Activation Of LX-2 Cells Research Articles

Dibutyl phthalate induces liver fibrosis via p38MAPK/NF-κB/NLRP3-mediated pyroptosis

Dibutyl phthalate (DBP) is one of the most employed plasticizers pervading the environment. DBP is a newly identified global organic pollutant that can activate NLRP3 inflammasomes and induce inflammatory liver injury. However, its hepatotoxicity remains poorly understood. The objective of this investigation was to investigate the probable pathways underlying DBP-induced liver injury. First, C57BL/6N mice were orally administered DBP at 10 and 50 mg/kg B.W. doses for 28 days. The observed results indicated a significant increase in liver collagen deposition and upregulated protein expression of fibrosis markers in mice. In addition, the p38MAPK/NF-κB signaling pathway and pyroptosis-related protein expression were upregulated. To establish a correlation between these changes, we conducted a conditioned medium co-culture of human hepatocellular carcinoma (HepG2) and human hepatic stellate (LX-2) cells. We performed inhibitor interventions to validate the mechanism of DBP-induced liver fibrosis in vitro. After treatment with p38MAPK (SB203580), NF-κB (PDTC), and NLRP3 (MCC950) inhibitors, the activation of LX-2 cells, the p38MAPK/NF-κB signaling pathway and pyroptosis due to DBP were alleviated. Therefore, DBP exposure leads to NLRP3-mediated pyroptosis of hepatocytes via the p38MAPK/NF-κB signaling pathway, activating LX-2 cells and causing liver fibrosis. Our findings offer a conceptual framework to understand the pathological underpinnings of DBP-induced liver injury while proposing novel ideas to prevent and treat DBP hepatotoxicity. Thus, targeting p38MAPK, NF-κB, and NLRP3 may prevent DBP-induced liver fibrosis.

PNPLA3-I148M Variant Promotes the Progression of Liver Fibrosis by Inducing Mitochondrial Dysfunction.

Patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 polymorphism (I148M) is strongly associated with non-alcoholic steatohepatitis and advanced fibrosis; however, the underlying mechanisms remain largely unknown. In this study, we investigated the effect of PNPLA3-I148M on the activation of hepatic stellate cell line LX-2 and the progression of liver fibrosis. Immunofluorescence staining and enzyme-linked immunosorbent assay were used to detect lipid accumulation. The expression levels of fibrosis, cholesterol metabolism, and mitochondria-related markers were measured via real-time PCR or western blotting. Electron microscopy was applied to analyze the ultrastructure of the mitochondria. Mitochondrial respiration was measured by a Seahorse XFe96 analyzer. PNPLA3-I148M significantly promoted intracellular free cholesterol aggregation in LX-2 cells by decreasing cholesterol efflux protein (ABCG1) expression; it subsequently induced mitochondrial dysfunction characterized by attenuated ATP production and mitochondrial membrane potential, elevated ROS levels, caused mitochondrial structural damage, altered the oxygen consumption rate, and decreased the expression of mitochondrial-function-related proteins. Our results demonstrated for the first time that PNPLA3-I148M causes mitochondrial dysfunction of LX-2 cells through the accumulation of free cholesterol, thereby promoting the activation of LX-2 cells and the development of liver fibrosis.

The lncRNA HOTAIR via miR-17-5p is involved in arsenite-induced hepatic fibrosis through regulation of Th17 cell differentiation.

Arsenic compounds are toxins that are widely distributed in the environment. Chronic exposure to low levels of these compounds can cause hepatic fibrosis and other damage. Th17 differentiation of CD4+ T cells and the secretion of IL-17 activates hepatic stellate cells (HSCs), which are involved in hepatic fibrosis, but their mechanisms in arsenic-induced hepatic fibrosis are unclear. We found, in arsenite-induced fibrotic livers of mice, increases of CD4+ T cell infiltration, Th17 cell nuclear receptor retinoic acid receptor-related orphan receptor γt (RORγt), and secretion of the pro-inflammatory cytokine IL-17. There were also elevated levels of the lncRNA, HOTAIR. For Jurkat cells, arsenite elevated levels of HOTAIR and protein levels of RORγt and IL-17A, decreased miR-17-5p, promoted Th17 cell differentiation, and released IL-17. The culture medium of arsenite-treated Jurkat cells activated LX-2 cells. Down-regulation of HOTAIR or up-regulation of miR-17-5p blocked arsenite-induced Th17 cell differentiation, which inhibited the LX-2 cell activation. However, down-regulation of HOTAIR and miR-17-5p reversed this inhibitory effect. For mice, silencing of HOTAIR diminished the hepatic levels of RORγt and IL-17A and alleviated arsenite-induced hepatic fibrosis. These results demonstrate that, for CD4+ T cells, arsenite promotes RORγt-mediated Th17 cell differentiation through HOTAIR down-regulation of miR-17-5p, and increases the secretion of cytokine IL-17A, which activates HSCs; the activated HSCs facilitate hepatic fibrosis. The findings reveal a new mechanism and a potential therapeutic target for arsenite-induced hepatic fibrosis.

Recombinant Schistosoma japonicum egg ribonuclease SjCP1412 inhibits the activation of LX-2 hepatic stellate cells in vitro

To investigate the effect of recombinant Schistosoma japonicum egg ribonuclease SjCP1412 (rSjCP1412) on proliferation, cell cycle, apoptosis and activation of human hepatic stellate cells LX-2 in vitro, and explore the underlying mechanisms. The rSjCP1412 protein was expressed in Escherichia coli BL21 by prokaryotic expression, and the highly purified soluble rSjCP1412 protein was prepared by Ni NTA affinity chromatography and urea gradient refolding dialysis. Yeast RNA was digested using 12.5, 25.0, 50.0 µg rSjCP1412 proteins at 37 °C for 2, 3, 4 h, and the enzymatic products were electrophoresed on 1.5% agarose gel to observe the RNAase activity of rSjCP1412 protein. The proliferation of LX-2 cells stimulated by different doses of rSjCP1412 protein for 48 hours was measured using CCK-8 assay, and the apoptosis of LX-2 cells stimulated by different doses of rSjCP1412 protein for 48 hours was detected using the Annexin V-FITC/PI double staining, while the percentage of LX-2 cells at G0/G1, S and G2/M phases of cell cycle following stimulation with different doses of rSjCP1412 protein for 48 h was detected by DAPI staining. The type I collagen, type III collagen and α-smooth muscle actin (α-SMA) mRNA expression was quantified using quantitative florescent real-time PCR (qPCR) assay and Western blotting at transcriptional and translational levels in LX-2 cells following stimulation with different doses of rSjCP1412 protein for 48 h, while soluble egg antigen (SEA) served a positive control and PBS without rSjCP1412 protein as a normal control in the above experiments. The expression of collagen I, α-SMA and Smad4 protein was determined using Western blotting in LX-2 cells following stimulation with rSjCP1412 protein, transforming growth factor-β1 (TGF-β1) alone or in combination, to examine the signaling for the effect of rSjCP1412 protein on LX-2 cells. The rSjCP1412 protein was successfully expressed and the highly purified soluble rSjCP1412 protein was prepared, which had a RNase activity. Compared with the normal group, the survival rates of LX-2 cells significantly decreased post-treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein and SEA for 48 h (F = 22.417 and 20.448, both P values < 0.05). The apoptotic rates of LX-2 cells significantly increased post-treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h (F = 11.350, P < 0.05), and treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h resulted in arrest of LX-2 cells in G0/G1 phase (F = 20.710, P < 0.05). Treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h caused a significant reduction in relative expression levels of collagen I (F = 11.340, P < 0.05), collagen III (F = 456.600, P < 0.05) and α-SMA mRNA (F = 23.100, P < 0.05) in LX-2 cells, and both rSjCP1412 protein and SEA treatment caused a significant reduction in collagen I (F = 1 302.000, P < 0.05), α-SMA (F = 49.750, P < 0.05) and Smad4 protein expression (F = 52.420, P < 0.05) in LX-2 cells. In addition, rSjCP1412 protein treatment inhibited collagen I (F = 66.290, P < 0.05), α-SMA (F = 31.300, P < 0.05) and Smad4 protein expression (F = 27.010, P < 0.05) in LX-2 cells activated by TGF-β1. rSjCP1412 protein may induce apoptosis of LX-2 cells and inhibit proliferation, cell cycle and activation of LX-2 cells through down-regulating Smad4 signaling molecules.

Diethyldithiocarbamate inhibits the activation of hepatic stellate cells via PPARα/FABP1 in mice with non-alcoholic steatohepatitis

Activation of hepatic stellate cells (HSCs) is the main course of liver fibrosis which is positively correlated with adverse clinical outcomes in non-alcoholic steatohepatitis (NASH). Diethyldithiocarbamate (DDC) attenuates NASH related liver fibrosis in mice, but its underlying mechanisms remains unclear. In this study, the data showed that DDC inhibited the activation of HSCs in high fat choline-deficient, L-amino acid-defined (CDAA) diet induced NASH. Double Immunofluorescence analysis showed that the baseline expression of peroxisome proliferator-activated receptor α (PPARα) is high in HSCs in normal mouse liver and notably decreases in the NASH liver, indicating that PPARα might be associated with the activation of HSCs. While, DDC upregulated PPARα in HSCs in the NASH liver. Mixture of free fatty acid was used to induce steatosis of hepatocytes. Human HSCs (LX-2 cells) were activated after co-cultured with steatotic hepatocytes, and DDC inhibited the activation of LX-2 cells. Meanwhile, DDC upregulated PPARα and FABP1, and promoted the accumulation of LDs in LX-2 cells. PPARα small interfering RNA blocked these effect of DDC. These findings suggest that PPARα is associated with the activation of HSCs in the context of NASH. DDC improves NASH related fibrosis through inhibiting the activation of HSCs via PPARα/FABP1.

A miR-340/SPP1 axis inhibits the activation and proliferation of hepatic stellate cells by inhibiting the TGF-β1/Smads pathway.

Hepatic fibrosis (HF) is a common pathological complication of liver cirrhosis which affects human health. It is well established that microRNAs (miRNAs) regulate the proliferation, activation and apoptosis of hepatic stellate cells (HSCs). To determine the function and molecular mechanism of miR-340-5p/secreted phosphoprotein 1 (SPP1) axis in HF and identify potential therapeutic targets. The HF model in cholestatic rats was induced by ligating the common bile duct. The histological sections of the liver tissues were stained with hematoxylin and eosin (H&E), Masson's trichrome or Sirius Red. The differential expression of mRNAs in the liver tissues was examined using the microarray analysis. The expression levels of miR-340-5p, SPP1, alpha-smooth muscle actin (α-SMA), Collagen I, phosphorylated Smad2 (p-Smad2), and p-Smad3 were determined using quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Cell proliferation was quantified using cell counting kit-8 (CCK-8) assays. The regulatory effect of miR-340-5p on SPP1 was determined with fluorescent reporter assay. The bile duct ligation (BDL) rat model was successfully induced, and SPP1 was upregulated in liver tissue from the BDL group compared to that of the sham group. The expression level of miR-340-5p was decreased in activated human primary normal fibroblasts (NFs) and activated LX-2 cells, and the mRNA and protein expression levels of SPP1 were increased in activated LX-2 cells. The SPP1 was the target of miR-340-5p, and the overexpression of SPP1 increased the proliferation of LX-2 cells, the expression of HF markers α-SMA and Collagen I, and key factors p-Smad2 and p-Smad3 (all p < 0.05). However, reverse results were obtained with the overexpression of miR-340-5p in LX-2 cells. Our findings provide evidence that SPP1 targeted by miR-340-5p promotes LX-2 cell proliferation and activation through the TGF-β1/Smads signaling pathway. Therefore, miR-340-5p and SPP1 may be possible therapeutic targets for HF.

MiRNA-21, which disrupts metabolic reprogramming to facilitate CD4+ T cell polarization toward the Th2 phenotype, accelerates arsenite-induced hepatic fibrosis

Elevated levels of arsenic may be present in groundwater, and long-term exposure to arsenic increases hepatic fibrosis. T helper 2 (Th2) cells are involved in the fibrotic cascade, and cell metabolism is a regulatory factor participating in CD4+ T cell differentiation and function. However, the mechanism for Th2 cell regulation of arsenite-induced hepatic fibrosis is not fully understood. In present study, for arsenite-fed mice, activated hepatic stellate cells may be involved in the infiltration of CD4+ T cells, accompanied by up-regulation of GATA3, a transcription factor, and IL-13, the major Th2 cytokine. Exposed to arsenite, Jurkat cells had increased aerobic glycolysis to promote the cell cycle and cell proliferation. Further, this process elevated levels of marker molecules, including those of the Th2 paradigm characterized by GATA3, IL-4, and IL-13. LX-2 cells were activated when treated with culture medium from Jurkat cells exposed to arsenite. miR-21 may be a therapeutic target for arsenite-induced hepatic fibrosis. In vitro, miR-21 knock-down caused inhibition of the PTEN/PI3K/AKT pathway induced by arsenite. It also reversed the elevated glycolysis and the accelerated cell cycle and cell proliferation. Indeed, this alteration led to diminished expression of GATA3, IL-4, and IL-13 in T cells differentiated under Th2 conditions, which inhibits activation of LX-2 cells. Consistent with the results in vitro, miR-21 knock-out in mice reversed hepatic fibrosis and attenuated the levels of GATA3 and IL-13 induced by arsenite. These findings indicate that miR-21 regulates the glycolysis of CD4+ T cells through the PTEN/PI3K/AKT pathway to accelerate the cell cycle, thereby facilitating CD4+ T cell polarization toward Th2 and releasing the fibrogenic factor IL-13, which participates in arsenite-associated hepatic fibrosis. Inhibition of Th2 polarization of CD4+T cells or miR-21 could be a therapeutic strategy to combat hepatic fibrosis caused by exposure to arsenic.

S100A8-Mediated NLRP3 Inflammasome-Dependent Pyroptosis in Macrophages Facilitates Liver Fibrosis Progression.

NLRP3 inflammasome-dependent pyroptosis has been implicated in liver fibrosis progression. However, the definite intrahepatic cell types that undergo pyroptosis and the underlying mechanism as well as the clinical importance remain unclear. Here, augmented levels of pyroptosis-related indicators GSDMD, IL-1β, and IL-18 were verified in both liver fibrosis patients and CCl4-induced fibrotic mouse model. Confocal imaging of NLRP3 with albumin, F4/80 or α-SMA revealed that enhanced NLRP3 was mainly localized to kupffer cells (KCs), indicating that KCs are major cell types that undergo pyroptosis. Targeting pyroptosis by inhibitor MCC950 attenuated the severity and ameliorated liver function in fibrosis models. In addition, elevated S100A8 in liver fibrosis patients was correlated with pyroptosis-related indicators. S100A8 stimulated pyroptotic death of macrophages, which resulted in activation of human hepatic stellate cell line LX-2 cells and increased collagen deposition. Mechanistically, S100A8 activated TLR4/NF-κB signaling and upregulated its target genes NLRP3, pro-IL-1β, and pro-IL-18 expression, and induced reactive oxygen (ROS) abundance to activate NLRP3 inflammasome, finally leading to pyroptotic cell death in macrophages. More importantly, circulating GSDMD had the optimal predicting value for liver fibrosis progression. In conclusion, S100A8-mediated NLRP3 inflammasome-dependent pyroptosis by TLR4/NF-κB activation and ROS production in macrophages facilitates liver fibrosis progression. The identified GSDMD has the potential to be a biomarker for liver fibrosis evaluation.

20-Hydroxytetraenoic acid induces hepatic fibrosis via the TGF-β1/Smad3 signaling pathway

Hepatic fibrosis is caused by excessive accumulation of extracellular matrix (ECM) due to repeated liver injury. Hepatic stellate cells (HSCs) play a key role in the pathogenesis and progression of hepatic fibrosis. A study showed that CYP4A14 gene defect can inhibit hepatic fibrosis, but the specific mechanism was not clear. In this experiment, patients with hepatic fibrosis, LX-2 cells (a human HSCs line), and mice with liver fibrosis induced by carbon tetrachloride (CCl4) were used to study the effect of 20-Hydroxytetraenoic acid (20-HETE), one of the main metabolites of arachidonic acid (AA) catalyzed by CYP4A enzyme, on hepatic fibrosis and its mechanism. Our experimental results showed that the 20-HETE of patients with hepatic fibrosis is significantly higher than that of normal people and is closely related to the degree of fibrosis. 20-HETE could induce activation of LX-2 cells and 20-HETE antagonist could inhibit the induction of 20-HETE. 20-HETE was significantly increased in CCl4-induced liver fibrosis mice and inhibition of 20-HETE production could attenuate hepatic fibrosis. 20-HETE induced hepatic fibrosis mainly via the TGF- β1/Smad3 signal pathway. In conclusion, the results suggest that 20-HETE plays an important role in hepatic fibrosis and may be a possible target for the clinical treatment of hepatic fibrosis.

Danshensu attenuated lipopolysaccharide-induced LX-2 and T6 cells activation through regulation of ferroptosis.

Liver fibrosis and cirrhosis are primarily caused by the activation of hepatic stellate cells (HSCs), regardless of their etiology. Collagen type I (collagen I) and connective tissue growth factor (CTGF) is produced more readily by activated HSCs. Consequently, identifying the molecular and cellular mechanisms responsible for HSCs activation is essential to better understand its mechanism of action and therapeutic potential. Cell death is caused by iron-dependent lipid peroxidation during ferroptosis. Ferroptosis plays an important role in the survival of activated HSCs and could contribute to the development of innovative prevention and treatment strategies for liver fibrosis. Danshensu (Dan) is a pure molecule extracted from the Salvia miltiorrhiza herb that protects against liver damage. However, Dan's effect on attenuating HSCs activation by regulating ferroptosis remains unclear. The results of this study indicated that lipopolysaccharide (LPS)-induced LX-2 and T6 cells activation occurs through the upregulation of collagen I, CTGF, Gpx4, and SLC7A11. Interestingly, Dan attenuated LPS-induced liver fibrosis in those cells by upregulating collagen I, CTGF, Gpx4, and SLC7A11 and by increasing lipid reactive oxygen species accumulation. Furthermore, the results also showed that the ferroptosis inhibitor liproxstatin attenuated the overproduction of lipid reactive oxygen species in LPS-activated LX-2 cells. We conclude that Dan attenuates LPS-induced HSC activation during liver fibrosis by regulating ferroptosis in LX-2 and T6 cells.

A novel matrine derivative, WM130, inhibits activation and movement of human hepatic stellate LX-2 cells by targeting cofilin 1.

The online version contains supplementary material available at 10.1007/s10616-022-00548-w.

Collagen-binding fibroblast growth factor ameliorates liver fibrosis in murine bile duct ligation injury

Cholestatic liver injury, characterized by liver fibrosis, has increasingly become a global health problem, with no effective treatment available. Hepatic stellate cells (HSCs) differentiate into myofibroblasts, leading to excessive deposition of the extracellular matrix (ECM), which is a feature of liver fibrosis. Basic fibroblast growth factor (bFGF) has proven antifibrotic effects in chronic liver disease; however, the lack of an effective delivery system to the injury site reduces its therapeutic efficacy. The aim of this study was to assess the therapeutic effect of collagen-binding bFGF (CBD-bFGF) for the treatment of liver fibrosis in a murine bile duct ligation (BDL) model. We found that CBD-bFGF treatment significantly alleviated liver injury in the early phase of BDL injury, and was associated with decreased necroptotic cell death and inflammatory response. Moreover, CBD-bFGF had enhanced therapeutic effects for liver fibrosis on day 7 after surgery compared to those obtained with native bFGF treatment. In vitro, CBD-bFGF treatment notably inhibited TGF-β1-induced LX-2 cell activation, migration, and contraction compared with native bFGF. In conclusion, CBD-bFGF may be a promising treatment for hepatic fibrosis.

Long noncoding RNA GAS5 inhibits LX-2 cells activation by suppressing NF-κB signalling through regulation of the miR-433-3p/TLR10 axis.

Liver fibrosis is a common disease that can lead to hepatic failure. Our aims were to reveal the role of GAS5 in the regulation of liver fibrosis. LX-2 human hepatic satellite cells (HSCs) were cultured and activated using TGF-β1 treatment. A CCK-8 assay was performed to assess cell viability. A luciferase assay was employed to monitor the interactions between miR-433-3p and GAS5 or toll-like receptor 10 (TLR10). Western blotting and real-time quantitative PCR (RT-qPCR) were applied to detect the expression levels of α-SMA, Col. I, PCNA-, MMP2-, MMP9-, TLR10-, and NF-κB-related molecules at the protein and RNA levels. GAS5 and TLR10 were decreased while miR-433-3p was upregulated in TGF-β1-activated LX-2 cells. Upregulation of GAS5 or downregulation of miR-433-3p suppressed HSC activation, and luciferase assays indicated that miR-433-3p binds with GAS5 and the 3'-UTR of TLR10. MiR-433-3p upregulation and TLR10 downregulation rescued the impacts of GAS5 overexpression or miR-433-3p knockdown on LX-2 cells. Upregulation of GAS5 also suppressed the phosphorylation of NF-κB through the miR-433-3p/TLR10 axis. LncRNA GAS5 exerts an inhibitory effect on HSC activation by suppressing NF-κB signalling through regulation of the miR-433-3p/TLR10 axis.

Mesenchymal stem cells improve liver fibrosis and protect hepatocytes by promoting microRNA-148a-5p-mediated inhibition of Notch signaling pathway

BackgroundMesenchymal stem cells (MSCs) are considered to be a potential therapeutic tool for liver fibrosis. Inhibiting the activation of hepatic stellate cells (HSCs) and protecting hepatocytes are important mechanisms for the anti-fibrotic effect of MSCs. However, how MSCs inhibit liver fibrosis by regulating the expression of microRNAs (miRNAs) has not been fully clarified.MethodsTransforming growth factor-β1 (TGF-β1)-activated HSCs LX-2 were single cultured or co-cultured with human umbilical cord mesenchymal stem cells (HUC-MSCs). High-throughput sequencing was used to evaluate the differentially expressed microRNAs (DEMs) between the two groups. Quantitative real-time PCR (qRT-PCR), Western blot, and transfection experiments were used to investigate and screen the most significantly up-regulated DEM. Bioinformatics analysis was used to predict the target mRNAs and the potential functions of the DEM. The possible mechanism of HUC-MSCs against liver fibrosis was analyzed by co-culture experiment of HUC-MSCs with LX-2 cells, and HUC-MSCs treatment of Bile duct ligation (BDL)-induced liver fibrosis in mice. Finally, the mechanism of the DEM regulating liver fibrosis was confirmed in human liver fibrosis specimens.ResultsMicroRNA-148a-5p (miR-148a-5p) was the most significantly up-regulated DEM in activated LX-2 cells co-cultured with HUC-MSCs compared with LX-2 cells single cultured. Up-regulation of the expression of miR-148a-5p in activated LX-2 cells could significantly inhibit the expression of hepatic fibrosis markers α-SMA and Col1α1. Notch2 was one target gene of miR-148a-5p. Co-cultured with HUC-MSCs could inhibit the activation of LX-2 cells by inhibiting the expression of the Notch2 and the Notch signaling pathway. In addition, HUC-MSCs treatment could up-regulate the expression of miR-148a-5p in liver tissue and hepatocytes, promote the proliferation and avoid the apoptosis of hepatocytes, and reduce the degree of fibrosis by inhibiting expression of the Notch2 and the Notch signaling pathway in BDL-induced liver fibrosis mice. Moreover, miR-148a-5p was down-regulated and Notch2 was up-regulated in fibrotic human liver tissues compared with the normal livers.ConclusionsHUC-MSCs treatment could inhibit HSCs activation, protect hepatocytes, and alleviate BDL-induced liver fibrosis in mice by up-regulating the expression of miR-148-5p and inhibiting the Notch signaling pathway. The down-regulation of miR-148-5p and up-regulation of Notch2 could be used as biomarkers to monitor the progression of liver fibrosis.

MiR-301a-3p promotes hepatic stellate cells activation and liver fibrogenesis via regulating PTEN/PDGFR-β

Hepatic fibrosis is an essential pathology of multiple chronicliverdiseases. The aim of this study was to investigate the role of miR-301a-3p in hepatic fibrosis. We found that miR-301a-3p was upregulated in hepatic fibrosis patients and in culture-activated human hepatic stellate cells (HSCs). Interestingly, miR-301a-3p expression was increased in hepatic fibrosis progression mice while decreased in hepatic fibrosis recovery mice, indicating that miR-301a-3p may participate in the hepatic fibrosis pathology. Functionally, the effects of miR-301a-3p both on hepatic fibrosis progression and regression were assessed in vivo. Inhibiting miR-301a-3p amelioratedmouse liver fibrogenesis and collagen deposition and suppressed HSC activation and fibrogenic factor expression. Whereas, in hepatic fibrosis regression, upregulating miR-301a-3p impaired mouse hepatic fibrosis recovery by inducing HSC activation and triggering inflammation. Consistently, gain-of-function and loss-of-function analysis of miR-301a-3p were performed to evaluate its effects on human HSCs LX-2 cell. We found that suppressing miR-301a-3p inhibited LX-2 cell activation and proliferation, and induced LX-2 cell apoptosis, accompaniedby decreased fibrotic mediators expression. Collectively, these findings suggest miR-301a-3p drives liver fibrogenesis and HSC activation in hepatic fibrosis. Mechanistically, we demonstrated miR-301a-3p binds directly to phosphatase and tensin homolog (PTEN) by luciferase reporter analysis, pull-down, and RIP assay. Indicating that miR-301a-3p plays a critical rolein promotingliverfibrogenesis viamodulating the PTEN/platelet derived growth factor β (PDGFR-β) pathway. In conclusion, our findings demonstrate that miR-301a-3p expression is closely correlated with hepatic fibrosis pathology, and that enhancing miR-301a-3p maintains the HSC profibrogenic phenotype, triggers inflammatoryresponses, promotes fibrogenic factor production, and further exacerbates liver fibrogenesis. These findings suggest that miR-301a-3p may serve as a promising diagnostic and prognosis biomarker for hepatic fibrosis treatment.

Dehydromevalonolactone ameliorates liver fibrosis and inflammation by repressing activation of NLRP3 inflammasome

Liver fibrosis is an important process in chronic liver disease and is strongly related to poor prognosis. Dehydromevalonolactone (C8) is a natural product isolated from a fungus of Fusarium sp. CPCC 401218, and its pharmacological activity has never been reported before. In this study, the potential of C8 as an anti-hepatic fibrosis agent was investigated. In human hepatic stellate cell (HSC) line LX-2, C8 suppressed the increased expression of COL1A1 and α-SMA induced by TGFβ1, which indicated that C8 could repress the activation of HSCs. In bile duct ligated rats, C8 administration (100 mg/kg, i.p.) markedly attenuated liver injury, fibrosis, and inflammation, and suppressed the expression of the macrophage surface marker F4/80. In terms of mechanism, C8 treatment blocked the activation of the NLRP3 inflammasome, which was stimulated by LPS and nigericin in bone marrow-derived macrophages (BMDMs) and companied by the release of active IL-1β. In addition, the activation of LX-2 cells induced by IL-1β released from BMDMs was also inhibited after C8 administration, which indicated that C8 repressed HSCs activation by inhibiting the activation of NLRP3 inflammasome in macrophages. Furthermore, C8 exhibited the effects of anti-fibrosis and inhibiting the expression of NLRP3 inflammasome in non-alcoholic steatohepatitis (NASH) mice. Finally, C8 can be commendably absorbed in vivo and was safe for mice at the concentration of 1000 mg/kg (p.o.). In summary, our study reveals that C8 ameliorates HSCs activation and liver fibrosis in cholestasis rats and NASH mice by inhibiting NLRP3 inflammasome in macrophages, and C8 might be a safe and effective candidate for the treatment of liver fibrosis.

Kinsenoside alleviates inflammation and fibrosis in experimental NASH mice by suppressing the NF-κB/NLRP3 signaling pathway

BackgroundNon-alcoholic steatohepatitis (NASH) has replaced viral hepatitis as the main driver of the rising morbidity and mortality associated with cirrhosis and liver cancer worldwide, while no FDA-approved therapies are currently known. Kinsenoside (KD), naturally isolated from Anoectochilus roxburghii, possesses multiple biological activities, including lipolysis, anti-inflammation, and hepatoprotection. However, the effects of KD on NASH remain unclear. PurposeThis study aimed to explore the roles of KD in NASH and its engaged mechanisms. MethodsTwo typical animal models of NASH, mice fed a methionine-choline-deficient (MCD) diet (representing non-obese NASH) and mice fed a high-fat and -fructose diet (HFFD) (representing obese NASH), were used to investigate the effect of KD on NASH in vivo. Transcriptome sequencing was performed to elucidate the underlying mechanisms of KD. Lipopolysaccharide (LPS)-stimulated THP-1 cells and transforming growth factor β1 (TGF-β1)-activated LX-2 cells were applied to further explore the effects and mechanisms of KD in vitro. ResultsThe intragastric administration of KD remarkably alleviated MCD/HFFD-induced murine NASH almost in a dose-dependent manner. Specifically, KD reduced lipid accumulation, inflammation, and fibrosis in the liver of NASH mice. KD ameliorated alanine aminotransferase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD), and malondialdehyde (MDA) abnormalities. In addition, it decreased the level of serum proinflammatory factors (IL-12p70, IL-6, TNF-α, MCP-1, IFN-γ) and the hepatic expression of typical fibrosis-related molecules (α-SMA, Col-I, TIMP-1). Mechanically, KD attenuated the MCD/HFFD-induced NASH through the inhibition of the NF-κB/NLRP3 signaling pathway. Consistently, KD reduced inflammation stimulated by LPS in THP-1 cells via suppressing the NF-κB/NLRP3 pathway. Furthermore, it prevented the activation of LX-2 cells directly, by inhibiting the proliferation stimulated by TGF-β1, and indirectly, by inactivating the NLRP3 inflammasome in macrophages. ConclusionFor the first time, the practical improvement of NASH by KD was revealed. Our study found that KD exerted its alleviative effects on NASH through the inhibition of the NF-κB/NLRP3 signaling pathway. Given its hepatoprotective and nontoxic properties, KD has the potential to be a novel and effective drug to treat NASH.

Human amniotic mesenchymal stem cells-derived IGFBP-3, DKK-3, and DKK-1 attenuate liver fibrosis through inhibiting hepatic stellate cell activation by blocking Wnt/β-catenin signaling pathway in mice

BackgroundLiver fibrosis is an outcome of restoring process in chronic liver injury. Human amniotic mesenchymal stem cells (hAMSCs) derived from amniotic membrane have multilineage differentiation, immunosuppressive, and anti-inflammatory potential which makes them suitable for treating liver fibrosis. This study aimed to explore the effect and mechanism of hAMSCs on liver fibrosis.MethodshAMSCs were transplanted into carbon tetrachloride (CCl4)-induced liver fibrosis mice via tail vein, and the effects of hAMSCs on hepatic fibrosis were assessed. The effects of hAMSCs and hAMSCs conditional medium (CM) on the activation of hepatic stellate cells (HSCs) were investigated in vivo and in vitro. Antibody array assay was used to identify the cytokines secreted by hAMSCs that may inhibit the activation of HSCs. Finally, the underlying mechanisms were explored by assessing IGF-1R/PI3K/AKT and GSK3β/β-catenin signaling pathways in the activated HSCs (LX-2) with hAMSCs and hAMSCs transfected with corresponding siRNAs.ResultsOur results showed that hAMSCs possessed the characterizations of mesenchymal stem cells. hAMSCs significantly reduced liver fibrosis and improved liver function in mice by inhibiting HSCs activation in vivo. Both hAMSCs and hAMSC-CM remarkably inhibited the collagen deposition and activation of LX-2 cells in vitro. Antibody array assay showed that insulin-like growth factor binding protein-3 (IGFBP-3), Dickkopf-3 (DKK-3), and Dickkopf-1 (DKK-1) were highly expressed in the co-culture group and hAMSC-CM group compared with LX-2 group. Western blot assay demonstrated that IGFBP-3, DKK-3, and DKK-1 derived from hAMSCs inhibit LX-2 cell activation through blocking canonical Wnt signaling pathway.ConclusionsOur results demonstrated that IGFBP-3, Dkk3, and DKK-1 secreted by hAMSCs attenuated liver fibrosis in mice through inhibiting HSCs activation via depression of Wnt/β-catenin signaling pathway, suggesting that hAMSCs or hAMSC-CM provides an alternative therapeutic approach for the treatment of liver fibrosis.

Upregulation of GLT25D1 in Hepatic Stellate Cells Promotes Liver Fibrosis via the TGF-β1/SMAD3 Pathway In Vivo and In vitro.

Collagen β(1-O) galactosyltransferase 25 domain 1 (GLT25D1) is associated with collagen production and glycosylation, and its knockout in mice results in embryonic death. However, its role in liver fibrosis remains elusive, particularly in hepatic stellate cells (HSCs), the primary collagen-producing cells associated with liver fibrogenesis. Herein, we aimed to elucidate the role of GLT25D1 in HSCs. Bile duct ligation (BDL)-induced mouse liver fibrosis models, primary mouse HSCs (mHSCs), and transforming growth factor beta 1 (TGF-β1)-stimulated LX-2 human hepatic stellate cells were used in in vivo and in vitro studies. Stable LX-2 cell lines with either GLT25D1 overexpression or knockdown were established using lentiviral transfection. RNA-seq was performed to investigate the genomic differences. HPLC-MS/MS were used to identify glycosylation sites. Scanning electronic microscopy (SEM) and second-harmonic generation/two-photon excited fluorescence (SHG/TPEF) were used to image collagen fibril morphology. GLT25D1 expression was upregulated in nonparenchymal cells in human cirrhotic liver tissues. Meanwhile, its knockdown attenuated collagen deposition in BDL-induced mouse liver fibrosis and inhibited mHSC activation. GLT25D1 was overexpressed in activated versus quiescence LX-2 cells and regulated in vitro LX-2 cell activation, including proliferation, contraction, and migration. GLT25D1 also significantly increased liver fibrogenic gene and protein expression. GLT25D1 upregulation promoted HSC activation and enhanced collagen expression through the TGF-β1/SMAD signaling pathway. Mass spectrometry showed that GLT25D1 regulated the glycosylation of collagen in HSCs, affecting the diameter of collagen fibers. Collectively, the upregulation of GLT25D1 in HSCs promoted the progression of liver fibrosis by affecting HSCs activation and collagen stability.

Synthetic peptides derived from the Schistosoma mansoni secretory protein Sm16 induce contrasting responses in hepatic stellate cells

Sm16 is a 16 KDa protein released by Schistosoma mansoni that modulates inflammatory responses in host cells. Sm16 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 Sm16 exposure on HSCs, two synthetic peptide derivatives of Sm16, 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 Sm16 in combatting fibrotic liver disease.