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Liver fibrosis is a common pathological process in the progression of several chronic liver diseases to cirrhosis and hepatocellular carcinoma. Therefore, the development of medications that can repress the progress of liver fibrosis is essential. We discovered that initially, 12β-(m-methyl-benzoyl)-11,12-dihydro oleanolic acid (12d-OA), a farnesoid X receptor (FXR) modulator, possessed potential anti-fibrotic properties. Through an in-depth study, we revealed that 12d-OA not only inhibited the expression of fibrogenic markers in the LX-2 cells and HSC-T6 cells but also exhibited significant protective effects against liver injury and liver fibrosis in bile duct ligation (BDL) rats. Further exploration of its molecular mechanism indicated that 12d-OA exerted antifibrotic activity by inhibiting the extracellular signal-regulated kinase (ERK)/stress-activated protein kinase (p38) signaling pathways. Consequently, the great effects of 12d-OA in vitro and in vivo suggest that it may be a good candidate for liver fibrosis. [ABSTRACT FROM AUTHOR]

Abstract Introduction and objectives In diseases characterized by fibrosis, hepatic stellate cells (HSCs) are mesenchymal cells that play an important role in liver fibrosis. circRNAs are involved in regulating hydatid exocyst formation through miRNA sponge adsorption. The mechanisms of hepatic cystic hydatid outer cyst formation, HSC, and liver fibrosis are unclear. Materials and methods Based on our sequencing data, we validated the mechanism by which circRNA-049637 regulated hepatic cystic hydatid growth and promoted outer fibrocystic wall formation. Results Our results revealed that circRNA_049637 silencing promoted the proliferation of LX-2 human HSCs, affected the cell cycle, and increased the mRNA and protein expression levels of liver fibrosis-related indicators such as α-SMA, COL1A1, COL3A1, and TGFβRII. Conclusions CircRNA_049637 may induce the formation of hepatic hydatid cysts by promoting hepatic fibrosis via HSC activation.

Hepatic stellate cells (HSCs) are responsible for liver fibrosis accompanied by its activation into myofibroblasts and the abundant production of extracellular matrix. However, the HSC contribution to progression of liver inflammation has been less known. We aimed to elucidate the mechanism in HSCs underlying the inflammatory response and the function of tumor necrosis factor α‐related protein A20 (TNFAIP3). We established A20 conditional knockout (KO) mice crossing Twist2‐Cre and A20 floxed mice. Using these mice, the effect of A20 was analyzed in mouse liver and HSCs. The human HSC line LX‐2 was also used to examine the role and underlying molecular mechanism of A20. In this KO model, A20 was deficient in >80% of HSCs. Spontaneous inflammation with mild fibrosis was found in the liver of the mouse model without any exogenous agents, suggesting that A20 in HSCs suppresses chronic hepatitis. Comprehensive RNA sequence analysis revealed that A20‐deficient HSCs exhibited an inflammatory phenotype and abnormally expressed chemokines. A20 suppressed JNK pathway activation in HSCs. Loss of A20 function in LX‐2 cells also induced excessive chemokine expression, mimicking A20‐deficient HSCs. A20 overexpression suppressed chemokine expression in LX‐2. In addition, we identified DCLK1 in the genes regulated by A20. DCLK1 activated the JNK pathway and upregulates chemokine expression. DCLK1 inhibition significantly decreased chemokine induction by A20‐silencing, suggesting that A20 controlled chemokine expression in HSCs via the DCLK1–JNK pathway. In conclusion, A20 suppresses chemokine induction dependent on the DCLK1–JNK signaling pathway. These findings demonstrate the therapeutic potential of A20 and the DCLK1–JNK pathway for the regulation of inflammation in chronic hepatitis. [ABSTRACT FROM AUTHOR]

Introduction and objectives: In diseases characterized by fibrosis, hepatic stellate cells (HSCs) are mesenchymal cells that play an important role in liver fibrosis. circRNAs are involved in regulating hydatid exocyst formation through miRNA sponge adsorption. The mechanisms of hepatic cystic hydatid outer cyst formation, HSC, and liver fibrosis are unclear. Materials and methods: Based on our sequencing data, we validated the mechanism by which circRNA-049637 regulated hepatic cystic hydatid growth and promoted outer fibrocystic wall formation. Results: Our results revealed that circRNA_049637 silencing promoted the proliferation of LX-2 human HSCs, affected the cell cycle, and increased the mRNA and protein expression levels of liver fibrosis-related indicators such as α-SMA, COL1A1, COL3A1, and TGFβRII. Conclusions: CircRNA_049637 may induce the formation of hepatic hydatid cysts by promoting hepatic fibrosis via HSC activation. [ABSTRACT FROM AUTHOR]

The role of peroxisome proliferator-activated receptor (PPAR)β/δ in hepatic fibrosis remains a subject of debate. Here, we examined the effects of a PPARβ/δ agonist on the pathogenesis of liver fibrosis and the activation of hepatic stellate cells (HSCs), the main effector cells in liver fibrosis, in response to the pro-fibrotic stimulus transforming growth factor-β (TGF-β). The PPARβ/δ agonist GW501516 completely prevented glucose intolerance and peripheral insulin resistance, blocked the accumulation of collagen in the liver, and attenuated the expression of inflammatory and fibrogenic genes in mice fed a choline-deficient high-fat diet (CD-HFD). The antifibrogenic effect of GW501516 observed in the livers CD-HFD-fed mice could occur through an action on HSCs since primary HSCs isolated from Ppard-/- mice showed increased mRNA levels of the profibrotic gene Col1a1. Moreover, PPARβ/δ activation abrogated TGF-β1-mediated cell migration (an indicator of cell activation) in LX-2 cells (immortalized activated human HSCs). Likewise, GW501516 attenuated the phosphorylation of the main downstream intracellular protein target of TGF-β1, suppressor of mothers against decapentaplegic (SMAD)3, as well as the levels of the SMAD3 co-activator p300 via the activation of AMP-activated protein kinase (AMPK) and the subsequent inhibition of extracellular signal-regulated kinase-1/2 (ERK1/2) in LX-2 cells. Overall, these findings uncover a new mechanism by which the activation of AMPK by a PPARβ/δ agonist reduces TGF-β1-mediated activation of HSCs and fibrosis via the reduction of both SMAD3 phosphorylation and p300 levels.

Abstract Background The carbazole skeleton is an important structural motif occurring naturally or synthesized chemically and has antihistaminic, antioxidant, antitumor, antimicrobial, and anti-inflammatory activities. Objectives This study aimed to design and synthesize a novel series of carbazole derivatives and evaluate their antiproliferative and antioxidant activities. Methods The synthesized compounds were characterized utilizing HRMS, 1H-, and 13CAPT-NMR, and assessed for their anticancer, antifibrotic, and antioxidant effects utilizing reference biomedical procedures. In addition, the AutoDock Vina application was used to perform in-silico docking computations. Results A series of carbazole derivatives were synthesized and characterized in the current study. Compounds 10 and 11 were found to have a stronger antiproliferative effect than compounds 2–5 against HepG2, HeLa, and MCF7 cancer cell lines with IC50 values of 7.68, 10.09, and 6.44 µM, respectively. Moreover, compound 9 showed potent antiproliferative activity against HeLa cancer cell lines with an IC50 value of 7.59 µM. However, except for compound 5, all of the synthesized compounds showed moderate antiproliferative activities against CaCo-2 with IC50 values in the range of 43.7–187.23 µM. All of these values were compared with the positive control anticancer drug 5-Fluorouracil (5-FU). In addition, compound 9 showed the most potent anti-fibrotic compound, and the cellular viability of LX-2 was found 57.96% at 1 µM concentration in comparison with the positive control 5-FU. Moreover, 4 and 9 compounds showed potent antioxidant activities with IC50 values of 1.05 ± 0.77 and 5.15 ± 1.01 µM, respectively. Conclusion Most of the synthesized carbazole derivatives showed promising antiproliferative, antioxidant, and antifibrotic biological effects, and further in-vivo investigations are needed to approve or disapprove these results.

Background: The carbazole skeleton is an important structural motif occurring naturally or synthesized chemically and has antihistaminic, antioxidant, antitumor, antimicrobial, and anti-inflammatory activities. Objectives: This study aimed to design and synthesize a novel series of carbazole derivatives and evaluate their antiproliferative and antioxidant activities. Methods: The synthesized compounds were characterized utilizing HRMS, 1H-, and 13CAPT-NMR, and assessed for their anticancer, antifibrotic, and antioxidant effects utilizing reference biomedical procedures. In addition, the AutoDock Vina application was used to perform in-silico docking computations. Results: A series of carbazole derivatives were synthesized and characterized in the current study. Compounds 10 and 11 were found to have a stronger antiproliferative effect than compounds 2–5 against HepG2, HeLa, and MCF7 cancer cell lines with IC50 values of 7.68, 10.09, and 6.44 µM, respectively. Moreover, compound 9 showed potent antiproliferative activity against HeLa cancer cell lines with an IC50 value of 7.59 µM. However, except for compound 5, all of the synthesized compounds showed moderate antiproliferative activities against CaCo-2 with IC50 values in the range of 43.7–187.23 µM. All of these values were compared with the positive control anticancer drug 5-Fluorouracil (5-FU). In addition, compound 9 showed the most potent anti-fibrotic compound, and the cellular viability of LX-2 was found 57.96% at 1 µM concentration in comparison with the positive control 5-FU. Moreover, 4 and 9 compounds showed potent antioxidant activities with IC50 values of 1.05 ± 0.77 and 5.15 ± 1.01 µM, respectively. Conclusion: Most of the synthesized carbazole derivatives showed promising antiproliferative, antioxidant, and antifibrotic biological effects, and further in-vivo investigations are needed to approve or disapprove these results. [ABSTRACT FROM AUTHOR]

Abstract Background Cancer is one of the most dangerous and widespread diseases in the world today and it has risen to the position of the leading cause of death around the globe in the last few decades. Due to the inherent resistance of many types of cancer to conventional radiotherapy and chemotherapy, it is vital to develop innovative anticancer medications. Recently, a strategy based on nanotechnology has been used to improve the effectiveness of both old and new cancer drugs. Objectives The present study aimed to design and synthesize a series of phenyl-isoxazole–Carboxamide derivatives, evaluate their anticancer properties, and improve the permeability of potent compounds into cancer cells by using a nano-emulgel strategy. Methods The coupling reaction of aniline derivatives and isoxazole–Carboxylic acid was used to synthesize a series of isoxazole–Carboxamide derivatives. IR, HRMS, 1H-NMR, and 13C-NMR spectroscopy techniques, characterized all the synthesized compounds. The in-vitro cytotoxic evaluation was performed by using the MTS assay against seven cancer cell lines, including hepatocellular carcinoma (Hep3B and HepG2), cervical adenocarcinoma (HeLa), breast carcinoma (MCF-7), melanoma (B16F1), colorectal adenocarcinoma (Caco-2), and colon adenocarcinoma (Colo205), as well as human hepatic stellate (LX-2) in addition to the normal cell line (Hek293T). A nano-emulgel was developed for the most potent compound, using a self-emulsifying technique. Results All synthesized compounds were found to have potent to moderate activities against B16F1, Colo205, and HepG2 cancer cell lines. The results revealed that the 2a compound has broad spectrum activity against B16F1, Colo205, HepG2, and HeLa cancer cell lines with an IC50 range of 7.55–40.85 µM. Moreover, compound 2e was the most active compound against B16F1 with an IC50 of 0.079 µM compared with Dox (IC50 = 0.056 µM). Nanoemulgel was used to increase the potency of the 2e molecule against this cancer cell line, and the IC50 was reduced to 0.039 µM. The antifibrotic activities were investigated against the LX-2 cell line, and it was found that our synthesized molecules showed better antifibrotic activities at 1 µM than 5-FU, and the cell viability values were 67 and 95%, respectively. Conclusion This study suggests that a 2e nano-formalized compound is a potential and promising anti-melanoma agent.

Background: Mesenchymal stem cells (MSCs) are the most promising tools for cell treatment and human tissue regeneration, e.g., in liver fibrosis. Mesenchymal stem cells repair tissue damage through paracrine mediators such as exosomes. Types and concentrations of inflammatory mediators, including transforming growth factor-beta (TGFβ1), in MSCs microenvironment can affect MSCs' function and therapeutic potency. Objectives: This experimental study aimed to explore the effects of Wharton jelly MSCs (WJ-MSCs) exosomes on fibrotic gene expression and Smad2/3 phosphorylation (phospho-Smad2/3 (p-Smad2/3)). Moreover, we further investigated whether WJ-MSCs pretreatment with different concentrations of TGFβ1 changes the anti-fibrotic properties of their exosomes. Methods: After isolation from the umbilical cord, WJ-MSCs were characterized by observing differentiation and measuring surface biomarkers using flowcytometry. The WJ-MSC-derived exosomes were extracted and identified using transmission electron microscopy (TEM), dynamic light scattering (DLS), and western blotting. Real-time PCR and western blot for extracellular matrix (ECM) and p-Smad2/3 expression detection were used to investigate the effect of exosomes from untreated and TGFβ1-pretreated WJ-MSCs on activated hepatic stellate cells (HSCs). Results: Phospho-Smad2/3, α-smooth muscle actin (α-SMA), and collagen1α1 levels were enhanced following treatment with TGFβ1, whereas E-cadherin was decreased. However, the outcomes were reversed after treatment with WJ-MSC-derived exosomes. Exosomes from TGFβ1-pretreated WJ-MSCs induced a significant decrease in p-Smad2/3 levels in activated HSCs, accompanied by the upregulation of E-cadherin gene expression and downregulation of α-SMA and collagen1α1 when compared to untreated WJ-MSC-derived exosomes. The p-Smad2/3 proteins were significantly decreased (fold change: 0.23, P-value < 0.0001) after exposure to low-dose TGFβ1-pretreated WJ-MSC-derived exosomes (0.1 ng/mL), showing the best effect on activated HSCs. Conclusions: Exosomes derived from untreated WJ-MSCs could regress TGFβ-Smad2/3 signaling and the expression of fibrotic markers in activated LX-2 cells. However, these effects were significantly profound with applying exosomes derived from 0.1 ng/mL TGFβ-pretreated WJ-MSCs. We also observed the dose-response effects of TGFβ on WJ-MSCs-derived exosomes. Therefore, exosomes derived from TGFβ-pretreated WJ-MSCs may be critical in improving fibrosis and benefit liver fibrosis patients. [ABSTRACT FROM AUTHOR]

The role of peroxisome proliferator-activated receptor (PPAR)β/δ in hepatic fibrosis remains a subject of debate. Here, we examined the effects of a PPARβ/δ agonist on the pathogenesis of liver fibrosis and the activation of hepatic stellate cells (HSCs), the main effector cells in liver fibrosis, in response to the pro-fibrotic stimulus transforming growth factor-β (TGF-β). The PPARβ/δ agonist GW501516 completely prevented glucose intolerance and peripheral insulin resistance, blocked the accumulation of collagen in the liver, and attenuated the expression of inflammatory and fibrogenic genes in mice fed a choline-deficient high-fat diet (CD-HFD). The antifibrogenic effect of GW501516 observed in the livers CD-HFD-fed mice could occur through an action on HSCs since primary HSCs isolated from Ppard -/- mice showed increased mRNA levels of the profibrotic gene Col1a1. Moreover, PPARβ/δ activation abrogated TGF-β1-mediated cell migration (an indicator of cell activation) in LX-2 cells (immortalized activated human HSCs). Likewise, GW501516 attenuated the phosphorylation of the main downstream intracellular protein target of TGF-β1, suppressor of mothers against decapentaplegic (SMAD)3, as well as the levels of the SMAD3 co-activator p300 via the activation of AMP-activated protein kinase (AMPK) and the subsequent inhibition of extracellular signal-regulated kinase-1/2 (ERK1/2) in LX-2 cells. Overall, these findings uncover a new mechanism by which the activation of AMPK by a PPARβ/δ agonist reduces TGF-β1-mediated activation of HSCs and fibrosis via the reduction of both SMAD3 phosphorylation and p300 levels. [Display omitted] • PPARβ/δ activation improves hepatic fibrosis in mice fed a choline-deficient high-fat diet (CD-HFD). • Primary hepatic stellate cells isolated from Ppard -/- mice show increased mRNA levels of Col1a1. • PPARβ/δ activation abrogates TGF-β1-mediated cell migration and SMAD3 activation in LX-2 cells. • The effects of PPARβ/δ were mediated via the activation of AMPK and the subsequent inhibition of ERK1/2. [ABSTRACT FROM AUTHOR]

Non-steroidal anti-inflammatory drugs (NSAIDs) are considered one of the most commonly used medications globally. Seventeen isoxazole-containing compounds with various functional groups were evaluated in this work to identify which one was the most potent and which group was most selective toward COX-1 and COX-2 by using an in vitro COX inhibition assay kit. Their cytotoxicity was evaluated on the normal hepatic cell line (LX-2) utilizing the MTS assay. Moreover, these molecules' antibacterial and antifungal activities were evaluated using a microdilution assay against several bacterial and fungal species. In addition, molecular docking studies were conducted to identify the possible binding interactions between these compounds and their biological targets by using the X-ray crystal structure of the human COX enzyme and different proteins of bacterial and fungal strains. At the same time, the QiKProp module was used for ADME-T analysis. The results showed that all evaluated isoxazole derivatives showed moderate to potent activities against COX enzymes. The most potent compound against COX-1 and COX-2 enzymes was A13, with IC50 values of 64 and 13 nM, respectively, and a significant selectivity ratio of 4.63. It was clear that the 3,4-dimethoxy substitution on the first phenyl ring and the Cl atom on the other phenyl pushed the 5-methyl-isoxazole ring toward the secondary binding pocket and created the ideal binding interactions with the COX-2 enzyme in comparison with the other compounds. Compound A8 showed antibacterial and antifungal activities against Pseudomonas aeruginosa, Klebsiella pneumonia, and Candida albicans with MIC values of 2 mg/ml. In fact, this compound showed possible binding interactions with the elastase in P. aeruginosa and KPC-2 carbapenemase in K. pneumonia. Furthermore, for better understanding, molecular dynamics simulations were undertaken to study the change in dynamicity of the protein backbone and ligand after the ligand binds to the protein and to ensure the stability of ligand–protein complexes. [ABSTRACT FROM AUTHOR]

In the present study, we aimed to confirm whether NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) could induce apoptosis of human hepatic stellate cells (HSCs) LX-2 and explore the potential pharmacological mechanism underlying these effects. In this study, LX-2 cells were cultured in vitro, and the experiment was divided into two groups, including the control and PDTC groups. The viability of LX-2 cells was measured by CCK8 assay after the cells were exposed to PDTC. The anti-apoptotic effect of PDTC was detected by AO/EB double assay staining kit. Additionally, the activities of NF-κB, Fas/FasL, apoptosis-related proteins, as well as the cellular localization of AIF, were determined by Western blotting analysis and immunofluorescence staining respectively. After PDTC treatment for 12 and 24 h, AO/EB dual staining showed typical apoptotic changes, such as cell volume reduction, cell shrinkage, nuclear fragmentation, and so on. PDTC at 60 μmol/L significantly increased the proliferation inhibition rate and decreased the secretion of collagen I, collagen III, and α-SMA in LX-2 cells. The Western blotting analysis and RT-PCR showed no significant difference in the expression of AIF between the control group and PDTC group, and the expressions of Fas and FasL were not observed in all groups (P > 0.05). Further results showed that PDTC could promote the displacement of AIF from mitochondria to the nucleus, activate the apoptotic signaling in the cell nucleus, and possibly participate in the apoptosis process of LX-2 cells. In conclusion, the pharmacological mechanism of PDTC against hepatic fibrosis might be to promote the displacement of AIF from mitochondria to the nucleus, then activate the apoptotic signaling in the cell nucleus, and finally induce the apoptosis of LX-2 cells. Meanwhile, these results also revealed that the Fas/FasL-mediated apoptosis pathway was not involved in the PDTC-induced apoptosis process of LX-2 cells. [ABSTRACT FROM AUTHOR]

Background: Cancer is one of the most dangerous and widespread diseases in the world today and it has risen to the position of the leading cause of death around the globe in the last few decades. Due to the inherent resistance of many types of cancer to conventional radiotherapy and chemotherapy, it is vital to develop innovative anticancer medications. Recently, a strategy based on nanotechnology has been used to improve the effectiveness of both old and new cancer drugs. Objectives: The present study aimed to design and synthesize a series of phenyl-isoxazole–Carboxamide derivatives, evaluate their anticancer properties, and improve the permeability of potent compounds into cancer cells by using a nano-emulgel strategy. Methods: The coupling reaction of aniline derivatives and isoxazole–Carboxylic acid was used to synthesize a series of isoxazole–Carboxamide derivatives. IR, HRMS, 1H-NMR, and 13C-NMR spectroscopy techniques, characterized all the synthesized compounds. The in-vitro cytotoxic evaluation was performed by using the MTS assay against seven cancer cell lines, including hepatocellular carcinoma (Hep3B and HepG2), cervical adenocarcinoma (HeLa), breast carcinoma (MCF-7), melanoma (B16F1), colorectal adenocarcinoma (Caco-2), and colon adenocarcinoma (Colo205), as well as human hepatic stellate (LX-2) in addition to the normal cell line (Hek293T). A nano-emulgel was developed for the most potent compound, using a self-emulsifying technique. Results: All synthesized compounds were found to have potent to moderate activities against B16F1, Colo205, and HepG2 cancer cell lines. The results revealed that the 2a compound has broad spectrum activity against B16F1, Colo205, HepG2, and HeLa cancer cell lines with an IC50 range of 7.55–40.85 µM. Moreover, compound 2e was the most active compound against B16F1 with an IC50 of 0.079 µM compared with Dox (IC50 = 0.056 µM). Nanoemulgel was used to increase the potency of the 2e molecule against this cancer cell line, and the IC50 was reduced to 0.039 µM. The antifibrotic activities were investigated against the LX-2 cell line, and it was found that our synthesized molecules showed better antifibrotic activities at 1 µM than 5-FU, and the cell viability values were 67 and 95%, respectively. Conclusion: This study suggests that a 2e nano-formalized compound is a potential and promising anti-melanoma agent. [ABSTRACT FROM AUTHOR]

Liver fibrosis (LF) is a pathological process of the liver that threatens human health. Currently, effective treatments are still lacking. Esculin, a prominent constituent found in the Fraxinus rhynchophylla. (bark), Aesculus hippocastanum. (bark), and Cichorium intybus. (herb), has been shown to possess significant anti-inflammatory, antioxidant, and antibacterial properties. However, to date, there have been no studies investigating its potential efficacy in the treatment of LF. The study aims to investigate the therapeutic effect of esculin on LF and elucidate its potential molecular mechanism. Carbon tetrachloride (CCl 4) was injected intraperitoneally to induce LF in mice, and transforming growth factor β1 (TGF-β1) was injected to induce LX-2 cells to investigate the improvement effect of esculin on LF. Kit, histopathological staining, immunohistochemistry (IHC), immunofluorescence (IF), polymerase chain reaction (PCR), and western blot (WB) were used to detect the expression of fiber markers and nuclear factor erythroid 2-related factor 2 (Nrf2)/glutathione peroxidase 4 (GPX4) signaling pathway in liver tissue and LX-2 cells. Finally, molecular docking, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) were used to verify the targeting between Nrf2 and esculin. Esculin significantly inhibited CCl 4 -induced hepatic fibrosis and inflammation in mice. This was evidenced by the improvement of liver function indexes, fibrosis indicators, and histopathology. Additionally, esculin treatment prominently reduced the levels of pro-inflammatory factors, oxidative stress, and liver Fe2+ in CCl 4 -induced mice. In vitro studies also showed that esculin treatment significantly inhibited TGF-β1-induced LX-2 cell activation and decreased alpha-smooth muscle actin (α-SMA) and collagen I expression. Mechanism experiments proved that esculin can activate the Nrf2/GPX4 signaling pathway and inhibit liver ferroptosis. However, when LX-2 cells were treated with the Nrf2 inhibitor (ML385), the therapeutic effect of esculin significantly decreased. This study is the first to demonstrate that esculin is a potential natural active ingredient in the treatment of LF, which can inhibit the activation of hepatic stellate cells (HSC) and improve LF. Its therapeutic effect is related to the activation of the Nrf2/GPX4 signaling pathway. Schematic diagram of the model for the effect of esculin on liver fibrosis. Esculin reduces hepatic stellate cell ferroptosis and inhibits its activation by activating the Nrf2/GPX4 signaling pathway, thereby reducing the severity of liver fibrosis. [Display omitted] [ABSTRACT FROM AUTHOR]

Liver fibrosis is a wound-healing response and the activation of the hepatic stellate cell (HSC) is the core of hepatic fibrosis. MicroRNAs (miRNAs) participate in the development of fibrosis. It is reported that histone deacetylases (HDAC2) tyrosine phosphorylation by cellular-Abelsongene (c-Abl) induces malignant growth of cells. Here, we investigated the effect of miR-122-5p on the proliferation and apoptosis of HSCs. Normal human HSC line LX-2 and LX-2 cells stimulated by transforming growth factor (TGF)-β1 for 24 h were cultured and assigned into the blank group and the TGF-β1 group. The miR-122-5p inhibitor and its negative control were transfected into LX-2 cells and miR-122-5p mimic and its negative control were transfected into LX-2 cells stimulated by TGF-β1. The result showed that miR-122-5p expression was decreased in TGF-β1-stimulated LX-2 cells. miR-122-5p overexpression reduced the mRNA and protein levels of collagen I and α-smooth muscle actin, inhibited cell proliferation, and accelerated cell apoptosis. miR-122-5p targeted c-Abl. Meanwhile, miR-122-5p overexpression inhibited HSC activation by suppressing the c-Abl/HDAC2 pathway. In summary, miR-122-5p overexpression exerted anti-fibrosis effect and inhibited HSC activation by suppressing the c-Abl/HDAC2 pathway. [ABSTRACT FROM AUTHOR]

Background: Hepatic stellate cells (HSCs) are liver-resident myofibroblast precursors responsible for the production of collagen and maintenance of the hepatic extracellular matrix (ECM). As such, they are generally associated with fibrotic liver diseases. HSCs become "activated" in response to tissue damage or pathogen invasion, a process most commonly driven by transforming growth factor-β1 (TGF-β1). Despite this, the full extent of TGF-β1 signalling in these cells is poorly understood. Clarifying the range and diversity of this signalling will further improve our understanding of the process of HSC activation. Methods and results: RNA sequencing was used to quantitate the transcriptomic changes induced in LX-2 cells, an activated human HSC line, following TGF-b1 treatment. In total, 5,258 genes were found to be significantly differentially expressed with a false discovery rate cut-off of < 0.1. The topmost deregulated of these genes included those with no currently characterised role in either HSC activation or fibrotic processes, including CIITA and SERPINB2. In silico analysis revealed the prominent signalling pathways downstream of TGF-β1 in LX-2 cells. Conclusions: In this study, we describe the genes and signalling pathways significantly deregulated in LX-2 cells following TGF-β1 treatment. We identified several highly deregulated genes with no currently characterised role in HSC activation, which may represent novel mediators of fibrotic responses in HSCs or the liver macroenvironment. This work may be of use in the identification of new markers of liver fibrosis and could provide insight into prospective genes or pathways that might be targeted for the amelioration of fibrotic liver disease in the future. [ABSTRACT FROM AUTHOR]

Background: Liver fibrosis (LF) is a pathological process of the liver that threatens human health. Currently, effective treatments are still lacking. Esculin, a prominent constituent found in the Fraxinus rhynchophylla. (bark), Aesculus hippocastanum. (bark), and Cichorium intybus. (herb), has been shown to possess significant anti-inflammatory, antioxidant, and antibacterial properties. However, to date, there have been no studies investigating its potential efficacy in the treatment of LF., Objective: The study aims to investigate the therapeutic effect of esculin on LF and elucidate its potential molecular mechanism., Methods: Carbon tetrachloride (CCl 4 ) was injected intraperitoneally to induce LF in mice, and transforming growth factor β1 (TGF-β1) was injected to induce LX-2 cells to investigate the improvement effect of esculin on LF. Kit, histopathological staining, immunohistochemistry (IHC), immunofluorescence (IF), polymerase chain reaction (PCR), and western blot (WB) were used to detect the expression of fiber markers and nuclear factor erythroid 2-related factor 2 (Nrf2)/glutathione peroxidase 4 (GPX4) signaling pathway in liver tissue and LX-2 cells. Finally, molecular docking, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) were used to verify the targeting between Nrf2 and esculin., Results: Esculin significantly inhibited CCl 4 -induced hepatic fibrosis and inflammation in mice. This was evidenced by the improvement of liver function indexes, fibrosis indicators, and histopathology. Additionally, esculin treatment prominently reduced the levels of pro-inflammatory factors, oxidative stress, and liver Fe 2+ in CCl 4 -induced mice. In vitro studies also showed that esculin treatment significantly inhibited TGF-β1-induced LX-2 cell activation and decreased alpha-smooth muscle actin (α-SMA) and collagen I expression. Mechanism experiments proved that esculin can activate the Nrf2/GPX4 signaling pathway and inhibit liver ferroptosis. However, when LX-2 cells were treated with the Nrf2 inhibitor (ML385), the therapeutic effect of esculin significantly decreased., Conclusion: This study is the first to demonstrate that esculin is a potential natural active ingredient in the treatment of LF, which can inhibit the activation of hepatic stellate cells (HSC) and improve LF. Its therapeutic effect is related to the activation of the Nrf2/GPX4 signaling pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier GmbH.)

Background: The short half-life of TRAIL (tumor necrosis factor–related apoptosis-inducing ligand) greatly limits its clinical application. This study was aimed to improve its potency on liver fibrosis through PEG (polyethylene glycol) modification prolonging the half-life of TRAIL.Methods: PEG, TRAIL, and the chemically synthesized complex PEG-TRAIL were used to treat 3T3 and LX-2 cells and liver fibrotic mice. In vitro, cell viability, apoptosis, and fibrosis were investigated using CCK-8 (cell counting kit-8) assay, flow cytometry, and Western blotting, respectively. In vivo, Sirius red staining, immunohistochemistry, and α-SMA (α-smooth muscle actin)/TUNEL (terminal deoxynucleotidyl transferase dUTP [2'-deoxyuridine 5'-triphosphate] nick end labeling) double-labeling immunofluorescence (IF) were performed after various treatments for liver fibrotic mice. The fibrotic liver was subjected to DR4 (death receptor 4)/TRAIL double-labeling IF to assess the retention of TRAIL enhanced by PEGylation.Results: The cells treated with PEG-TRAIL showed lower cell viability, higher apoptosis level, and stronger anti-fibrotic effect compared with PEG or TRAIL treatment. In vivo, PEGylated TRAIL exhibited a longer circulation than TRAIL did. Compared with TRAIL treatment, PEG-TRAIL caused a significant reduction of α-SMA and a markedly increase of apoptotic aHSCs. PEGylation is more likely to prolong the retention of TRAIL in circulation and enhance the possibility to target aHSCs and DR4-positive (DR4+) cells in the liver.Conclusion: PEG-TRAIL presents better anti-fibrotic and proapoptotic effects, for which, the prolonged circulation half-life in vivo may account. The PEG-TRAIL may serve as a new clinical therapeutic for liver fibrosis in the future.

Liver fibrosis (LF) occurs as a result of persistent liver injury and can be defined as a pathologic, chronic, wound‐healing process in which functional parenchyma is progressively replaced by fibrotic tissue. As a phenomenon involved in the majority of chronic liver diseases, and therefore prevalent, it exerts a significant impact on public health. This impact becomes even more patent given the lack of a specific pharmacological therapy, with LF only being ameliorated or prevented through the use of agents that alleviate the underlying causes. Hepatic stellate cells (HSCs) are fundamental mediators of LF, which, activated in response to pro‐fibrotic stimuli, transdifferentiate from a quiescent phenotype into myofibroblasts that deposit large amounts of fibrotic tissue and mediate pro‐inflammatory effects. In recent years, much effort has been devoted to understanding the mechanisms through which HSCs are activated or inactivated. Using cell culture and/or different animal models, numerous studies have shown that autophagy is enhanced during the fibrogenic process and have provided specific evidence to pinpoint the fundamental role of autophagy in HSC activation. This effect involves – though may not be limited to – the autophagic degradation of lipid droplets. Several hepatoprotective agents have been shown to reverse the autophagic alteration present in LF, but clinical confirmation of these effects is pending. On the other hand, there is evidence that implicates autophagy in several anti‐fibrotic mechanisms in HSCs that stimulate HSC cell cycle arrest and cell death or prevent the generation of pro‐fibrotic mediators, including excess collagen accumulation. The objective of this review is to offer a comprehensive analysis of published evidence of the role of autophagy in HSC activation and to provide hints for possible therapeutic targets for the treatment and/or prevention of LF related to autophagy. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Abstract Background: High morbidity and limited therapies of hepatic fibro genesis are important factor for better understanding the molecular mechanisms of the disease. Advances in the understanding of the molecular behavior of hepatic stellate cells (HSC) allow the progress of a field dedicated to anti-fibrotic therapy. Melanoma differentiation associated gene-7 (IL-24/mda-7) as a gene induced during terminal differentiation in human melanoma cells, but the inflammatory response of cells to IL-24/mda-7 is not entirely cleared. Materias and Methods: LX-2 cells (a human hepatic stellate cell) were treated by leptin (positive control), media (control negative), or were transfected by empty plasmid and pcDNA3.1/mda-7. The inflammatory state was evaluated through measuring the mRNA expression level of inflammatory molecule, IL-1β. The role of IL-24/mda-7 modulation on inflammatory response was assayed using SOCS1 and SOCS3 gene expressions. Results: The expression levels of IL-1β, SOCS1 and SOCS3 were compared in LX-2 cell line groups. The expression of the IL-1β in the transfected cells was higher than the control cell, but it was not significant. The results indicated that the expressions of SOCS1 and SOCS3 were up-regulated following pcDNA 3.1/mda-7 transfection into LX-2 cells compared to control plasmids (p=0.0179, p=0.0428). Conclusion: The endogenous IL-24/mda-7 exhibited a significant modulatory effect on stellate cells. Therefore, IL-24/mda-7 and relevant signaling pathways could be employed as a target for fibrosis treatment.

Background: Experimental and clinical evidence suggests that liver fibrosis is potentially reversible. Hepatic stellate cells (HSCs) play a key role in the development of hepatic fibrosis. Previous clinical applications and researches showed that Siniruangan recipe (SNRG) reversed liver fibrosis and even liver cirrhosis. This experimental study aimed to elucidate the effects of SNRG on the proliferation, apoptosis and activation of HSCs. Methods: The human HSCs line LX-2 was cultured with normal culture medium and multi-dose SNRG water decoction for 48 h. Cell Counting Kit-8 assay was used to detect the proliferation and cytotoxicity of LX-2 cells. Annexin V-FITC/PI double staining was performed to identify apoptotic cells. Immunofluorescence staining was used to determine the relative content of cleaved caspase-3, tissue inhibitor of metalloproteinase-1 (TIMP-1) and transforming growth factor-β1 (TGF-β1) in LX-2 cells. Western blot was used to detect the relative content of Bcl-2, Bax, α-smooth muscle actin, β-catenin and TIMP-1 protein expression in LX-2 cells. Results: The SNRG inhibited the proliferation of LX-2 and induced cell apoptosis through caspase-dependent and mitochondrial-dependent pathways. SNRG may inhibit the activation of LX-2 through the β-catenin pathway. The decrease in TIMP-1 and TGF-β1 protein induced by SNRG promoted the degradation of the extracellular matrix (ECM). Conclusions: SNRG induced LX-2 cell apoptosis, inhibited cell proliferation, decreased LX-2 cell activity and promoted the degradation of ECM in vitro, which may be important mechanisms for reversing liver fibrosis and liver cirrhosis. [ABSTRACT FROM AUTHOR]

Amniotic membranes (AM) have anti-fibrotic activity. Exosomes (nano-sized vesicles) function as conduits for intercellular transfer and contain all the necessary components to induce the resolution of fibrosis. In this study, we tested the hypothesis that the anti-fibrotic activity of AM is mediated by exosomes. AM-derived exosomes or amniotic stromal cell-derived exosomes were isolated and characterized. Anti-fibrotic activity of exosomes was evaluated using human hepatic stellate cells (LX-2), an in vitro model of fibrosis. Exosomes isolated from AM tissue-conditioned media had an average size of 75 nm. Exosomes significantly inhibited the proliferation of TGFβ1-activated LX-2 but had no effect on the proliferation of non-activated LX-2 cells. Exosomes also reduced the migration of LX-2 in a scratch wound assay. Furthermore, exosomes reduced the gene expression of pro-fibrotic markers such as COL1A1, ACTA, and TGFβ1 in LX-2 cells. Interestingly, exosomes isolated from AM tissue under hypoxic conditions seemed to show a stronger anti-fibrotic activity than exosomes isolated from tissue under normoxic conditions. Exosomes released by in vitro cultured AM stromal cells were smaller in size compared with tissue exosomes and also showed anti-fibrotic activity on LX-2 cells. In conclusion, AM-tissue-released exosomes contribute to the anti-fibrotic activity of AM. This is the first report of isolation, characterization, and functional evaluation of exosomes derived from amniotic tissues with the direct comparison between tissue-derived exosomes and cultured cell-derived exosomes.

There is a growing body of studies that show the important role of NS3 protein from hepatitis C virus in fibrosis. However, mechanisms of the effects of this protein on immune modulation of stellate cells remain to be investigated. Herein, the effect of NS3 protein on the expression level of suppressor of cytokine signaling (SOCS)1/3 and interleukin-24 (IL-24)-related genes was investigated in hepatic stellate cell (HSC), LX-2. Recombinant NS3 protein was added to LX-2 HSC culture. Leptin and standard medium treatments were also included in experiments as positive and negative controls, respectively. Total RNA was extracted from each well at 6, 12, and 24 h after NS3 addition. The expression levels of the fibrotic (transforming growth factor beta 1 [TGF-β], alpha-smooth muscle actin [α-SMA], and COL1A1), inflammatory (IL-6 and IL-24), IL-20R, IL-22R, and immunosuppressive genes (SOCS1 and SOCS3) were evaluated by real-time polymerase chain reaction (PCR). Recombinant NS3 protein induced activated phenotypes of LX-2 with a significant increase in the expression level of α-SMACOL1A1 (p < 0.0001) and TGF-β. Moreover, this exposure led to a meaningful elevation in the expression of IL-6. Furthermore, compared with leptin (control), after the stellate cell treatment with NS3, SOCS1 and SOCS3 gene expression induced at a comparable level. Compared with the control sample, the NS3 protein significantly increased the expression level of IL-24 and its related receptors, IL-20R and IL-22R. This study not only confirmed the previously proved inflammatory and fibrotic effect of this protein but also indicated that high expression levels of SOCS1, SOCS3, and IL-24 have a significant effect on HSC activation. Therefore, these two molecules can be used as a potential therapeutic target candidate. [ABSTRACT FROM AUTHOR]

Induction of apoptosis or quiescent hepatic stellate cells (HSCs) can be an attractive molecular strategy due to the importance of activation of HSCs during hepatic fibrogenesis. Interleukin-24/melanoma differentiation-associated gene-7 (IL-24/mda-7) is a cytokine that has attracted a great deal of attention in the tumor killing as well as pathophysiology of the diseases. In this study, the Pro-apoptotic and senescence inductive properties of IL-24/mda-7 were assessed in human-derived HSCs. Three plasmids expressing natural mda-7, peptide modified version, mda-7-RGD genes beside a recombinant IL-24 protein, were added or transfected into activated LX-2 cells. Cell viability and the amount of apoptosis were analyzed using MTT and Annexin V staining method, respectively. Hence, the expression levels of apoptotic genes and PPARγ in different groups were also compared by real-time PCR analysis. Furthermore, the senescence effect of IL-24/mda-7 by a β-galactosidase (SA-β-gal) senescence assay, was evaluated. The viability assessment showed that pmda-7-RGD had the most significant growth inhibitory effect when compared to the control group, pcDNA3.1 (P = 0.0002). The apoptosis analysis also revealed a significant impact of different mda-7 forms in apoptosis induction. The measuring of cell senescence also indicated that IL-24/mda-7 in plasmid and protein forms exhibited a senescence inductive activity as determined by an increase in PPARγ gene expression and beta-galctosidase activity. In conclusion, our findings demonstrated that both endogenous and soluble forms of IL-24/mda-7 induced apoptosis and senescence in activated LX-2 cells and more importantly, fusion of RGD peptide to this cytokine enhanced these activities. So, RGD-modified IL-24/mda-7 could be a suitable candidate for further molecular therapy of fibrosis. [ABSTRACT FROM AUTHOR]

A fibrose hepática é uma condição clínica presente na maioria das doenças hepáticas, caracterizada pelo aumento na síntese dos componentes da matriz extracelular na tentativa de cicatrização do tecido lesionado. A principal célula responsável pelo desenvolvimento da fibrose hepática é a célula estrelada hepática, localizada no espaço de Disse. Esta apresenta dois fenótipos: o quiescente e o ativado. O metabolismo de lipídeo na célula estrelada está relacionado com o fornecimento de energia para a iniciação e perpetuação do seu estado ativado. Dentro deste contexto a ácido graxo sintase (FASN), proteína chave da síntese de ácido graxo de novo torna-se alvo do estudo, uma vez que o metabolismo de lipídeos está intimamente relacionado ao processo fibrosante em células estreladas hepáticas. Sabe-se que o fármaco triclosan (TCS) é inibidor da proteína FASN na síntese de ácido graxo de novo, com potencial alteração do processo fibrosante em células estreladas hepáticas. Sendo assim, avaliamos os aspectos celulares e moleculares do efeito do fármaco TCS na modulação do metabolismo de lipídeo através da inibição da FASN em modelo de células estreladas hepáticas LX-2. Os resultados demonstraram que o TCS na concentração 50 µM em cultura celular LX-2 crescida em 10% de soro bovino fetal (SBF) – estado ativado- promove alterações morfofuncionais nas células; tanto pela despolimerização dos filamentos de actina quanto pela formação de estruturas globulares de actina no citoesqueleto celular. Desta maneira, o fármaco TCS apresentou características possivelmente citotóxicas em células LX-2 com 10% de SBF. Por outro lado, o estudo da inibição da proteína FASN demonstrou resultados que sugerem uma transdiferenciação celular correlacionada com a reversão do quadro pró-fibrosante hepático em células LX-2, com o aumento das gotículas de lipídeo no citoplasma e aumento da expressão dos genes relacionados ao metabolismo de lipídeos. Estes resultados requerem maiores investigações da modulação do fármaco TCS na patogenia investigada.

Background: The aim of this study was to characterize the effects of the coagulation proteinase thrombin on proteomic level in human hepatic stellate LX-2 cells. Methods: Proteomic analyses were performed using surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS). The protein profiles obtained from LX-2 cell lysates using strong anion exchanger Q10 ProteinChip arrays were statistically analyzed. Results: The peak intensities of 50 protein/peptide clusters were identified as being different between nonstimulated and LX-2 cells treated with thrombin for 6 h and 24 h, respectively. As the most significantly enhanced single signal in LX-2 cells stimulated with thrombin, a protein with a molecular mass of 13.560 kDa has been identified that corresponds exactly to calcium dependent phospholipase 2 (cPLA2). Thrombin-induced increase in the cPLA2 protein expression in LX-2 cells was confirmed by using the Western blotting technique. Conclusions: Together with the finding that thrombin induced phosphorylating activation of cPLA2 in LX-2 cells, our data point to an important function of the thrombin-mediated modulation of cytosolic phospholipase A2 in hepatic stellate cells.

Hepatitis C virus (HCV) infection is considered one of the most important causes of chronic liver diseases. Many reports have shown that the proteins of the HCV via interactions with gene expression regulatory networks such as cellular pathways and microRNAs can contribute to the development of chronic liver diseases. The present study aimed to investigate the effects of the HCV NS3 protein on the expression of miR-150 miR-199a, miR-335, miR-194, miR-27a in a cell culture model. Plasmids expressing the full length of the HCV NS3 protein were transfected into the LX-2 cell line, while at the same time a plasmid expressing empty GFP (green fluorescent protein) was used as a negative control group. Subsequently, total RNA was extracted and real-time PCR was performed to measure microRNA expression levels. Additionally, the trypan blue exclusion test was performed to examine the effect of the expressing NS3 protein plasmid on cellular viability. The analysis of microRNA gene expression in LX-2 cells indicated that the NS3 protein, which is endogenous to HCV, can significantly upregulate the expression of miR-27a and downregulate the expression of miR-335 and miR-150 in comparison with the control plasmid expressing GFP and normal cells (p < 0.01). These results suggest that the HCV NS3 protein may play a role in the pathogenesis of chronic hepatic diseases such as liver fibrosis via interaction with cellular microRNAs and modulation of microRNA gene expressions. [ABSTRACT FROM AUTHOR]

Activation of hepatic stellate cells (HSCs) is the pivotal event during liver fibrosis. Interleukin (IL)-24/mela­noma differentiation-associated gene-7 (mda-7) has attracted attention in the pathophysiology of some diseases, while its role in activation/suppression of human HSCs is still unclear. It is important to elucidate whether the expression levels of the IL-24/mda-7 protein and its receptors in HSC cells are changed following activation. LX-2 cells, a human hepatic stellate cell line were activated by a combination of leptin and serum starvation. The activation state was evaluated through measuring the mRNA expression of profibrotic molecules, collagen-I, TIMP metalloproteinase inhibitor-1 and transforming growth factor-β. The expression of IL-24/mda-7 was assessed in mRNA and protein levels by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and ELISA methods, respectively. Hence, the amount of IL-22R1 and IL-20R2 subunit expression was also compared in activated and normal LX-2 cells by RT-qPCR. The expression level of IL-24/mda-7 and its cognate receptors was detectable both in the normal and activated LX-2 cell line. Furthermore, in activated LX-2, a significant increase of IL24 expression either on IL-22R1 and IL-20R2 subunits was also noticeable in comparison to normal cells. The activation state of LX-2 cells caused significant changes of IL-24/mda-7 and its receptors expression. In addition, the elevation in IL-24/mda-7 during LX-2 cell activation, suggested that IL-24/mda-7 and its cognate receptors serve a possible role in the development of the fibrosis process. Therefore, IL-24/mda-7 and relevant signaling pathways may be employed as a target for fibrosis treatment. [ABSTRACT FROM AUTHOR]

The development of liver fibrosis is associated with inflammatory responses resulting from chronic liver disease. Immature dendritic cells (imDCs) play an important role in modulating the inflammatory environment of the liver. This study investigated the effects of imDCs on the regulation of hepatic stellate cells (HSCs) during liver fibrosis. We isolated and induced imDCs from monocytes of healthy volunteers, activated LX-2 cells with TGF-β to establish in vivo liver fibrosis HSCs model, and then set up a cell co-culture system with transwell membranes. imDC surface markers and apoptosis rates of LX-2 cells were detected by flow cytometry. The concentration of IL-10 secreted by imDC was measured through ELISA. The expression of α-SMA in LX-2 after co-culture was examined by qRT‑PCR. Proliferation of LX-2 cells were detected by CCK-8. The western blot was used to illustrate the LX-2 activation-related proteins such as Smad3/7 and TGF-β1. The imDCs co-culture group and the interleukin-10 (IL-10) treatment group had similar results, as they were both able to increase apoptosis, inhibit proliferation, downregulate α-SMA mRNA, and reduce TGF-β1 and Smad3 protein expression in LX-2 cells. Additionally, the Smad7 protein level was increased after treatment with imDC and IL-10. However, the results in the IL-10 antagonist group showed the opposite trend to that of imDCs and IL-10 groups. Thus, these results suggest that imDC secretion of IL-10 negatively regulates activated LX-2 cells, probably via inhibition of the TGF-β1/Smad3 pathway and increased expression of Smad7 protein. This may be a potential therapeutic target for liver fibrosis. [ABSTRACT FROM AUTHOR]

Background: Previous studies have established that proteinase-activated receptor 2 (PAR2) promotes migration and invasion of hepatocellular carcinoma (HCC) cells, suggesting a role in HCC progression. Here, we assessed the impact of PAR2 in HCC stromal cells on HCC growth using LX-2 hepatic stellate cells (HSCs) and Hep3B cells as model. Methods: PAR2 expression and function in LX-2 cells was analysed by RT-PCR, confocal immunofluorescence, electron microscopy, and [Ca2+]i measurements, respectively. The impact of LX-2-expressed PAR2 on tumour growth in vivo was monitored using HCC xenotransplantation experiments in SCID mice, in which HCC-like tumours were induced by coinjection of LX-2 cells and Hep3B cells. To characterise the effects of PAR2 activation in LX-2 cells, various signalling pathways were analysed by immunoblotting and proteome profiler arrays. Results: Following verification of functional PAR2 expression in LX-2 cells, in vivo studies showed that these cells promoted tumour growth and angiogenesis of HCC xenografts in mice. These effects were significantly reduced when F2RL1 (encoding PAR2) was downregulated by RNA interference (RNAi). In vitro studies confirmed these results demonstrating RNAi mediated inhibition of PAR2 attenuated Smad2/3 activation in response to TGF-β1 stimulation in LX-2 cells and blocked the pro-mitotic effect of LX-2 derived conditioned medium on Hep3B cells. Furthermore, PAR2 stimulation with trypsin or a PAR2-selective activating peptide (PAR2-AP) led to activation of different intracellular signalling pathways, an increased secretion of pro-angiogenic and pro-mitotic factors and proteinases, and an enhanced migration rate across a collagen-coated membrane barrier. Silencing F2RL1 by RNAi or pharmacological inhibition of Src, hepatocyte growth factor receptor (Met), platelet-derived growth factor receptor (PDGFR), p42/p44 mitogen activated protein kinase (MAPK) or matrix-metalloproteinases (MMPs) blocked PAR2-AP-induced migration. Conclusion: PAR2 in HSCs plays a crucial role in promoting HCC growth presumably by mediating migration and secretion of pro-angiogenic and pro-mitotic factors. Therefore, PAR2 in stromal HSCs may have relevance as a therapeutic target of HCC. [ABSTRACT FROM AUTHOR]

It was reported that O glycosylation is associated with hepatic stellate cell activation and regulates collagen expression. In this study, we aimed to investigate the effect of O glycosylation on the activation of human hepatic stellate cells. We found that the inhibitor of O glycosylation, benzyl-α-GalNAc (2 and 4 mM), could significantly inhibit cells proliferation in a dose-dependent manner. Moreover, benzyl-α-GalNAc decreased the expressions of α-smooth muscle actin, collagen I, and collagen III. The results indicate that O glycosylation is involved in the activation of hepatic stellate cells. [ABSTRACT FROM AUTHOR]

Hepatic fibrosis (HF) is a worldwide health problem for which there is no medically effective drug treatment at present, and which is characterized by activation of hepatic stellate cells (HSCs) and excessive extracellular matrix (ECM) deposition. The HF model in cholestatic rats by ligating the common bile duct was induced and the differentially expressed miRNAs in the liver tissues were analyzed by microarray, which showed that miR-22-3p and miR-29a-3p were significantly downregulated in bile-duct ligation (BDL) rat liver compared with the sham control. The synergistic anti-HF activity and molecular mechanism of miR-22-3p and miR-29a-3p by targeting AKT serine/threonine kinase 3 (AKT3) in HSCs were explored. The expression levels of miR-22-3p and miR-29a-3p were downregulated in activated LX-2 and human primary normal hepatic fibroblasts (NFs), whereas AKT3 was found to be upregulated in BDL rat liver and activated LX-2 cells. The proliferation, colony-forming, and migration ability of LX-2 were inhibited synergistically by miR-22-3p and miR-29a-3p. In addition, cellular senescence was induced and the expressions of the LX-2 fibrosis markers COL1A1 and α-SMA were inhibited by miR-22-3p and miR-29a-3p synergistically. Subsequently, these two miRNAs binding to the 3'UTR of AKT3 mRNA was predicted and evidenced by the luciferase reporter assay. Furthermore, the proliferation, migration, colony-forming ability, and the expression levels of COL1A1 and α-SMA were promoted and cellular senescence was inhibited by AKT3 in LX-2 cells. Thus, miR-22-3p/miR-29a-3p/AKT3 regulates the activation of HSCs, providing a new avenue in the study and treatment of HF.

Sm16 is a 16 KDa protein released by Schistosoma mansoni that modulates inflammatory responses in host cells. Sm 16 is expressed by several life cycle stages of S. mansoni , including the egg stage. Schistosome eggs are known to provoke chronic schistosomiasis pathology, which involves the development of liver fibrosis. Hepatic stellate cells (HSCs), which are responsible for this fibrosis, are susceptible to immunomodulation by S. mansoni whole egg secretions. To define the effects of Sm 16 exposure on HSCs, two synthetic peptide derivatives of Sm 16, coined "KS-84″ and "KS-66″, were tested against LX-2 cells, an immortalised human HSC line, and RNA sequencing was used to assess the transcriptional changes induced by each peptide. In total, 78 and 798 genes were found to be significantly differentially expressed by KS-84 and KS-66 treatment, respectively. In silico pathway analysis of these genes revealed that KS-84 reduced LX-2 cell activation and fibrotic potential, whereas KS-66 increased both processes. Reduced transforming growth factor-β1 (TGF-β1) signalling was identified as a potential mechanism of KS-84-induced inhibition of LX-2 activation. Taken together, these findings indicate a potential role for Sm 16 in combatting fibrotic liver disease. [Display omitted] • Peptide derivatives of the S. mansoni protein Sm 16 alter the responses of HSCs. • KS-84 was predicted to inhibit HSC activation and fibrotic potential. • Reduced TGF-β signaling was identified as a possible mechanism for this inhibition. • Sm 16-derived peptides may have a role in combatting fibrotic liver disease. [ABSTRACT FROM AUTHOR]

Background: Sorafenib, which is approved for treatment of HCC, has also shown promising antifibrotic activity, and therefore refinement of its dosing requirements and window of efficacy are important goals prior to antifibrotic clinical trials. Aim: The purpose of this study was to determine the minimal effective dose and optimal timing of sorafenib therapy in cultured human stellate cells and in rats with experimental hepatic fibrosis. Methods: Effects of sorafenib were assessed in a human stellate cell line (LX-2). In vivo, rats were treated for 8 weeks with TAA three times per week (150 mg/kg IP), and with either PBS or sorafenib administered daily at doses of 1.25, 5 or 7 mg/kg/day gavage either at the beginning of TAA administration for 8 weeks, during weeks 4-8, or from weeks 8-12. Results: Sorafenib treatment significantly inhibited LX-2 proliferation by >75 % (7.5 or 15 μM). Treatment with 7.5-μM sorafenib for 12 h markedly inhibited expression of TGFβ1, TIMP-1, collagen I, and MMP2 mRNAs, but not of β-PDGFR or type I TGFβR. In vivo, sorafenib significantly inhibited liver fibrosis when started concurrently with TAA and during weeks 4-8 with TAA. In contrast, there was no significant effect of sorafenib on fibrogenic gene expression or fibrosis when begun after cirrhosis was already established. Conclusion: Sorafenib is anti-proliferative and antifibrotic towards human HSCs in culture, and is a potent antifibrotic agent in TAA-induced hepatic fibrosis in rats. The drug is effective at relatively low doses at the early stage of liver fibrosis, but is not effective when cirrhosis is already established. [ABSTRACT FROM AUTHOR]

Background and Aims: Radiation-induced liver fibrosis (RILF), delayed damage to the liver (post-irradiation) remains a major challenge for the radiotherapy of liver malignancies. This study investigated the potential function and mechanism of circTUBD1 in the development of RILF., Methods: By using a dual luciferase assay, RNA pull-down assays, RNA sequencing, chromatin immunoprecipitation (known as ChIP) assays, and a series of gain- or loss-of-function experiments, it was found that circTUBD1 regulated the activation and fibrosis response of LX-2 cells induced by irradiation via a circTUBD1/micro-203a-3p/Smad3 positive feedback loop in a 3D system., Results: Knockdown of circTUBD1 not only reduced the expression of α-SMA, as a marker of LX-2 cell activation, but also significantly decreased the levels of hepatic fibrosis molecules, collagen type I alpha 1 (COL1A1), collagen type III alpha 1 (COL3A1), and connective tissue growth factor (CTGF) in a three-dimensional (3D) culture system and RILF model in vivo . Notably, knockdown of circTUBD1 alleviated early liver fibrosis induced by irradiation in mice models., Conclusions: This study is the first to reveal the mechanism and role of circTUBD1 in RILF via a circTUBD1/micro-203a-3p/Smad3 feedback loop, which provides a novel therapeutic strategy for relieving the progression of RILF., Competing Interests: The authors have no conflict of interests related to this publication., (© 2022 Authors.)

Abstract: The LX-2 cell line has characteristics of hepatic stellate cells (HSCs), which are considered pericytes of the hepatic microcirculatory system. Recent studies have suggested that HSCs might have mesenchymal origin. We have performed an extensive characterization of the LX-2 cells and have compared their features with those of mesenchymal cells. Our data show that LX-2 cells have a phenotype resembling activated HSCs as well as bone marrow-derived mesenchymal stem cells (BM-MSCs). Our immunophenotypic analysis showed that LX-2 cells are positive for activated HSC markers (αSMA, GFAP, nestin and CD271) and classical mesenchymal makers (CD105, CD44, CD29, CD13, CD90, HLA class-I, CD73, CD49e, CD166 and CD146) but negative for the endothelial marker CD31 and endothelial progenitor cell marker CD133 as well as hematopoietic markers (CD45 and CD34). LX-2 cells also express the same transcripts found in immortalized and primary BM-MSCs (vimentin, annexin 5, collagen 1A, NG2 and CD140b), although at different levels. We show that LX-2 cells are capable to differentiate into multilineage mesenchymal cells in vitro and can stimulate new blood vessel formation in vivo. LX-2 cells appear not to possess tumorigenic potential. Thus, the LX-2 cell line behaves as a multipotent cell line with similarity to BM-MSCs. This line should be useful for further studies to elucidate liver regeneration mechanisms and be the foundation for development of hepatic cell-based therapies. [Copyright &y& Elsevier]

Abstract: We have demonstrated that gene polymorphisms within the N-terminal leader sequence of TGF-β1 contribute to the outcome of hepatic fibrogenesis. In addition, the polymorphism at codon 25 affects TGF-β1 production in peripheral blood leukocytes. Therefore, it is general assumed that these polymorphisms influence cellular secretion of this cytokine. In the present study, we analysed if this widespread hypothesis is true. We cloned FLAG-tagged CMV-driven human full-length TGF-β1 expression constructs of the different allelic variations (i.e. 10Leu/25Arg, 10Pro/25Pro and 10Pro/25Arg) and transfected them into the immortal hepatic stellate cell line LX-2 and Chinese Hamster Ovary cells. Surprisingly, the allelic variants carrying a proline either in codon 10 or 25 showed overall reduced expression as assessed by Western blot and quantitative ELISA. We conclude that the allelic variations within the signal sequence influence the expression and not secretion of TGF-β1. Detailed RNA structure prediction analysis further suggests that the individual variants form different secondary structures. [Copyright &y& Elsevier]

Hepatic stellate cells (HSCs) play a significant role in the development of chronic liver diseases. Hepatic damage activates HSCs and results in hepatic fibrosis. The functions of activated HSCs require an increase in the cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ). However, the regulatory mechanisms underlying Ca 2+ signaling in activated HSCs remain largely unknown. In the present study, functional analyses of Ca 2+ -sensing receptors (CaSRs) were performed using activated human HSCs, LX-2. Expression analyses revealed that CaSR proteins were expressed in α-smooth muscle actin-positive LX-2 cells. Extracellular Ca 2+ restoration (from 0 to 2.2 mM) increased [Ca 2+ ] cyt in these cells. The extracellular Ca 2+ -induced increase in [Ca 2+ ] cyt was reduced by the CaSR antagonists, NPS2143 and Calhex 231. Furthermore, the growth of LX-2 cells was blocked by NPS2143 and Calhex 231 in concentration-dependent manners (IC 50  = 6.0 and 9.5 μM, respectively). LX-2 cell proliferation was also attenuated by NPS2143 and Calhex 231. In conclusion, CaSRs are functionally expressed in activated HSCs and regulate Ca 2+ signaling and cell proliferation. The present results provide insights into the molecular mechanisms underlying hepatic fibrosis and will contribute to the development of potential therapeutic targets.

Long non-coding (lnc)RNAs serves an important role in the occurrence and development of hepatic fibrosis. lncRNA AK021443 is highly expressed in hepatocellular carcinoma (HCC) and promotes HCC cell proliferation, invasion and migration. The present study aimed to investigate the effect of AK021443 on hepatic fibrosis. AK021443 was overexpressed in the human LX-2 hepatic stellate cell (HSC) line using a plasmid to observe its effect on hepatic fibrosis in vitro. A Cell Counting Kit-8 assay was performed to assess cell proliferation, whereas cell cycle distribution and related proteins were analyzed via flow cytometry and western blotting, respectively. The protein expression levels of epithelial-mesenchymal transition (EMT)-associated and extracellular matrix (ECM) proteins were also analyzed via western blotting. Immunofluorescence was conducted to observe the generation of collagen1, and the activity of inflammatory factors and reactive oxygen species (ROS) was also analyzed. Compared with the pcDNA group, AK021443 overexpression significantly promoted cell proliferation, enhanced the transition of cells from G1 to S phase and increased the expression of cyclin-dependent kinase 2 and cyclin D1, but reduced the p21 protein expression levels. In addition, EMT capabilities, ECM deposition and the generation of collagen1 were increased by AK021443 overexpression compared with the pcDNA group. Moreover, AK021443 overexpression significantly increased the release of inflammatory cytokines, including TGF-β, interleukin-1β, platelet derived growth factor, epidermal growth factor and ROS, compared with the pcDNA group. In conclusion, the present study suggested that AK021443 overexpression increased HSC proliferation, activation and the proinflammatory response, indicating the potential role of AK02144 in aggravating hepatic fibrosis. [ABSTRACT FROM AUTHOR]

Amniotic membranes (AM) have anti-fibrotic activity. Exosomes (nano-sized vesicles) function as conduits for intercellular transfer and contain all the necessary components to induce the resolution of fibrosis. In this study, we tested the hypothesis that the anti-fibrotic activity of AM is mediated by exosomes. AM-derived exosomes or amniotic stromal cell-derived exosomes were isolated and characterized. Anti-fibrotic activity of exosomes was evaluated using human hepatic stellate cells (LX-2), an in vitro model of fibrosis. Exosomes isolated from AM tissue-conditioned media had an average size of 75 nm. Exosomes significantly inhibited the proliferation of TGFβ1-activated LX-2 but had no effect on the proliferation of non-activated LX-2 cells. Exosomes also reduced the migration of LX-2 in a scratch wound assay. Furthermore, exosomes reduced the gene expression of pro-fibrotic markers such as COL1A1, ACTA, and TGFβ1 in LX-2 cells. Interestingly, exosomes isolated from AM tissue under hypoxic conditions seemed to show a stronger anti-fibrotic activity than exosomes isolated from tissue under normoxic conditions. Exosomes released by in vitro cultured AM stromal cells were smaller in size compared with tissue exosomes and also showed anti-fibrotic activity on LX-2 cells. In conclusion, AM-tissue-released exosomes contribute to the anti-fibrotic activity of AM. This is the first report of isolation, characterization, and functional evaluation of exosomes derived from amniotic tissues with the direct comparison between tissue-derived exosomes and cultured cell-derived exosomes. [ABSTRACT FROM AUTHOR]

Objective: To investigate the effects of ceramide pathway on the inhibition of artesunate (Art) to hepatic fibrosis. Methods: LX-2 cells were divided into control group, Art treated group with 350 μmol/L, fumonisin B1 (FB1) treated group with 6 μmol/L, and Co-administration group of artesunate 350 μmol/L and fumonisin B1 6 μmol/L. There were 7 compound holes in each group. After 24 hours of treatment, the cells and supernatant were collected and detected. The expressions of homo sapiens longevity assurance homologue 2 (LASS2), peroxisome proliferators-activated receptors-γ (PPAR-γ) and Caspase-3 were evaluated by Western blot, the content of ceramide was evaluated by HPLC-FLD method, MTT assay was adopted to measure the rate of proliferation of LX-2 cells. The content of hydroxyproline was determined by digestive method. Results: Compared with the control group, the expression of ceramide synthase protein and the ceramide content were increased significantly, the proliferation of LX-2 cells was inhibited significantly, the expressions of PPAR-γ and Caspase-3 protein were up-regulated and the secretion of hydroxyproline was inhibited in Art treated group (P＜0.05). In FB1 treated group, the protein expression of ceramide synthase and the ceramide content were decreased significantly, the proliferation of LX-2 cells was increased significantly, the expressions of PPAR-γ and Caspase-3 protein were down-regulated, and the secretion of hydroxyproline was increased (P＜0.05). Compared with the Art alone group, the combination of the two drugs could significantly reduce the effects of Art on the expression of ceramide synthase protein and the increase of ceramide content, and attenuate the effects of Art on the cell proliferation , PPAR-γ, Caspase-3 protein expression and hydroxyproline level of LX-2 cells (P＜0.05). Conclusion: Artesunate could inhibit hepatic fibrosis by increasing the content of ceramide through the ceramide synthase-ceramide pathway.

To determine the correlation between QKI and pancreatic cancer tissues, the QKI expression of pancreatic cancer cells and fibroblasts in the tumor-surrounding microenvironment were detected. Then, QKI overexpression and interference with QKI short hairpin RNA in LX-2 (a fibroblast cell line) were established in vitro. Meanwhile, to observe the cell proliferation, invasion, migration, and other changes, QKI, and related epithelial-mesenchymal transition (EMT) molecules were detected by a polymerase chain reaction and Western blot analysis. In addition, an in vivo tumorigenicity test in node mice was performed to confirm whether QKI expression can promote the proliferation, invasion, and metastasis of pancreatic cancer ductal epithelial cells. Finally, the autophagy levels of fibroblasts with QKI overexpression were observed by electron microscopy to further explore the QKI pathogenic mechanism. It was found that cell proliferation, invasion, migration, and EMT-related markers were increased in QKI-overexpressed fibroblasts LX-2. Furthermore, in vivo, liver and peritoneal metastasis decreased overall survival rate and increasing autophagy levels in QKI-overexpressing nude mice were observed. Meanwhile, knock down QKI with small interfering RNA can reverse all the above effects. QKI can promote the proliferation, metastasis, and invasion of pancreatic cancer through activating fibroblasts surrounding pancreatic cancer and accelerating EMT and increasing the autophagy in pancreatic cancer. QKI may become a potential target for the treatment of pancreatic cancer., (© 2019 Wiley Periodicals, Inc.)