosis, TGF-β1 expression and TIMP-1, while promoting Smad7 expression, MMP-1 and MMP-2 in fibrotic mice. 16S rRNA analysis indicated that metformin significantly ameliorated the Bacteroides, Helicobacter, Parabacteroides and Parasutterella imbalance. We identified 385 differential metabolites between the metformin and model groups. Prevotella abundance significantly decreased in the metformin group and positively correlated with decreased taurocholic acid levels. Metformin potentially reverses liver fibrosis by inhibiting inflammation, mitigating oxidative stress damage and suppressing hepatocyte apoptosis via intestinal flora metabolite regulation. Metformin also regulates the TGF-β/Smads and TIMP-1/MMPs signalling pathways. This study provides a theoretical basis for the clinical use of metformin in patients with liver fibrosis.

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.

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 and Type I collagen 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. 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.

Isolation and Biological Evaluation of Alfa-Mangostin as Potential Therapeutic Agents against Liver Fibrosis.

The increased proliferation and activation of hepatic stellate cells (HSCs) are associated with liver fibrosis development. To date, there are no FDA-approved drugs for the treatment of liver cirrhosis. Augmentation of HSCs apoptosis is one of the resolutions for liver fibrosis. In this study, we extracted α-mangostin (1,3,6-trihydroxy-7-methoxy-2,8-bis(3-methyl-2-butenyl)-9H-xanthen-9-one) from the fruit waste components of mangosteen pericarp. The isolated α-mangostin structure was determined and characterized with nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS) and compared with those known compounds. The intracellular signaling pathway activities of α-mangostin on Transforming growth factors-beta 1 (TGF-β1) or Platelet-derived growth factor subunit B (PDGF-BB) induced HSCs activation and were analyzed via Western blot and Real-time Quantitative Polymerase Chain Reaction (Q-PCR). α-Mangostin-induced mitochondrial dysfunction and apoptosis in HSCs were measured by seahorse assay and caspase-dependent cleavage. The in vivo anti-fibrotic effect of α-mangostin was assessed by carbon tetrachloride (CCl4) treatment mouse model. The data showed that α-mangostin treatment inhibited TGF-β1-induced Smad2/3 phosphorylation and alpha-smooth muscle actin (α-SMA) expression in HSCs in a dose-dependent manner. Regarding the PDGF-BB-induced HSCs proliferation signaling pathways, α-mangostin pretreatment suppressed the phosphorylation of extracellular-signal-regulated kinase (ERK) and p38. The activation of caspase-dependent apoptosis and dysfunction of mitochondrial respiration (such as oxygen consumption rate, ATP production, and maximal respiratory capacity) were observed in α-mangostin-treated HSCs. The CCl4-induced liver fibrosis mouse model showed that the administration of α-mangostin significantly decreased the expression of the fibrosis markers (α-SMA, collagen-a2 (col1a2), desmin and matrix metalloproteinase-2 (MMP-2)) as well as attenuated hepatic collagen deposition and liver damage. In conclusion, this study demonstrates that α-mangostin attenuates the progression of liver fibrosis through inhibiting the proliferation of HSCs and triggering apoptosis signals. Thus, α-mangostin may be used as a potential novel therapeutic agent against liver fibrosis.

Human Induced Hepatocytes-Derived Extracellular Vesicles Ameliorated Liver Fibrosis in Mice via Suppression of TGF-β1/Smad Signaling and Activation of Nrf2/HO-1 Signaling.

Liver fibrosis is a wound-healing response caused by persistent liver injury and often occurs in chronic liver diseases. Effective treatments for liver fibrosis are still pending. Recent studies have revealed that extracellular vesicles (EVs) derived from primary hepatocytes (Hep-EVs) have therapeutic potential for multiple liver diseases. However, Hep-EVs are difficult to manufacture in bulk because of the limited sources of primary hepatocytes. Human-induced hepatocytes (hiHep) are hepatocyte-like cells that can expand in vitro, and their cell culture supernatant is thus an almost unlimited resource for EVs. This study aimed to investigate the potential therapeutic effects of EVs derived from hiHeps. hiHep-EVs inhibited the expression of inflammatory genes and the secretion of inflammation-related cytokines, and suppressed the activation of hepatic stellate cells by inhibiting the TGF-β1/Smad signaling pathway. The anti-inflammatory and antifibrotic effects of hiHep-EVs were similar to those of mesenchymal stem cell (MSC)-EVs. Furthermore, the administration of hiHep-EVs ameliorated oxidative stress, inflammation, and fibrosis in a CCl4-induced liver fibrosis mouse model. The expression of α smooth muscle actin (α-SMA), collagen I, and collagen III was reduced, which may be attributed to the regulation of metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 by hiHep-EVs, and the protein expression of Nrf2, HO-1, and NQO1 was increased. Taken together, our results suggested that hiHep-EVs alleviated liver fibrosis by activating the Nrf2/HO-1 signaling pathway and inhibiting the TGF-β1/Smad signaling pathway. This study revealed the hepatoprotective effect of hiHep-EVs, and provided a new approach to treating liver fibrosis.

Anti-liver fibrosis activity of curcumin/chitosan-coated green silver nanoparticles

Liver fibrosis results from the hepatic accumulation of the extracellular matrix accompanied by a failure of the mechanisms responsible for matrix dissolution. Pathogenesis of liver fibrosis is associated with many proteins from different cell types. In the present study, in silico molecular docking analysis revealed that curcumin may inhibit the fibrosis-mediating proteins PDGF, PDGFRB, TIMP-1, and TLR-9 by direct binding. Nano-formulation can overcome curcumin problems, increasing the efficacy of curcumin as a drug by maximizing its solubility and bioavailability, enhancing its membrane permeability, and improving its pharmacokinetics, pharmacodynamics and biodistribution. Therefore, green silver nanoparticles (AgNPs) were synthesized in the presence of sunlight by means of the metabolite of Streptomyces malachiticus, and coated with curcumin-chitosan mixture to serve as a drug delivery tool for curcumin to target CCl4-induced liver fibrosis mouse model. Fibrosis induction significantly increased hepatic gene expression of COL1A1, α-SMA, PDGFRB, and TIMP1, elevated hepatic enzymes, increased histopathological findings, and increased collagen deposition as determined by Mason’s trichrome staining. Treatment with naked AgNPs tended to increase these inflammatory effects, while their coating with chitosan, similar to treatment with curcumin only, did not prevent the fibrogenic effect of CCl4. The induction of liver fibrosis was reversed by concurrent treatment with curcumin/chitosan-coated AgNPs. In this nano form, curcumin was found to be efficient as anti-liver fibrosis drug, maintaining the hepatic architecture and function during fibrosis development. This efficacy can be attributed to its inhibitory role through a direct binding to fibrosis-mediating proteins such as PDGFRB, TIMP-1, TLR-9 and TGF-β.

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

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

Exosomal miR-500 Derived From Lipopolysaccharide-Treated Macrophage Accelerates Liver Fibrosis by Suppressing MFN2.

Liver fibrosis is an outcome of chronic hepatic injury, which can eventually result in cirrhosis, liver failure, and even liver cancer. The activation of hepatic stellate cell (HSC) is a prominent driver of liver fibrosis. Recently, it has been found that the crosstalk between HSCs and immune cells, including hepatic macrophages, plays an important role in the initiation and development of liver fibrosis. As a vital vehicle of intercellular communication, exosomes transfer specific cargos into HSCs from macrophages. Here, we show that exosomes derived from lipopolysaccharide (LPS)-treated macrophages has higher expression level of miR-500. And overexpression or inhibition of miR-500 in macrophage exosomes could promote or suppress HSC proliferation and activation. Treatment of exosomes with miR-500 overexpression can accelerate liver fibrosis in CCl4-induced liver fibrosis mouse model. miR-500 promotes HSC activation and liver fibrosis via suppressing MFN2. Moreover, miR-500 in serum exosomes could be a biomarker for liver fibrosis. Taken together, exosomal miR-500 derived from LPS-activated macrophages promotes HSC proliferation and activation by targeting MFN2 in liver fibrosis.

Ethyl Acetate Fraction of Dicliptera chinensis (L.) Juss. Ameliorates Liver Fibrosis by Inducing Autophagy via PI3K/AKT/mTOR/p70S6K Signaling Pathway.

To investigate the molecular mechanism underlying the anti-hepatic fibrosis activity of ethyl acetate fraction Dicliptera chinensis (L.) Juss. (EDC) in human hepatic stellate cells (HSCs) in vitro and in a carbon tetrachloride (CCl4)-induced hepatic fibrosis mouse model in vivo. For in vitro study, HSCs were pre-treated with platelet-derived growth factor (10 ng/mL) for 2 h to ensure activation and treated with EDC for 24 h and 48 h, respectively. The effect of EDC on HSCs was assessed using cell counting kit-8 assay, EdU staining, transmission electron microscopy, immunofluorescence staining, and Western blot, respectively. For in vivo experiments, mice were intraperitoneally injected with CCl4 (2 ° L/g, adjusted to a 25% concentration in olive oil), 3 times per week for 6 weeks, to develop a hepatic fibrosis model. Forty 8-week-old male C57BL/6 mice were divided into 4 groups using a random number table (n=10), including control, model, positive control and EDC treatment groups. Mice in the EDC and colchicine groups were intragastrically administered EDC (0.5 g/kg) or colchicine (0.2 mg/kg) once per day for 6 weeks. Mice in the control and model groups received an equal volume of saline. Biochemical assays and histological examinations were used to assess liver damage. Protein expression levels of α -smooth muscle actin (α -SMA) and microtubule-associated protein light chain 3B (LC3B) were measured by Western blot. EDC reduced pathological damage associated with liver fibrosis, downregulated the expression of α -SMA and upregulated the expression of LC3B (P<0.05), both in HSCs and the CCl4-induced liver fibrosis mouse model. The intervention of bafilomycin A1 and rapamycin in HSCs strongly supported the notion that inhibition of autophagy enhanced α -SMA protein expression levels (P<0.01). The results also found that the levels of phosphoinositide (PI3K), p-PI3K, AKT, p-AKT, mammalian target of rapamycin (mTOR), p-mTOR, and p-p70S6K all decreased after EDC treatment (P<0.05). EDC has anti-hepatic fibrosis activity by inducing autophagy and might be a potential drug to be further developed for human liver fibrosis therapy.

Matrix metalloproteinase-1 decorated polymersomes, a surface-active extracellular matrix therapeutic, potentiates collagen degradation and attenuates early liver fibrosis

Liver fibrosis affects millions of people worldwide and is rising vastly over the past decades. With no viable therapies available, liver transplantation is the only curative treatment for advanced diseased patients. Excessive accumulation of aberrant extracellular matrix (ECM) proteins, mostly collagens, produced by activated hepatic stellate cells (HSCs), is a hallmark of liver fibrosis. Several studies have suggested an inverse correlation between collagen-I degrading matrix metalloproteinase-1 (MMP-1) serum levels and liver fibrosis progression highlighting reduced MMP-1 levels are associated with poor disease prognosis in patients with liver fibrosis. We hypothesized that delivery of MMP-1 might potentiate collagen degradation and attenuate fibrosis development. In this study, we report a novel approach for the delivery of MMP-1 using MMP-1 decorated polymersomes (MMPsomes), as a surface-active vesicle-based ECM therapeutic, for the treatment of liver fibrosis. The storage-stable and enzymatically active MMPsomes were fabricated by a post-loading of Psomes with MMP-1. MMPsomes were extensively characterized for the physicochemical properties, MMP-1 surface localization, stability, enzymatic activity, and biological effects. Dose-dependent effects of MMP-1, and effects of MMPsomes versus MMP-1, empty polymersomes (Psomes) and MMP-1 + Psomes on gene and protein expression of collagen-I, MMP-1/TIMP-1 ratio, migration and cell viability were examined in TGFβ-activated human HSCs. Finally, the therapeutic effects of MMPsomes, compared to MMP-1, were evaluated in vivo in carbon-tetrachloride (CCl4)-induced early liver fibrosis mouse model. MMPsomes exhibited favorable physicochemical properties, MMP-1 surface localization and improved therapeutic efficacy in TGFβ-activated human HSCs in vitro. In CCl4-induced early liver fibrosis mouse model, MMPsomes inhibited intra-hepatic collagen-I (ECM marker, indicating early liver fibrosis) and F4/80 (marker for macrophages, indicating liver inflammation) expression. In conclusion, our results demonstrate an innovative approach of MMP-1 delivery, using surface-decorated MMPsomes, for alleviating liver fibrosis.

Carvacrol alleviates liver fibrosis by inhibiting TRPM7 and modulating the MAPK signaling pathway

Liver fibrosis is a compensatory response to the tissue repair process. The activation and proliferation of hepatic stellate cells (HSCs) are thought to be related to the occurrence of hepatic fibrosis. Therefore, inhibiting the activation and proliferation of HSCs is a key step in alleviating liver fibrosis. As a non-specific inhibitor of transient receptor potential melastatin 7 (TRPM7), carvacrol has anti-tumor, anti-inflammatory and anti-hepatic fibrosis activities. This study aimed to explore the protective effect of carvacrol on liver fibrosis and related molecular mechanisms. A CCl4-induced liver fibrosis mouse model and platelet-derived growth factor (PDGF-BB)-activated HSC-T6 cells (a rat hepatic stellate cell line) were employed for in vivo and in vitro experiments. C57BL/6J mice were orally administered different concentrations of carvacrol every day for 6 weeks during the development of CCl4-induced liver fibrosis. The results show that carvacrol could effectively reduce liver damage and the progression of liver fibrosis in mice, which are expressed as fibrotic markers levels were reduced and histopathological characteristics were improved. Moreover, carvacrol inhibited the proliferation and activation of HSC-T6 cells induced by PDGF-BB. In addition, it was found that carvacrol inhibits the expression of TRPM7 and mediated through mitogen-activated protein kinases (MAPK). Collectively, our study shows that carvacrol can reduce liver fibrosis by inhibiting the activation and proliferation of hepatic stellate cells, and the MAPK signaling pathway might be involved in this process.

SB431542-Loaded Liposomes Alleviate Liver Fibrosis by Suppressing TGF-β Signaling.

Liver fibrosis is a common outcome of most chronic liver diseases, but there is no clinically approved drug for its treatment. Previous studies have reported the potential of SB431542 as an inhibitor of TGF-β signaling in the treatment of liver fibrosis, but it shows poor water solubility and low bioavailability. Here, we improve these characteristics of SB431542 by loading it into liposomes (SB-Lips) with two FDA-approved excipients: soya phosphatidyl S100 and Solutol HS15. In vitro, SB-Lips had stronger inhibitory effects on the proliferation and activation of hepatic stellate cells LX-2 than free SB. After an intravenous injection in a CCl4-induced liver fibrosis mouse model, SB-Lips accumulated preferentially in the liver, its area under the concentration-time curve was significantly higher than that of free SB431542, and it alleviated hepatic fibrosis significantly more than free drug, which was associated with greater inhibition of TGF-β signaling. Furthermore, SB-Lips did not cause significant injury to other organs. These results suggest that our liposomal system is safe and effective for delivering SB431542 to fibrotic liver.

Hsa_circ_0004018 suppresses the progression of liver fibrosis through regulating the hsa-miR-660-3p/TEP1 axis.

Efforts have been made in the prevention and treatment of liver fibrosis. The inhibition or depletion of the hepatic stellate cells (HSCs) has been considered as a potential approach. Recently, there are numbers of studies about the role of the circular RNA in the disease progression. However, the role of circular RNA in the regulation of HSCs and the progression of liver fibrosis remained elusive. In this study, we constructed a CCl4-induced liver fibrosis mouse model and overexpressed hsa_circ_0004018 in HSCs. Then, salvianolic acid B was used to treat HSCs in vitro. We found that hsa_circ_0004018 is downregulated in liver fibrogenesis. Luciferase reporter assay was performed to verify the interaction of hsa_circ_0004018, hsa-miR-660-3p and TEP1. It showed that hsa_circ_0004018 may act as a sponge of hsa-miR-660-3p, which can target and downregulate the expression of TEP1. hsa_circ_0004018 expressing lentivirus was used to investigate the in-vivo function of hsa_circ_0004018 in CCl4-induced liver fibrosis mice. We also reveal that the hsa_circ_0004018/hsa-miR-660-3p/TEP1 axis contributes to the proliferation and activation of HSCs. In addition, the overexpression of hsa_circ_0004018 alleviated the progression of liver fibrosis. In conclusion, our study highlights hsa_circ_0004018 as a potential biomarker and therapeutic target for liver fibrosis.

A model study for the manufacture and validation of clinical-grade deciduous dental pulp stem cells for chronic liver fibrosis treatment

BackgroundHuman deciduous pulp stem cells (hDPSCs) have remarkable stem cell potency associated with cell proliferation, mesenchymal multipotency, and immunosuppressive function and have shown beneficial effects in a variety of animal disease models. Recent studies demonstrated that hDPSCs exhibited in vivo anti-fibrotic and anti-inflammatory action and in vivo hepatogenic-associated liver regeneration, suggesting that hDPSCs may offer a promising source with great clinical demand for treating liver diseases. However, how to manufacture ex vivo large-scale clinical-grade hDPSCs with the appropriate quality, safety, and preclinical efficacy assurances remains unclear.MethodsWe isolated hDPSCs from human deciduous dental pulp tissues formed by the colony-forming unit-fibroblast (CFU-F) method and expanded them under a xenogeneic-free and serum-free (XF/SF) condition; hDPSC products were subsequently stored by two-step banking including a master cell bank (MCB) and a working cell bank (WCB). The final products were directly thawed hDPSCs from the WCB. We tested the safety and quality check, stem cell properties, and preclinical potentials of final hDPSC products and hDPSC products in the MCB and WCB.ResultsWe optimized manufacturing procedures to isolate and expand hDPSC products under a XF/SF culture condition and established the MCB and the WCB. The final hDPSC products and hDPSC products in the MCB and WCB were validated the safety and quality including population doubling ability, chromosome stability, microorganism safety, and stem cell properties including morphology, cell surface marker expression, and multipotency. We also evaluated the in vivo immunogenicity and tumorigenicity and validated in vivo therapeutic efficacy for liver regeneration in a CCl4-induced chronic liver fibrosis mouse model in the final hDPSC products and hDPSC products in the WCB.ConclusionThe manufacture and quality control results indicated that the present procedure could produce sufficient numbers of clinical-grade hDPSC products from a tiny deciduous dental pulp tissue to enhance clinical application of hDPSC products in chronic liver fibrosis.

Alogliptin alleviates liver fibrosis via suppression of activated hepatic stellate cell

Liver fibrosis occurs in most types of chronic liver diseases. The understanding of the pathogenesis of liver fibrosis has grown considerably, but the effective treatments are still lacking. Alogliptin, a classical Dipeptidyl peptidase-4 (DPP4) inhibitor with great effects on type 2 diabetes, has shown the potential to protect liver, but its effects on the progression of liver fibrosis have not been clarified. Herein, we explored the anti-fibrosis effects of alogliptin. In vitro, we demonstrated that alogliptin suppressed the activation of LX-2 upon transforming growth factor-β (TGF-β) challenge. In vivo, chronic treatment with alogliptin alleviated hepatic steatosis and protected from the liver injury in ob/ob mice, which delayed the progression of liver fibrosis. Furthermore, alogliptin significantly relieved the hepatic fibrosis in CCl4-induced liver fibrosis mouse model. In conclusion, our results demonstrate that negatively modulation of alogliptin on hepatic stellate cell (HSC) activation might contribute to liver fibrosis alleviation. Our research provides the potential possibility of alogliptin on the application for liver fibrosis therapy and suggests that DPP4 may be a novel target for liver fibrosis therapy.

Methylation of RCAN1.4 mediated by DNMT1 and DNMT3b enhances hepatic stellate cell activation and liver fibrogenesis through Calcineurin/NFAT3 signaling.

Background: Liver fibrosis is characterized by extensive deposition of extracellular matrix (ECM) components in the liver. RCAN1 (regulator of calcineurin 1), an endogenous inhibitor of calcineurin (CaN), is required for ECM synthesis during hypertrophy of various organs. However, the functional role of RCAN1 in liver fibrogenesis has not yet been addressed.Methods: We induced experimental liver fibrosis in mice by intraperitoneal injection of 10 % CCl4 twice a week. To investigate the functional role of RCAN1.4 in the progression of liver fibrosis, we specifically over-expressed RCAN1.4 in mice liver using rAAV8-packaged RCAN1.4 over-expression plasmid. Following the establishment of the fibrotic mouse model, primary hepatic stellate cells were isolated. Subsequently, we evaluated the effect of RCAN1.4 on hepatic fibrogenesis, hepatic stellate cell activation, and cell survival. The biological role and signaling events for RCAN1 were analyzed by protein-protein interaction (PPI) network. Bisulfite sequencing PCR (BSP) was used to predict the methylated CpG islands in the RCAN1.4 gene promoter. We used the chromatin immunoprecipitation (ChIP assay) to investigate DNA methyltransferases which induced decreased expression of RCAN1.4 in liver fibrosis.Results: Two isoforms of RCAN1 protein were expressed in CCl4-induced liver fibrosis mouse model and HSC-T6 cells cultured with transforming growth factor-beta 1 (TGF-β1). RCAN1 isoform 4 (RCAN1.4) was selectively down-regulated in vivo and in vitro. The BSP analysis indicated the presence of two methylated sites in RCAN1.4 promoter and the downregulated RCAN1.4 expression levels could be restored by 5-aza-2'-deoxycytidine (5-azadC) and DNMTs-RNAi transfection in vitro. ChIP assay was used to demonstrate that the decreased RCAN1.4 expression was associated with DNMT1 and DNMT3b. Furthermore, we established a CCl4-induced liver fibrosis mouse model by injecting the recombinant adeno-associated virus-packaged RCAN1.4 (rAAV8-RCAN1.4) over-expression plasmid through the tail vein. Liver- specific-over-expression of RAN1.4 led to liver function recovery and alleviated ECM deposition. The key protein (a member of the NFAT family of proteins) identified on PPI network data was analyzed in vivo and in vitro. Our results demonstrated that RCAN1.4 over-expression alleviates, whereas its knockdown exacerbates, TGF-β1-induced liver fibrosis in vitro in a CaN/NFAT3 signaling-dependent manner.Conclusions: RCAN1.4 could alleviate liver fibrosis through inhibition of CaN/NFAT3 signaling, and the anti-fibrosis function of RCAN1.4 could be blocked by DNA methylation mediated by DNMT1 and DNMT3b. Thus, RCAN1.4 may serve as a potential therapeutic target in the treatment of liver fibrosis.

DNA Methylation of PTGIS Enhances Hepatic Stellate Cells Activation and Liver Fibrogenesis.

The activation of hepatic stellate cells (HSCs) is a central event in the progression of liver fibrosis. Multiple studies proved that DNA methylation might accelerate HSCs activation. However, the specific pathogenesis of liver fibrosis remains not fully addressed. Our laboratory performed Genome methylation screening to find out the methylated gene in mice with liver fibrosis. The pilot experiments showed that the promoter of prostacyclin synthase (PTGIS) gene was hypermethylated in CCl4-induced liver fibrosis mouse model. Moreover, the down-regulated PTGIS expression can be restored by DNMTs-RNAi and 5-aza-2-deoxycytidine (5-azadC), an inhibitor of DNA methyltransferase (DNMTs). Methylation-specific PCR (MSP) showed that the methylation status of PTGIS in HSC-T6 cells cultures with TGF-β1 (10 ng/mL) was elevated compared with control group. Chromatin immunoprecipitation (ChIP) assay indicated that PTGIS methylation was mainly induced by DNMT1 and DNMT3b. We further investigated the function of PTGIS in liver fibrosis by Recombinant Hepatic-adeno-associated virus (rAAV8)-PTGIS overexpression. The data indicated that overexpression of PTGIS in mouse liver accompanied by elevated apoptosis-related proteins expression in primary HSCs. Conversely, PTGIS silencing mediated by RNAi enhanced the expression of α-SMA and COL1a1 in vitro. Those results illustrated that adding PTGIS expression inhibits the activation of HSCs and alleviates liver fibrosis. Therefore, our study unveils the role of PTGIS in HSCs activation, which may provide a possible explanation for CCl4-mediated liver fibrosis.

ω-3 PUFAs ameliorate liver fibrosis and inhibit hepatic stellate cells proliferation and activation by promoting YAP/TAZ degradation.

Elevated levels of the transcriptional regulators Yes-associated protein (YAP) and transcriptional coactivators with PDZ-binding motif (TAZ), key effectors of the Hippo pathway, have been shown to play essential roles in controlling liver cell fate and the activation of hepatic stellate cells (HSCs). The dietary intake of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) has been positively associated with a number of health benefits including prevention and reduction of cardiovascular diseases, inflammation and cancers. However, little is known about the impact of ω-3 PUFAs on liver fibrosis. In this study, we used CCl4-induced liver fibrosis mouse model and found that YAP/TAZ is over-expressed in the fibrotic liver and activated HSCs. Fish oil administration to the model mouse attenuates CCl4-induced liver fibrosis. Further study revealed that ω-3 PUFAs down-regulate the expression of pro-fibrogenic genes in activated HSCs and fibrotic liver, and the down-regulation is mediated via YAP, thus identifying YAP as a target of ω-3 PUFAs. Moreover, ω-3 PUFAs promote YAP/TAZ degradation in a proteasome-dependent manner. Our data have identified a mechanism of ω-3 PUFAs in ameliorating liver fibrosis.

The interplay of the Notch signaling in hepatic stellate cells and macrophages determines the fate of liver fibrogenesis.

Hepatic stellate cells (HSCs) known as “master producers” and macrophages as “master regulators”, are the key cell types that strongly contribute to the progression of liver fibrosis. Since Notch signaling regulates multiple cellular processes, we aimed to study the role of Notch signaling in HSCs differentiation and macrophages polarization and to evaluate its implication in liver fibrogenesis. Notch pathway components were found to be significantly upregulated in TGFβ-activated HSCs, inflammatory M1 macrophages, and in mouse and human fibrotic livers. Interestingly, inhibition of Notch using a selective γ-secretase inhibitor, Avagacestat, significantly inhibited TGFβ-induced HSC activation and contractility, and suppressed M1 macrophages. Additionally, Avagacestat inhibited M1 driven-fibroblasts activation and fibroblasts-driven M1 polarization (nitric oxide release) in fibroblasts and macrophages co-culture, and conditioned medium studies. In vivo, post-disease treatment with Avagacestat significantly attenuated fibrogenesis in CCl4-induced liver fibrosis mouse model. These effects were attributed to the reduction in HSCs activation, and inhibition of inflammatory M1 macrophages and upregulation of suppressive M2 macrophages. These findings suggest that Notch signaling plays a crucial role in HSC activation and M1/M2 polarization of macrophages in liver fibrosis. These results provide new insights for the development of novel therapies against liver fibrosis through modulation of Notch signaling.