Inhibitory effect of oestradiol on activation of rat hepatic stellate cells in vivo and in vitro.
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ABSTRACT:
SUBMITTER: Shimizu I
PROVIDER: S-EPMC1760074 | biostudies-other | 1999 Jan
REPOSITORIES: biostudies-other
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Project description:BackgroundAnti-inflammation via inhibition of NF-κB pathways in hepatic stellate cells (HSCs) is one therapeutic approach to hepatic fibrosis. Tanshinone IIA (C19H18O3, Tan IIA) is a lipophilic diterpene isolated from Salvia miltiorrhiza Bunge, with reported anti-inflammatory activity. We tested whether Tan IIA could inhibit HSC activation.Materials and methodsThe cell line of rat hepatic stellate cells (HSC-T6) was stimulated with lipopolysaccharide (LPS) (100 ng/ml). Cytotoxicity was assessed by MTT assay. HSC-T6 cells were pretreated with Tan IIA (1, 3 and 10 µM), then induced by LPS (100 ng/ml). NF-κB activity was evaluated by the luciferase reporter gene assay. Western blotting analysis was performed to measure NF-κB-p65, and phosphorylations of MAPKs (ERK, JNK, p38). Cell chemotaxis was assessed by both wound-healing assay and trans-well invasion assay. Quantitative real-time PCR was used to detect gene expression in HSC-T6 cells.ResultsAll concentrations of drugs showed no cytotoxicity against HSC-T6 cells. LPS stimulated NF-κB luciferase activities, nuclear translocation of NF-κB-p65, and phosphorylations of ERK, JNK and p38, all of which were suppressed by Tan IIA. In addition, Tan IIA significantly inhibited LPS-induced HSCs chemotaxis, in both wound-healing and trans-well invasion assays. Moreover, Tan IIA attenuated LPS-induced mRNA expressions of CCL2, CCL3, CCL5, IL-1β, TNF-α, IL-6, ICAM-1, iNOS, and α-SMA in HSC-T6 cells.ConclusionOur results demonstrated that Tan IIA decreased LPS-induced HSC activation.
| S-EPMC4116159 | biostudies-literature
Project description:Activation of hepatic stellate cells during liver fibrosis is a major event facilitating an increase in extracellular matrix deposition. The up-regulation of smooth muscle alpha-actin and collagen type I is indicative of the activation process. The involvement of cysteine cathepsins, a class of lysosomal cysteine proteases, has not been studied in conjunction with the activation process of hepatic stellate cells. Here we report a nuclear cysteine protease activity partially attributed to cathepsin F, which co-localizes with nuclear speckles. This activity can be regulated by treatment with retinol/palmitic acid, known to reduce the hepatic stellate cell activation. The treatment for 48 h leads to a decrease in activity, which is coupled to an increase in cystatin B and C transcripts. Cystatin B knockdown experiments during the same treatment confirm the regulation of the nuclear activity by cystatin B. We demonstrate further that the inhibition of the nuclear activity by E-64d, a cysteine protease inhibitor, results in a differential regulation of smooth muscle alpha-actin and collagen type I transcripts. On the other hand, cathepsin F small interfering RNA transfection leads to a decrease in nuclear activity and a transcriptional down-regulation of both activation markers. These findings indicate a possible link between nuclear cathepsin F activity and the transcriptional regulation of hepatic stellate cell activation markers.
| S-EPMC2555962 | biostudies-literature
Project description:BackgroundProgressive liver fibrosis leads to cirrhosis and end-stage liver disease. This disease is a consequence of strong interactions between matrix-producing hepatic stellate cells (HSCs) and resident and infiltrating immune cell populations. Accumulated experimental evidence supports the involvement of adult-derived human liver mesenchymal stem/progenitor cells (ADHLSCs) in liver regeneration. The aim of the present study was to evaluate the influence of ADHLSCs on HSCs, both in vitro and in vivo.MethodsActivated human HSCs were co-cultured with ADHLSCs or ADHLSC-conditioned culture medium. The characteristics of the activated human HSCs were assessed by microscopy and biochemical assays, whereas proliferation was analyzed using flow cytometry and immunocytochemistry. The secretion profile of activated HSCs was evaluated by ELISA and Luminex. ADHLSCs were transplanted into a juvenile rat model of fibrosis established after co-administration of phenobarbital and CCl4.ResultsWhen co-cultured with ADHLSCs or conditioned medium, the proliferation of HSCs was inhibited, beginning at 24 h and for up to 7 days. The HSCs were blocked in G0/G1 phase, and showed decreased Ki-67 positivity. Pro-collagen I production was reduced, while secretion of HGF, IL-6, MMP1, and MMP2 was enhanced. Neutralization of HGF partially blocked the inhibitory effect of ADHLSCs on the proliferation and secretion profile of HSCs. Repeated intrahepatic transplantation of cryopreserved/thawed ADHLSCs without immunosuppression inhibited the expression of markers of liver fibrosis in 6 out of 11 rats, as compared to their expression in the vehicle-transplanted group.ConclusionsThese data provide evidence for a direct inhibitory effect of ADHLSCs on activated HSCs, which supports their development for the treatment of liver fibrosis.
| S-EPMC5460523 | biostudies-literature
Project description:BACKGROUND: To investigate the potential mechanisms underlying the protective effects of 18? Glycyrrhizin (GL) on rat hepatic stellate cells (HSCs) and hepatocytes in vivo and in vitro. MATERIAL/METHODS: Sprague-Dawley (SD) rats were randomly divided into 5 groups: normal control group, liver fibrosis group, high-dose 18? GL group (25 mg/ kg/d), intermediate-dose 18? GL group (12.5 mg/kg/d) and low-dose 18? GL group (6.25 mg/ kg/d). The rat liver fibrosis model was induced by carbon tetrachloride (CCl4). The expressions of alpha-smooth muscle actin (?SMA) and NF-kappaB were determined by real-time PCR and immunohistochemistry. RESULTS: 18?GL dose-dependently inhibited the CCl4-induced liver fibrosis. There were significant differences in the mRNA and protein expressions of ?SMA between the fibrosis group and 18?-GL treatment groups, suggesting that 18? GL can suppress the proliferation and activation of HSCs. Few HSCs were apoptotic in the portal area and fibrous septum in the liver fibrosis group. However, the double-color staining of a-SMA and TUNEL showed that 18?-GL treatment groups increased HSC apoptosis. NF-kappaB was mainly found in the nucleus in the fibrosis group, while cytoplasmic expression of NF-kappaB was noted in the 18?GL groups. In the in vitro experiments, 18? GL promoted the proliferation of hepatocytes, but inhibited that of HSCs. HSCs were arrested in the G2/M phase following 18? GL treatment and were largely apoptotic. CONCLUSIONS: 18?-GL can suppress the activation of HSCs and induce the apoptosis of HSCs by blocking the translocation of NF-kappaB into the nucleus, which plays an important role in the protective effect of 18?-GL on liver fibrosis.
| S-EPMC3560665 | biostudies-literature
Project description:AimTo understand which and how different miRNAs are implicated in the process of hepatic stellate cell (HSC) activation.MethodsWe used microarrays to examine the differential expression of miRNAs during in vitro activation of primary HSCs (pHSCs). The transcriptome changes upon stable transfection of rno-miR-146a into an HSC cell line were studied using cDNA microarrays. Selected differentially regulated miRNAs were investigated by quantitative real-time polymerase chain reaction during in vivo HSC activation. The effect of miRNA mimics and inhibitor on the in vitro activation of pHSCs was also evaluated.ResultsWe found that 16 miRNAs were upregulated and 26 were downregulated significantly in 10-d in vitro activated pHSCs in comparison to quiescent pHSCs. Overexpression of rno-miR-146a was characterized by marked upregulation of tissue inhibitor of metalloproteinase-3, which is implicated in the regulation of tumor necrosis factor-α activity. Differences in the regulation of selected miRNAs were observed comparing in vitro and in vivo HSC activation. Treatment with miR-26a and 29a mimics, and miR-214 inhibitor during in vitro activation of pHSCs induced significant downregulation of collagen type I transcription.ConclusionOur results emphasize the different regulation of miRNAs in in vitro and in vivo activated pHSCs. We also showed that miR-26a, 29a and 214 are involved in the regulation of collagen type I mRNA.
| S-EPMC3122263 | biostudies-literature
Project description:IntroductionThe tumor microenvironment is involved in acquiring tumor malignancies of colorectal liver metastasis (CRLM). We have reported that TU-100 (Daikenchuto) suppresses hepatic stellate cell (HSC) activation in obstructive jaundice. In this study, we report new findings as the direct and indirect inhibitory effects of TU-100 on cancer cell growth through the suppression of HSC activation.Materials and methodsThe HSCs (LX2) were cultured in colon cancer cells (HCT116 and HT29)-conditioned medium (CM) with or without TU-100 treatment (90, 270, 900 μg/ml). Activated HSCs (aHSCs) were detected by α-SMA and IL-6 mRNA expressions and cytokine arrays of HSC's culture supernatants. Cancer cell growth was analyzed for proliferation and migration ability, compared with TU-100 treatment. To investigate the direct anti-tumor effect of TU-100, cancer cells were cultured in the presence of aHSC-CM and TU-100 (90, 270, 900) or aHSC-CM alone, and assessed autophagosomes, conversion to LC3-II protein, and Beclin-1 mRNA expression.ResultsColon cancer-CM significantly increased α-SMA and IL-6 mRNA expressions of aHSC. α-SMA and IL-6 mRNA expressions of aHSC, and IL-6 secretions from aHSCs were significantly decreased with TU-100 (270, 900) treatment, compared to colon cancer-CM alone. Compared with normal culture medium, aHSC-CM led to a significantly increased cell number and modified HSC-CM (TU-100; 270, 900) significantly suppressed cancer cell growth and migration. TU-100 (900) treatment induced autophagy and significantly promoted the autophagic cell death.ConclusionsTU-100 inhibited colon cancer cell malignant potential by both suppressing HSC activation and inducing directly autophagy of cancer cells.
| S-EPMC7733620 | biostudies-literature
Project description:Reversal of activated hepatic stellate cells (HSCs) to a quiescent state and apoptosis of activated HSCs are key elements in the reversion of hepatic fibrosis. CCAAT/enhancer binding protein ? (C/EBP-?) has been shown to inhibit HSC activation and promote its apoptosis. This study aims to investigate how C/EBP-? acetylation affects the fate of activated HSCs. Effects of a histone deacetylation inhibitor trichostatin A (TSA) on HSC activation were evaluated in a mouse model of liver fibrosis caused by carbon tetrachloride (CCl4) intoxication. TSA was found to ameliorate CCl4-induced hepatic fibrosis and improve liver function through increasing the protein level and enhancing C/EBP-? acetylation in the mouse liver. C/EBP-? acetylation was determined in HSC lines in the presence or absence of TSA, and the lysine residue K276 was identified as a main acetylation site in C/EBP-? protein. C/EBP-? acetylation increased its stability and protein level, and inhibited HSC activation. The present study demonstrated that C/EBP-? acetylation increases the protein level by inhibiting its ubiquitination-mediated degradation, and may be involved in the fate of activated HSCs. Use of TSA may confer an option in minimizing hepatic fibrosis by suppressing HSC activation, a key process in the initiation and progression of hepatic fibrosis.
| S-EPMC5849734 | biostudies-literature
Project description:Background and aimsAdenine is a uric acid pathway metabolite of no known function, and has recently been identified as a ligand for a rat G protein-coupled receptor. Due to the known role of other uric acid pathway metabolites in HSC biology, we tested the ability of adenine to induce HSC differentiation.MethodsRT-PCR was performed for adenine receptor expression in T-6 and primary rat HSC. T-6 and primary rats HSC were cultured with and without adenine, and stellation examined. Next, we examined inhibition of calcium signaling using caged IP(3). To test if adenine inhibits HSC chemotaxis T-6 cells and rat HSCs were cultured with or without adenine for 24 h in a transwell assay with PDGF as the chemoattractant. cDNA was prepared from T-6 and primary HSC for quantification of collagen 1 mRNA using real-time PCR.ResultsWe found that mRNA for the adenine receptor is expressed in T-6 cells and primary rat HSC. Also, adenine induces HSC stellation and adenine inhibits IP(3) mediated increase in cytosolic [Ca(2+)](i) and inhibits chemotaxis in T-6 cells and primary rat HSC. Adenine was also shown to up-regulate α-SMA and collagen 1, and this effect is lost by using specific si-RNA for the adenine receptor. Finally, adenine inhibits endothelin-1-induced gel contraction.ConclusionsThe adenine receptor is present in T-6 cells and primary rats HSC. Adenine, via the adenine receptor, induces morphological change, and cytosolic calcium signaling, inhibits chemotaxis, and up-regulates collagen 1 mRNA in HSCs.
| S-EPMC3760729 | biostudies-literature
Project description:At present, no satisfactory anti-liver fibrosis drugs have been used clinically due to the poor targeting ability and short half-life period. This study aimed to explore the effects of a new TRAIL (TNF-related apoptosis-inducing ligand) preparation that can target aHSCs (activated hepatic stellate cells) on liver fibrosis and explain the possible underlying mechanism. Using our self-made drug carrier pPB-SSL that specifically targets aHSCs, recombinant human TRAIL (rhTRAIL) protein was embedded in (named as pPB-SSL-TRAIL) and applied to treat liver fibrotic mice as well as 3T3 fibroblast cells and aHSCs. Through in vitro and in vivo experiments, we found that, compared with the groups treated with TRAIL (free rhTRAIL) and SSL-TRAIL (rhTRAIL capsulated within unmodified liposome), the group treated with pPB-SSL-TRAIL nanoparticles showed significantly lower cell viability and higher cell apoptosis in vitro. The targeting delivering system pPB-SSL also significantly enhanced the anti-fibrotic effect, apoptosis induction and long circulation of rhTRAIL. After the treatment with pPB-SSL-TRAIL, apoptosis of aHSCs was notably increased and hepatic fibrosis in mice was remarkably alleviated. In vitro, pPB-SSL-TRAIL nanoparticles were mainly transported and located on membrane or into cytoplasm, but the particles were distributed mainly in mouse fibrotic liver and most on the cell membrane of aHSCs. In conclusion, rhTRAIL carried by pPB-SSL delivering system has prolonged circulation in blood, be able to target aHSCs specifically, and alleviate fibrosis both in vitro and in vivo. It presents promising prospect in the therapy of liver fibrosis, and it is worthwhile for us to develop it for clinical use.
| S-EPMC6378220 | biostudies-literature
Project description:During liver tissue repair, hepatic stellate cells (HSCs), a pericyte-like nonparenchymal liver cell population, transform from a quiescent status (resting HSCs) into myofibroblast like cells (activated HSCs); the latter is the principal matrix-synthesizing cell of the liver. Although several factors have been shown to be involved in this important process, the molecular mechanisms regulating HSC activation are still under investigation. To identify key regulatory proteins involved in the HSC activation process, we used different mRNA display technologies, with cDNAs prepared from HSCs at different stages of in vitro activation. With the latter technique, the transcription factor Ets-1 was detected through its down-regulation during activation. As confirmed by Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, mRNAs coding for Ets-1 were present in the highest amounts in freshly isolated HSCs and in HSCs 2 days after plating (classified as resting HSCs/early activated HSCs) and were diminished in HSCs 7 days after plating (activated cells). Ets-1 protein was present in HSC-lysates, as assessed by Western blot, and bound to an oligonucleotide containing the Ets-1 consensus cis-acting motif, as demonstrated by electrophoretic mobility shift assay. Ets-1 binding activity peaked in nuclear extracts prepared from resting/early activated cells and was diminished in extracts derived from fully activated cells. In contrast, binding activity of the transcription factors TFIID, AP-1, and SP-1 was highest in activated HSCs and only barely detectable in resting/early activated HSCs. By Northern blot and RT-PCR analysis, Ets-1-specific transcripts were present in parenchymal and other nonparenchymal liver cells too, illustrating that hepatic Ets-1 expression is not specific or restricted to HSCs. However, the unique pattern of Ets-1 binding activity present in resting versus activated HSCs and its known implications for cellular differentiation and tissue remodeling suggest that Ets-1 could be of crucial importance for HSC activation and hepatic tissue repair.
| S-EPMC1866949 | biostudies-literature