Liver inflammation leads to the activation of hepatic stellate cells (HSCs), resulting in the development of liver fibrosis. The present study aimed to investigate the effects of prostaglandin E2 ...(PGE2), which is biosynthesized by Kupffer cells, hepatocytes, and HSCs during inflammation, on HSC activation, including its combinatory effect with caffeine.
HSCs isolated from mice were activated by culturing in a medium supplemented with 10% fetal bovine serum for 7 days on plastic plates. The activation of HSCs was evaluated by immunofluorescence of α-smooth muscle actin in HSCs. Comprehensive gene expression analysis was performed using mRNA-sequencing to compare HSCs cultured for 1 or 7 days, with or without PGE2, caffeine, or both.
PGE2 (1 μM) facilitated the activation of HSCs but inhibited the HSC activation in the presence of caffeine (3 mM). Comprehensive gene expression analysis revealed that HSCs treated with PGE2 in the presence of caffeine were classified in the same class as HSCs cultured for 1 day, i.e., quiescent HSCs. In contrast, PGE2 did not exhibit an inhibitory effect on HSC activation when co-treated with any isoform-specific phosphodiesterase inhibitors. Although the adenylate cyclase inhibitor 2′,5′-dideoxyadenosine suppressed the elevation of intracellular cAMP level induced by PGE2 in the presence of caffeine, it had no effect on the inhibition of HSC activation by PGE2 plus caffeine.
The effect of PGE2 on HSC activation is changed from facilitatory to inhibitory when combined with caffeine, suggesting that caffeine may effectively suppress liver fibrosis during inflammation.
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•Prostaglandin E2 (PGE2) facilitated the activation of mouse hepatic stellate cells (HSCs) induced by fetal bovine serum.•In the presence of caffeine, PGE2 rather suppressed HSC activation.•RNA-sequencing analysis showed that HSCs treated with PGE2 plus caffeine were classified in the same class as quiescent HSCs.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The increased incidence of obesity in the global population has increased the risk of several chronic inflammation-related diseases, including non-alcoholic steatohepatitis (NASH)-hepatocellular ...carcinoma (HCC). The progression from NASH to HCC involves a virus-independent liver carcinogenic mechanism; however, we currently lack effective treatment and prevention strategies. Several reports have suggested that fecal volatile organic compounds (VOCs) are strongly associated with NASH-HCC; therefore, we explored the biomarkers involved in its pathogenesis and progression. Fecal samples collected from control and NASH-HCC model STAM mice were subjected to headspace autosampler gas chromatography-electron ionization-mass spectrometry. Non-target profiling analysis identified diacetyl (2,3-butandione) as a fecal VOC that characterizes STAM mice. Although fecal diacetyl levels were correlated with the HCC in STAM mice, diacetyl is known as a cytotoxic/tissue-damaging compound rather than genotoxic or mutagenic; therefore, we examined the effect of bioactivity associated with NASH progression. We observed that diacetyl induced several pro-inflammatory molecules, including tumor necrosis factor-α, cyclooxygenase-2, monocyte chemoattractant protein-1, and transforming growth factor-β, in mouse macrophage RAW264.7 and Kupffer KPU5 cells. Additionally, we observed that diacetyl induced α-smooth muscle actin, one of the hallmarks of fibrosis, in an ex vivo cultured hepatic section, but not in in vitro hepatic stellate TWNT-1 cells. These results suggest that diacetyl would be a potential biomarker of fecal VOC in STAM mice, and its ability to trigger the macrophage-derived inflammation and fibrosis may partly contribute to NASH-HCC carcinogenesis.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Differentiation-inducing factor-1 (DIF-1), a morphogen produced by the cellular slime mold Dictyostelium discoideum, is a natural product that has attracted considerable attention for its antitumor ...properties. Here, we report a novel inhibitory effect of DIF-1 on the activation of hepatic stellate cells (HSCs) responsible for liver fibrosis. DIF-1 drastically inhibited transdifferentiation of quiescent HSCs into myofibroblastic activated HSCs in a concentration-dependent manner, thus conferring an antifibrotic effect against in the liver. Neither SQ22536, an adenylate cyclase inhibitor, nor ODQ, a guanylate cyclase inhibitor, showed any effect on the inhibition of HSC activation by DIF-1. In contrast, TWS119, a glycogen synthase kinase 3β (GSK3β) inhibitor, attenuated the inhibitory effect of DIF-1. Moreover, the level of inactive GSK3β (phosphorylated at Ser9) was significantly reduced by DIF-1. DIF-1 also inhibited nuclear translocation of β-catenin and reduced the level of non-phospho (active) β-catenin. These results suggest that DIF-1 inhibits HSC activation by disrupting the Wnt/β-catenin signaling pathway through dephosphorylation of GSK3β. We propose that DIF-1 is a possible candidate as a therapeutic agent for preventing liver fibrosis.
•A cellular slime mold product DIF-1 blocked hepatic stellate cell (HSC) activation.•An inhibitor of glycogen synthase kinase (GSK) 3β attenuated the effect of DIF-1.•The level of inactive GSK3β was significantly reduced by DIF-1.•DIF-1 reduced nuclear translocation of β-catenin and the level of active β-catenin.•DIF-1 is a possible candidate for liver fibrosis therapy to target HSC activation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Liver fibrosis is a major consequence of chronic liver disease, where excess extracellular matrix is deposited, due caused by the activation of hepatic stellate cells (HSCs). The suppression of ...collagen production in HSCs is therefore regarded as a therapeutic target of liver fibrosis. The present study investigated effects of harmine, which is a β-carboline alkaloid and known as an inhibitor of dual-specificity tyrosine-regulated kinases (DYRKs), on the production of collagen in HSCs. LX-2 cells, a human HSC cell line, were treated with harmine (0–10 μM) for 48 h in the presence or absence of TGF-β1 (5 ng/ml). The expression of collagen type I α1 (COL1A1) and DYRK isoforms was investigated by Western blotting, quantitative RT-PCR, or immunofluorescence. The influence of knockdown of each DYRK isoform on the COL1A1 expression was further investigated. The expression of COL1A1 was markedly increased by treating with TGF-β1 for 48 h in LX-2 cells. Harmine (10 μM) significantly inhibited the increased expression of COL1A1. LX-2 cells expressed mRNAs of DYRK1A, DYRK1B, DYRK2, and DYRK4, although the expression of DYRK4 was much lower than the others. Knockdown of DYRK1B, but not DYRK1A or DYRK2, with siRNA significantly suppressed TGF-β1-induced increase in COL1A1 expression. These results suggest that harmine suppresses COL1A1 expression via inhibiting DYRK1B in HSCs and therefore might be effective for the treatment of liver fibrosis.
•Harmine suppressed the production of collagen induced by TGF-β1 in LX-2 cells.•LX-2 cells expressed mRNAs of DYRK1A, DYRK1B, and DYRK2.•DYRK1B knockdown suppressed the production of collagen 1A1 induced by TGF-β1 in LX-2 cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Currently, there is no effective treatment for liver fibrosis. In vitro studies on the activation of hepatic stellate cells (HSCs), which is responsible for liver fibrosis, have been used as a drug ...screening system for it. However, even if some drug shows an inhibitory effect on HSC activation, its anti-fibrotic effect is required to be confirmed in liver fibrosis animal models, which further requires a lot of time and cost. In the present study, we tried to establish an ex vivo model of liver fibrosis using precision-cut liver slices (PCLSs) to solve such problems. PCLSs of 250 µm thickness were prepared from male C57BL/6J mice using a vibratome and cultured in RPMI medium in a 5% CO2 incubator. Although cellular ATP content was decreased on day 1 compared to day 0, it was then maintained until day 5, suggesting that the ex vivo model is viable for at least 5 days. Treatment with Et-OH (50, 100 mM), one of the liver injury stimuli, for 5 days increased mRNA expression of Acta2 and Col1a1, liver fibrosis markers, in PCLSs. DIF-1 (50, 100 µM), which has an anti-fibrotic effect, significantly suppressed the Et-OH-induced increases in the markers. These results suggest that the ex vivo model using PCLSs is useful as a drug screening system for the development of drugs for treatment of liver fibrosis.
Liver fibrosis is a significant consequence of chronic liver diseases, where excess deposition of extracellular matrix is caused by the activation of hepatic stellate cells (HSCs). The suppression of ...HSC activation is therefore regarded as a therapeutic target of liver fibrosis. The present study investigated the involvement of protein arginine methyltransferase 5 (PRMT5), which mediates genome organization and cell cycle regulation, in HSC activation. LX-2 cells, a human HSC cell line, were treated with TGF- β1 for 48 h in the presence of PRMT5 inhibitors (EPZ015666 and JNJ64619178). The expression of α-smooth muscle actin (α-SMA) and type I collagen α1 (COL1A1), activated HSC markers, were markedly increased by the TGF-β1 treatment. PRMT5 inhibitors suppressed the increased expression of α-SMA and COL1A1 in a concentration-dependent manner. Knockdown of PRMT5 also suppressed the TGF-β1-induced COL1A1 expression in LX-2 cells. RNA-sequencing analysis showed that GO terms related to ECM production and SMAD signaling were enriched with RNA of LX-2 cells treated with the PRMT5 inhibitor JNJ64619178. These results suggest that PRMT5 promotes HSC activation, possibly depending on the SMAD signaling pathway, and therefore might be a target for the prevention and treatment of liver fibrosis.
Liver inflammation leads to the activation of hepatic stellate cells (HSCs), resulting in the development of liver fibrosis. The present study aimed to investigate the effects of prostaglandin E
(PGE
...), which is biosynthesized by Kupffer cells, hepatocytes, and HSCs during inflammation, on HSC activation, including its combinatory effect with caffeine.
HSCs isolated from mice were activated by culturing in a medium supplemented with 10% fetal bovine serum for 7 days on plastic plates. The activation of HSCs was evaluated by immunofluorescence of α-smooth muscle actin in HSCs. Comprehensive gene expression analysis was performed using mRNA-sequencing to compare HSCs cultured for 1 or 7 days, with or without PGE
, caffeine, or both.
PGE
(1 μM) facilitated the activation of HSCs but inhibited the HSC activation in the presence of caffeine (3 mM). Comprehensive gene expression analysis revealed that HSCs treated with PGE
in the presence of caffeine were classified in the same class as HSCs cultured for 1 day, i.e., quiescent HSCs. In contrast, PGE
did not exhibit an inhibitory effect on HSC activation when co-treated with any isoform-specific phosphodiesterase inhibitors. Although the adenylate cyclase inhibitor 2',5'-dideoxyadenosine suppressed the elevation of intracellular cAMP level induced by PGE
in the presence of caffeine, it had no effect on the inhibition of HSC activation by PGE
plus caffeine.
The effect of PGE
on HSC activation is changed from facilitatory to inhibitory when combined with caffeine, suggesting that caffeine may effectively suppress liver fibrosis during inflammation.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Hepatic stellate cells (HSCs), located in the gap of hepatocytes and sinusoidal endothelial cells, transdifferentiate from quiescent form (qHSCs) into myofibroblast-like activated one (aHSCs) during ...liver injury. The expression of α-smooth muscle actin (α-SMA) and the production of type Ⅰ collagen are up-regulated in aHSCs. Therefore, the activation of HSCs is responsible for liver fibrosis and inhibiting the activation can be a novel therapeutic target for the fibrosis. In the present study, we show that differentiation-inducing factor-1 (DIF-1) that is a low molecular weight compound derived from the cellular slime mold, Dictyostelium discoideum, has a suppressive effect on HSC activation. qHSCs were isolated from ddY mice and cultured in DMEM supplemented with 10% FBS. We treated qHSCs with DIF-1 on the next day after isolation and analyzed the effect of DIF-1 on HSC activation. DIF-1 significantly suppressed the up-regulation of α-SMA. However, the effect of DIF-1 was abolished in the presence of TWS119, an activator of Wnt/β-catenin signal pathway. DIF-1 reduced the levels of non-phosphorylated β-catenin (activated β-catenin) and phosphorylated GSK3β. These results suggest that DIF-1 inhibits the Wnt/β-catenin signal pathway through dephosphorylating GSK3β, thereby suppressing HSC activation.
Hepatic stellate cells (HSCs) are known to play a central role in liver fibrosis (LF), and considered to be a target for LF therapy. We have previously reported that caffeine (CAF) suppresses HSC ...activation via inhibition of adenosine receptors, while also observing that prostaglandin E2 (PGE2) facilitates the inhibitory effect of CAF on HSC activation. However, the mechanism remains to be elucidated. In the present study, we aimed to elucidate the molecular mechanism of the inhibitory effect of PGE2 and CAF co-treatment. Despite sharing a common pathway for cAMP, intracellular cAMP levels showed no direct correlation with the inhibitory effect of PGE2 and CAF co-treatment on HSC activation. RNA-seq analysis identified the transcription factor NRF2 as a candidate gene involved in the inhibitory effect of PGE2 and CAF co-treatment on HSC activation. Reporter assays and immunostaining exhibited that the combination of PGE2 and CAF resulted in a significant augmentation of NRF2 transcription activity and enhanced nuclear translocation of NRF2. These results suggest that PGE2 facilitates the inhibitory effect of CAF on HSC activation by amplifying the transcriptional activity of NRF2.
Hepatic stellate cells (HSCs) are activated in response to liver injury and secrete huge amounts of collagen, the primary cause of liver fibrosis (LF). Thus, the regulation of trans-differentiation ...of HSCs, both activation of quiescent HSCs and reversion of activated HSCs, is crucial for therapeutic strategy for LF. However, few compounds have been reported to have such effects and no definitive therapy is available. Here, we elucidate the effect of DIF-1, a compound inhibiting HSC activation we previously reported, on activated HSC and LF mouse model. DIF-1 reduced the expression of type I collagen α 1 (Col1a1) and α-smooth muscle actin, markers of activated HSCs, even when treated after HSC activation. We further performed in silico analysis utilizing the relation between structural transition and HSC reversion effect of several DIF-1 analogs to identify molecular target of DIF-1. DIF-1 reduced the expression of activated HSC marker genes (Acta2, Col1a1, Pdgfrb), while it increased that of a quiescent HSC marker gene (Lrat) in thioacetamide-induced LF mouse model. Moreover, DIF-1 reduced the amount of collagen fiber in liver tissue. Taken together with our previous report, we propose that DIF-1 is a useful compound for LF treatment, acting on both quiescent and activated HSCs.
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