Human Hepatic Stellate Cell Line Research Articles

Stanniocalcin 1 is a serum biomarker and potential therapeutic target for HBV-associated liver fibrosis.

Stanniocalcin 1 (STC1), a secreted protein, is upregulated in human cancers including hepatocellular carcinoma (HCC). While most HCCs develop from chronic liver disease, which involves progressive parenchymal injury and fibrosis, the role of STC1 in this preneoplastic stage remains poorly understood. In this study we investigated the clinical relevance and functional significance of secreted STC1 in liver fibrosis. To this end, the STC1 level was determined in the serum samples of chronic hepatitis B patients and correlated with the degree of liver fibrosis. Diagnostic performance of STC1 was analysed by area under the receiver operating characteristic curve (AUROC), sensitivity, specificity, positive predictive value, and negative predictive value. The results were compared with other well-characterised serum biomarkers for liver fibrosis: Aspartate transaminase to Platelet Ratio Index (APRI) and Fibrosis-4 (FIB-4). The functional role of STC1 was interrogated by in vitro experiments using cell line models. Expression of fibrogenic markers was quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. Our results showed that the serum STC1 level in chronic hepatitis B patients was positively correlated with the degree of liver fibrosis and showed a stepwise increase in accordance with the severity of fibrosis. The AUROCs for detecting significant fibrosis (>9.0kPa) and cirrhosis (>12.0kPa) was 0.911 and 0.880, respectively. STC1 demonstrated a superior specificity and positive predictive value when compared to APRI and FIB-4. Consistent with this, STC1 was elevated in the liver tissues and sera of CCl4 -treated mice showing marked liver fibrosis. In vitro, STC1 was secreted by the human hepatic stellate cell line LX2. Human recombinant STC1 (rhSTC1) induced expression of fibrogenic markers in LX2 cells. The profibrogenic phenotype conferred by rhSTC1 or TGF-β1 in LX2 cells could be attenuated using anti-STC1 antibody. Taken together, STC1 is a specific serum biomarker for HBV-associated liver fibrosis. STC1 functionally promotes liver fibrogenesis and is a potential actionable target. © 2022 The Pathological Society of Great Britain and Ireland.

Role of long non-coding RNA-adducin 3 antisense RNA1 in liver fibrosis of biliary atresia

ABSTRACT Biliary atresia (BA) is a devastating liver disease in neonates. Liver fibrosis is regarded as a universal and prominent feature of BA. Studies have revealed that long non-coding RNAs (lncRNAs) regulate cellular processes during the development of liver fibrosis in BA. Long non-coding RNA-adducin 3 antisense RNA1 (lnc-ADD3-AS1) has been shown to increase susceptibility to BA. However, the role of lnc-ADD3-AS1 in liver fibrosis in BA remains unclear. Here, we investigated the role of lnc-ADD3-AS1 in the proliferation, migration, and apoptosis of the immortalized human hepatic stellate cell (HSC) line, LX-2. We successfully overexpressed and silenced lnc-ADD3-AS1 in LX-2 cells using adenovirus vectors and evaluated the proliferation of transfected cells using the Cell Counting Kit-8 (CCK8) assay. Cell apoptosis was detected using annexin V–fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining and flow cytometry. We then analyzed cell migration by performing wound-scratch and transwell migration assays. Our results show that lnc-ADD3-AS1 significantly promoted LX-2 cell proliferation and attenuated apoptosis. More importantly, lncRNA-ADD3-AS1 significantly accelerated the migration of LX-2 cells. Our data indicated that lncRNA-ADD3-AS1 plays a role in the pathogenesis of liver fibrosis in patients with BA and may serve as a potential diagnostic marker for monitoring liver fibrosis in BA or as a therapeutic target for the disease.

Increased Levels of CHI3L1 and HA Are Associated With Higher Occurrence of Liver Damage in Patients With Obstructive Sleep Apnea.

BackgroundObstructive sleep apnea-hypopnea syndrome (OSA) may cause liver fibrosis, and liver fibrosis serum biomarkers plays an important role on the diagnosis of liver fibrosis. In addition, this study aimed to observe the changes of 4 serum markers and Chitinase 3-like protein 1 (CHII3L1) levels in OSA patients with different disease severity and explore their interactions. And then, we examined whether intermittent hypoxia (IH) exposure can activate hepatic stellate cell.Methods74 OSA patients in Second Xiangya hospital from January 2021 to October 2021 was selected and categorized into mild, moderate, and severe groups according to AHI. In addition, 20 subjects were selected as the control group. Serum levels of liver fibrosis markers were determined by electrochemiluminescence immunoassay. Hepatic stellate cells were exposed to intermittent IH or normoxia (RA). Results were analyzed using the SPSS software.ResultsThere was a significant increase in serum hyaluronic acid (HA), collagen type IV (CIV) and CHI3L1 levels in OSA patients compared with control group. Specifically, serum liver fibrosis markers HA, CIV and CHI3L1 levels were positively correlated with apnea-hypopnea index (AHI), but negatively correlated with the lowest saturation oxygen (LSaO2) respectively. The LX-2 cells (human hepatic stellate cell line) exposed to IH showed significant increases in fibrotic protein expression.ConclusionOSA might either directly or indirectly trigger or exacerbate liver fibrosis, possibly via IH-related pathways.

Effects of praziquantel isomers on the proliferation and activation of the LX-2 human hepatic stellate cell line

To compare the effects of levo-praziquantel (L-PZQ) and dextro-praziquantel (D-PZQ) on the proliferation and activation of the human hepatic stellate cell line LX-2 in vitro. LX-2 cells were stimulated with transforming growth factor-β (TGF-β). LX-2 cell proliferation was measured using the CCK-8 assay after 24 h stimulation with 0 to 50 μg/mL concentrations of praziquantel, and the gene and protein expression of type Ⅰ collagen (collagen Ⅰ), type Ⅲ collagen (collagen Ⅲ) and α-smooth muscle actin (α-SMA) was quantified in LX-2 cells using quantitative real-time PCR (qPCR) and Western blotting assays 24 h and 48 h following stimulation with 15 μg/mL praziquantel to detect LX-2 cell activation. There were significant differences in the survival rate of LX-2 cells between L-PZQ and D-PZQ treatments at all concentrations (F = 6.119 and 79.180, both P values < 0.05). Either L-PZQ or D-PZQ at a concentration of < 30 μg/mL showed no remarkableeffectsonthe LX-2 cell proliferation (both P values > 0.05), and L-PZQ at a concentration of > 50 μg/mL and D-PZQ at a concentration of > 40 μg/mL inhibited the LX-2 cell proliferation (both P values < 0.05), while D-PZQ at concentrations of 40 μg/mL and 50 μg/mL showed greater inhibition on LX-2 cell proliferation than L-PZQ (t = 3.419 and 8.776, both P values < 0.05). There were significant differences in the collagen Ⅰ, collagen Ⅲ and α-SMA expression in LX-2 cells at both transcriptional (F = 21.55, 79.99 and 46.70, all P values < 0.05) and translational levels (F = 20.12, 30.29 and 32.93, all P values < 0.05) among the blank control group, TGF-β stimulation group, L-PZQ treatment group and D-PZQ treatment group. L-PZQ treatment resulted in remarkable inhibition on collagen Ⅲ and α-SMA gene expression in LX-2 cells (both P values < 0.05); however, the treatment showed no remarkable inhibition collagen Ⅰ gene expression or collagen Ⅰ, collagen Ⅲ or α-SMA protein expression in LX-2 cells (all P values > 0.05). In addition, D-PZQ treatment resulted in significant inhibition on collagen Ⅰ, collagen Ⅲ and α-SMA expression in LX-2 cells at both translational and transcriptional levels (all P values < 0.05), and D-PZQ showed higher inhibition on collagen Ⅰ, collagen Ⅲ and α-SMA gene expression in LX-2 cells than L-PZQ (all P values < 0.05). Both L-PZQ and D-PZQ inhibit the proliferation and activation of LX-2 cells, and D-PZQ shows a higher inhibitory activity than L-PZQ.

TJ-M2010-5, A self-developed MyD88 inhibitor, attenuates liver fibrosis by inhibiting the NF-κB pathway

Liver fibrosis is the result of most chronic inflammatory liver damage and seriously endangers human health. However, no drugs have been approved to treat this disease. Previous studies showed that the Toll‐like receptors (TLRs)/myeloid differentiation factor-88 (MyD88)/nuclear factor-κB (NF-κB) pathway plays a key role in liver fibrosis. TJ-M2010-5 is a self-developed small molecule MyD88 inhibitor, which has been proven to have a good protective effect in a variety of inflammatory disease models. In the present study, to investigate the anti-fibrotic effect of TJ-M2010-5, mice were injected with carbon tetrachloride (CCl4) in vivo and LX2 cells (a human hepatic stellate cell line) were treated with TGF-β1 in vitro to induce liver fibrosis. In vivo studies showed that TJ-M2010-5 attenuated the CCl4-induced liver damage, collagen accumulation, and the activation of hepatic stellate cells by inhibiting the nuclear transfer of NF-κB. Moreover, in vitro experiments of LX2 cells stimulated with TGF-β1 further indicated that the NF-κB pathway is involved in the development of liver fibrosis. TJ-M2010-5 significantly inhibited the proliferation and activation of LX2 cells. In addition, TJ-M2010-5 upregulated the expression of bone morphogenetic protein and membrane-bound inhibitor (BAMBI) in LX2 cells by blocking the activation of MyD88/NF-κB, thereby inhibiting the phosphorylation of Smad2/3 and the expression of collagen I (COL1A1) induced by TGF-β1. In conclusion, this study illustrates the anti-hepatic fibrosis effect of TJ-M2010-5 and provides a new treatment method for liver fibrosis.

The Invasive Potential of Hepatoma Cells Induced by Radiotherapy is Related to the Activation of Hepatic Stellate Cells and Could be Inhibited by EGCG Through the TLR4 Signaling Pathway.

Post-radiotherapy recurrence and metastasis of liver cancer were thought to arise from the invasion and metastasis of residual hepatocellular carcinoma cells, but it has now been shown to be closely related to the increased metastatic potential of residual liver cancer cells mediated by radiotherapy. The changes of liver microenvironment after radiotherapy also provide a favorable condition for promoting the metastatic potential of hepatocellular carcinoma. Studies have shown that radiation-induced activation of hepatic stellate cells (HSCs) is one of the main changes in the microenvironment of hepatocellular carcinoma. Therefore, we hypothesized that activated HSCs are involved in regulating the metastatic capacity of residual cancer cells after radiotherapy. The present study observed that 48 h co-culture of three human hepatoma cell lines (MHCC97-L, Hep-3B, LM3) with a irradiated human HSC line (LX-2) in a transwell chamber could significantly improve the invasion of the human hepatoma cells; and the culture supernatant of activated HSCs could also enhance the invasion of the hepatoma cells. In contrast, co-culture with irradiated hepatoma cells enhanced the invasion of LX-2 cells. In vitro, irradiation enhanced the activation phenotype and the toll like receptor 4 (TLR4) signaling pathway of LX-2 cells or primary mouse HSCs, which upregulated intercellular cell adhesion molecule-1 (ICAM1), laminin receptor (67 LR), Interleukin- 6 (IL-6), and CX3C chemokine ligand 1 (CX3CL1) and downregulated toll-interacting proteins. The compound (-)-epigallocatechin-3-gallate (EGCG) inhibited signal transduction of activated TLR4 and radiation-induced invasion of LX-2 cells by binding to 67 LR. These observations indicated that the enhancement of the metastatic potential of hepatoma cells after irradiation was relevant to the activation of HSCs, and the activation of TLR4 signaling pathway was involved in this process, which was inhibited by EGCG. Our results will help enhance the therapeutic efficacy of liver cancer stereotactic body radiation therapy to prevent and decrease the risks of post-radiotherapy recurrence and metastasis.

Involvement of TREK1 channels in the proliferation of human hepatic stellate LX-2 cells

Activation of hepatic stellate cells (HSCs) causes hepatic fibrosis and results in chronic liver diseases. Although activated HSC functions are facilitated by an increase in the cytosolic Ca2+ concentration ([Ca2+]cyt), the pathophysiological roles of ion channels are largely unknown. In the present study, functional analyses of the two-pore domain K+ (K2P) channels, which regulate the resting membrane potential and [Ca2+]cyt, were performed using the human HSC line, LX-2. Expression analyses revealed that TREK1 (also known as KCNK2 and K2P2.1) channels are expressed in LX-2 cells. Whole-cell K+ currents were activated by 10 μM arachidonic acid and the activation was abolished by 100 μM tetrapentylammonium, which are pharmacological characteristics of TREK1 channels. The siRNA knockdown of TREK1 channels caused membrane depolarization and reduced [Ca2+]cyt. In addition, TREK1 knockdown downregulated the gene expression of collage type I and platelet-derived growth factor. Furthermore, TREK1 knockdown inhibited the proliferation of LX-2 cells. In conclusion, the activity of TREK1 channels determines the resting membrane potential and [Ca2+]cyt, which play a role in extracellular matrix production and cell proliferation in HSCs. This study may help elucidate the molecular mechanism underlying hepatic fibrosis in HSCs and provide a potential therapeutic target for hepatic fibrosis.

Neuropeptide Calcitonin Gene-related Peptide Signaling via RAMP1 Stimulates Liver Fibrosis and Regulates Yes-associated Protein Activity during Chronic Liver Damage

Background & aims: Chronic liver damage can ultimately lead to liver cirrhosis. Sensory nerves innervating the liver secrete the neuropeptide calcitonin gene-related peptide (CGRP), which binds to receptor activity-modifying protein (RAMP)1. We investigated whether the loss of RAMP1 affects liver fibrosis in chronic liver inflammation. Methods: Wild-type and RAMP1-deficient mice were injected with carbon tetrachloride (CCl4) to induce liver injury. CGRP/RAMP1 levels in CCl4-treated livers were determined by RT-PCR. Fibrosis was assessed by Sirius red staining and Western blot (α-SMA and collagen). The influence of CGRP/RAMP1 on the Hippo signaling pathway was assessed by Western blot. In addition, we stimulated the human hepatic stellate cell line (LX-2) in vitro with CGRP. The expression of α-SMA, collagen and YAP proteins was quantified by Western blot. Results: Chronic liver injury resulted in upregulation of hepatic CGRP/RAMP1 mRNA expression. In the absence of RAMP1, murine livers exhibited less fibrosis. In addition, expression of markers for hepatocyte proliferation was decreased. Also, the expression of YAP was diminished in RAMP1-deficient mice. At the same time, phosphorylation of YAP on Ser127, which promotes inactivation of YAP, was increased in RAMP1-deficient livers. In vitro, stimulation of LX-2 cells with CGRP promoted hepatic stellate cell activation. Moreover, treatment with CGRP resulted in inactivation of YAP, confirming our in vivo results. Conclusion: RAMP1/CGRP signaling promotes liver fibrosis and controls YAP activity, as well as the activation of LX-2 cells in vitro. The CGRP receptor RAMP1 may serve as a potential target of antifibrotic therapy in liver disease.

Non-heat-stressed Method to Isolate Hepatic Stellate Cells From Highly Steatotic Tumor-bearing Liver Using CD49a.

Non-heat-stressed Method to Isolate Hepatic Stellate Cells From Highly Steatotic Tumor-bearing Liver Using CD49a.

MicroRNA-122-5p prevents proliferation and promotes apoptosis of hepatic stellate cells by suppressing the cellular-Abelsongene/histone deacetylases 2 pathway.

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 24h 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.

Mannan-Binding Lectin via Interaction With Cell Surface Calreticulin Promotes Senescence of Activated Hepatic Stellate Cells to Limit Liver Fibrosis Progression.

Background & AimsLiver fibrosis represents a hallmark of most chronic liver diseases (CLD) triggered by recurrent liver injury and subsequent myofibroblast transdifferentiations of resident hepatic stellate cells (HSCs). Mannan-binding lectin (MBL) is potentially involved in hepatic fibrosis in CLD through unclear mechanisms. Therefore, we investigated the crosstalk between MBL and HSCs, and the consequent effects on fibrosis progression.MethodsSamples from patients with liver cirrhosis were collected. MBL deficiency (MBL-/-) and wild-type (WT) C57BL/6J mice were used to construct a CCl4-induced liver fibrosis model. Administration of MBL-expressing, liver-specific, adeno-associated virus was performed to restore hepatic MBL expression in MBL-/- mice. The human HSC line LX-2 was used for in vitro experiments.ResultsMBL levels in patients with liver cirrhosis were correlated with disease severity. In the CCl4-induced liver fibrosis model, MBL-/- mice showed severer liver fibrosis accompanied by reduced senescent activated HSCs in liver tissue compared with WT mice, which could be inhibited by administering MBL-expressing, liver-specific, adeno-associated virus. Moreover, depleting senescent cells with senolytic treatment could abrogate these differences owing to MBL absence. Furthermore, MBL could interact directly with calreticulin associated with low-density lipoprotein receptor-related protein 1 on the cell surface of HSCs, which further promotes senescence in HSCs by up-regulating the mammalian target of rapamycin/p53/p21 signaling pathway.ConclusionsMBL as a newfound senescence-promoting modulator and its crosstalk with HSCs in the liver microenvironment is essential for the control of hepatic fibrosis progression, suggesting its potential therapeutic use in treating CLD associated with liver fibrosis.

Purification and Isolation of Hepatic Stellate Cells.

Quiescent human hepatic stellate cells (HSCs) serve as important reservoirs of fat-soluble vitamins in the body, namely vitamin A. In an activated form, HSCs are the drivers of fibrosis following chronic liver injury. In non-alcoholic steatohepatitis (NASH) specifically, activated HSCs are drivers of induction and progression of fibrogenesis. Isolation and purification of HSCs from donor liver samples provides an avenue to study HSCs and their fibrotic capabilities. Manual and chemical digestion of donor liver via dissection and Pronase, collagenase, and DNAse treatment creates a suspension of non-parenchymal liver cells. Quiescent HSCs can be further isolated from this suspension by density-gradient centrifugation in a 6%, 8%, 12%, and 15% arabinogalactan medium. After collection of HSCs from the low-density layers of the gradient, they can be grown on uncoated plastic. Rodent HSCs can also be isolated via density-gradient centrifugation. To isolate activated HSCs, liver tissue explants or established immortalized HSC lines can be utilized. Here, we described protocols for isolation of human and rodent HSCs.

Hepatoprotective effect of genistein against dimethylnitrosamine-induced liver fibrosis in rats by regulating macrophage functional properties and inhibiting the JAK2/STAT3/SOCS3 signaling pathway.

Liver fibrosis is a dysregulated wound-healing process in response to diverse liver injuries, and an effective drug therapy is not yet available. Genistein, which is one of the most active natural flavonoids mainly derived from soybean products (e.g., Cordyceps sinensis mycelium), exhibits various biological effects, including hepatoprotective and anti-inflammatory properties. However, the anti-hepatic fibrosis mechanisms of genistein are poorly understood. The aim of our research is to explore the effect and the possible mechanism of genistein against liver fibrosis. Cell counting kit-8, EdU, and flow cytometry assays were applied to evaluate the effects of genistein on cell viability, proliferation, and cell cycle arrest in human hepatic stellate cell (HSC) line LX2 cells. HSC activation was induced by transforming growth factor-β1 in LX2 cells and liver fibrosis model was established by the intraperitoneal injection of dimethylnitrosamine (DMN) in rats to assess the anti-fibrosis effects of genistein in vivo and in vitro models. HSC activation was assessed by qRT-PCR, Western blot, immunohistochemistry, and immunofluorescent assay. Liver injury and collagen deposition were evaluated by histopathological assay, serum biochemistry, and hepatic hydroxyproline content assays. The mRNA expressions of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and inflammation related-factors were assessed by qRT-PCR assay. Furthermore, the functional properties of macrophage in the liver were assessed by immunohistochemistry assay. The expression levels of the JAK2/STAT3/SOCS3 signaling pathway related-protein were assessed by Western blot analysis. Genistein significantly inhibited cell viability and proliferation and induced cell cycle arrest at G0/G1 phase in LX2 cells, respectively. Furthermore, oral administration of genistein significantly ameliorated liver injury and the collagen deposition in rats with DMN-induced fibrosis model. Genistein suppressed the expression levels of HSC activation marker α-smooth muscle actin and collagen type I alpha 1 in vivo and in vitro. Genistein significantly decreased the mRNA expression levels of extracellular matrix degradation genes MMP2/9 and TIMP1 in rats. Genistein alleviated the mRNA expression levels of IL-1β, IL-6, TNF-α, and MCP-1 and regulated the protein expressions of CD68, CD163, and CD206 in the liver. Moreover, genistein attenuated the expressions of p-JAK2/JAK2, p-STAT3/STAT3, and SOCS3 protein both in vivo and in vitro. Taken together, our results showed that genistein could be improved liver fibrosis both in vivo and in vitro, probably through regulating the functional properties of macrophage and inhibiting the JAK2/STAT3/SOCS3 signaling pathway.

Tetrahydropalmatine attenuates liver fibrosis by suppressing endoplasmic reticulum stress in hepatic stellate cells.

Tetrahydropalmatine attenuates liver fibrosis by suppressing endoplasmic reticulum stress in hepatic stellate cells.

Autophagy of hepatic stellate cell induced by Clonorchis sinensis.

Clonorchis sinensis was a food-borne zoonotic parasite in the worldwide and also an important risk factor of hepatic fibrosis. Excretory/secretion products of C. sinensis (CsESPs) are involved in parasite-host interactions and contribute to the development of hepatic damage. The aim of the present study was to investigate whether CsESPs and CsTP (adult protein) could induce autophagy of hepatic stellate cells (HSCs) and further activate HSCs so as to participate in the pathogenesis of hepatic fibrosis. The human hepatic stellate cell line LX-2 was stimulated by CsESPs and CsTP. CsESPs showed the effect on cell proliferation in methyl thiazolyl tetrazolium (MTT) assay while CsTP failed. Autophagosomes and autolysosomes were observed after the transmission mRFP-EGFP-LC3 plasmid into the LX-2 cells. CsESPs had more powerful to induce the accumulation of autophagosomes and autolysosomes to enhance autophagic flux compared with CsTP. Western-blotting analysis confirmed that the ratio of LC3-II/I in LX-2 cells was up-regulated after CsESPs treatment for 6h, which further proved that CsESPs could induce autophagy in LX-2 cells. Meanwhile, q-PCR results showed that the mRNA levels of collagen I, collagen III and α-SMA decreased in LX-2 cells after treatment with autophagy inhibitor chloroquine, whereas they increased when combination with CsESPs. These results suggested that CsESPs-induced autophagy might be involved in the activation of HSCs, and consequently participate in the pathogenesis of hepatic fibrosis caused by C. sinensis infection.

Effect of miR-183-5p on Cholestatic Liver Fibrosis by Regulating Fork Head Box Protein O1 Expression.

Liver fibrosis is a common pathological feature of end-stage liver disease and has no effective treatment. MicroRNAs (miRNAs) have been found to modulate gene expression in liver disease. But the potential role of miRNA in hepatic fibrosis is still unclear. The objective of this research is to study the potential mechanism and biological function of miR-183-5p in liver fibrosis. In this study, we used high-throughput sequencing to find that miR-183-5p is upregulated in human fibrotic liver tissues. In addition, miR-183-5p was upregulated both in rat liver fibrosis tissue induced by bile-duct ligation (BDL) and activated LX-2 cells (human hepatic stellate cell line) according to the result of quantitative real-time PCR (RT-qPCR). Moreover, the inhibition of miR-183-5p alleviated liver fibrosis, decreased the fibrotic biomarker levels in vitro and in vivo, and led toLX-2 cell proliferation inhibition and, apoptosis induction. The result of dual-luciferase assay revealed that miR-183-5p suppressed fork head box protein O1 (FOXO1) expression by binding to its 3′UTR directly. Next, we used lentivirus to overexpress FOXO1 in LX-2 cells, and we found that overexpression of FOXO1 reversed the promotion of miR-183-5p on liver fibrosis, reducing the fibrotic biomarker levels inLX-2 cells, inhibitingLX-2 cell proliferation, and promoting apoptosis. Furthermore, overexpression of FOXO1 prevented the activation of the transforming growth factor (TGF)-β signaling pathway in TGF-β1-induced LX-2 cells according to the result of western blotting. In conclusion, the findings showed thatmiR-183-5p might act as a key regulator of liver fibrosis, and miR-183-5p could promote cholestatic liver fibrosis by inhibiting FOXO1 expression through the TGF-β signaling pathway. Thus, inhibition of miR-183-5pmay be a new way to prevent and improve liver fibrosis.

Human Liver Stem Cell-Derived Extracellular Vesicles Target Hepatic Stellate Cells and Attenuate Their Pro-fibrotic Phenotype.

Liver fibrosis occurs in response to chronic liver injury and is characterized by an excessive deposition of extracellular matrix. Activated hepatic stellate cells are primarily responsible for this process. A possible strategy to counteract the development of hepatic fibrosis could be the reversion of the activated phenotype of hepatic stellate cells. Extracellular vesicles (EVs) are nanosized membrane vesicles involved in intercellular communication. Our previous studies have demonstrated that EVs derived from human liver stem cells (HLSCs), a multipotent population of adult stem cells of the liver with mesenchymal-like phenotype, exert in vivo anti-fibrotic activity in the liver. However, the mechanism of action of these EVs remains to be determined. We set up an in vitro model of hepatic fibrosis using a human hepatic stellate cell line (LX-2) activated by transforming growth factor-beta 1 (TGF-β1). Then, we investigated the effect of EVs obtained from HLSCs and from human bone marrow-derived mesenchymal stromal cells (MSCs) on activated LX-2. The incubation of activated LX-2 with HLSC-EVs reduced the expression level of alpha-smooth muscle actin (α-SMA). Conversely, MSC-derived EVs induced an increase in the expression of pro-fibrotic markers in activated LX-2. The analysis of the RNA cargo of HLSC-EVs revealed the presence of several miRNAs involved in the regulation of fibrosis and inflammation. Predictive target analysis indicated that several microRNAs (miRNAs) contained into HLSC-EVs could possibly target pro-fibrotic transcripts. In particular, we demonstrated that HLSC-EVs shuttled miR-146a-5p and that treatment with HLSC-EVs increased miR-146a-5p expression in LX-2. In conclusion, this study demonstrates that HLSC-EVs can attenuate the activated phenotype of hepatic stellate cells and that their biological effect may be mediated by the delivery of anti-fibrotic miRNAs, such as miR-146a-5p.

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma.

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroidmodel which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 µL media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 µL media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Discovery of 1,8-naphthalidine derivatives as potent anti-hepatic fibrosis agents via repressing PI3K/AKT/Smad and JAK2/STAT3 pathways

Liver fibrosis is one of the most common pathological consequences of chronic liver diseases (CLD). To develop effective antifibrotic strategies, a novel class of 1-(substituted phenyl)-1,8-naphthalidine-3-carboxamide derivatives were designed and synthesized. By means of the collagen type I α 1 (COL1A1)-based screening and cytotoxicity assay in human hepatic stellate cell (HSC) line LX-2, seven compounds were screened out from total 60 derivatives with high inhibitory effect and relatively low cytotoxicity for further COL1A1 mRNA expression analysis. It was found that compound 17f and 19g dose-dependently inhibited the expression of fibrogenic markers, including α-smooth muscle actin (α-SMA), matrix metalloprotein 2 (MMP-2), connective tissue growth factor (CTGF) and transforming growth factor β1 (TGFβ1) on both mRNA and protein levels. Further mechanism studies indicated that they might suppress the hepatic fibrogenesis via inhibiting both PI3K/AKT/Smad and non-Smad JAK2/STAT3 signaling pathways. Furthermore, 19g administration attenuated hepatic histopathological injury and collagen accumulation, and reduced fibrogenesis-associated protein expression in liver tissues of bile duct ligation (BDL) rats, showing significant antifibrotic effect in vivo. These findings identified 1,8-naphthalidine derivatives as potent anti-hepatic fibrosis agents, and provided valuable information for further structure optimization.