OBJECTIVETo investigate the effects of exogenous TGF-β3 on the expression of endogenous TGF-b3 in hepatic stellate cell (HSC).

METHODSHSCs were cultured and divided into two groups: TGF-β3 group and blank control group, the cells of TGF-β3 group were exposed to TGF-b3 (10 ng/ml), whereas the blank control group was not treated. The cells were incubated in the presence of exogenous TGF-β3 and then (1) were harvested at 0h, 1h, 2h, 4h, 12h, 24h, and real time PCR was performed to detect the mRNA expression of endogenous TGF-β3. (2) The cells were collected at 0h, 1h, 6h, 12h, and western-blot was used to detect the protein synthesis of endogenous TGF-β3 in HSC; (3) The cell culture supernatant was harvested at 0h, 1h, 2h, 4h, 8h, 14h, 24h, and ELISA was performed to measure the total protein of extracellular TGF-β3; HSCs were treated with TGF-β3 (10 ng/ml) for 2h. The cells were then incubated in serum-free medium and the cell culture supernatant was harvested at 2.25h, 2.5h, 3h, 4h, 6h, 10h and 14h. ELISA was used to detect the extracellular secret ion of endogenous TGF-β3 by HSCs.

RESULTS(1) Exogenous TGF-β3 treatment induced a marked increase in TGF-β3 mRNA expression. By 2h of exogenous TGF-β3 treatment, maximal TGF-β3 mRNA expression levels (2.796 ± 0.518) of 2.74 fold above control values (1.022 ± 0.038) was reached (P < 0.05). Thereafter, TGF-β3 mRNA expression level declined, and the expression level was maintained at level of 1.45-fold for at least 10h and was 1.18-fold above control values by 24h TGF-β3 treatment (P < 0.05); (2) No significant difference about the intracellular protein expression level of endogenous TGF-β3 was found between two groups. (P > 0.05); (3) The total expression level of TGF-β3 reached a peak [(18.931 ± 2.904) ng/ml] at 4h after TGF-β3 treatment (1.89-fold higher than basic TGF-β3 (10 ng/ml). After that, it slowly declined. The expression peak [(0.835 ± 0.027) ng/ml] induction of extracellular secreted TGF-β3 was at 3h (32.12-fold higher than control [(0.026 ± 0.022) ng/ml], (P < 0.05). Thereafter, TGF-β3 slowly decreased after the peak time, and their expressions were still statistically significant as compared to the control (P < 0.05).

CONCLUSIONExogenous TGF-β3 could increase the expression of endogenous TGF-β3 mRNA and extracellular secreted TGF-β3 protein obviously.

Animals ; Cells, Cultured ; Hepatic Stellate Cells ; drug effects ; secretion ; Rats ; Transforming Growth Factor beta3 ; metabolism ; pharmacology

OBJECTIVESTo investigate the effects of norepinephrine (NE) on the proliferation and apoptosis of hepatic stellate cells (HSCs).

METHODSCultured HSCs were used in 6 groups: (1) a control group; (2) a NE group; (3) a phentolamine plus propranolol group; (4) a phentolamine (an alpha-AR antagonist) group; (5) a CGP20712A (a beta1-AR antagonist) group; and (6) a ICI118551(a beta2-AR antagonist) group. After NE and the antagonists of adrenoceptor subtypes were administered to the cultured HSCs, MTT assay was used to evaluate the cell proliferation at 24 h, 48 h, and 72 h. Terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) assay and flow cytometry were used to detect cell apoptosis. An inverted microscope was used to observe the morphological changes of HSCs.

RESULTS(1) MTT assay indicated that NE significantly induced HSCs proliferation in a time-dependent manner, which were reduced by antagonist of alpha-AR, beta1-AR and beta2-AR. (2) At 24 h after HSCs exposure to NE, apoptosis rates decreased significantly compared with that of the control group (6.60%+/-3.05% vs 12.60%+/-4.76%). In the antagonists of adrenoceptor subtypes groups, especially of a and beta2 adrenoceptor subtypes, the apoptosis was less. (3) Apoptosis rate of the NE group was significantly lower than that of the control group (2.29%+/-0.22% vs 3.06%+/-0.57%). In the antagonists of alpha and b2 adrenoceptor groups the apoptosis was less. (4) No obvious morphological changes of HSCs were found after administration of NE.

CONCLUSIONSSympathetic neurotransmitter NE can induce proliferation and inhibit apoptosis of the cultured HSCs.

Animals ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Hepatic Stellate Cells ; cytology ; drug effects ; Norepinephrine ; pharmacology ; Rats

Cell Line ; Hepatic Stellate Cells ; drug effects ; metabolism ; Humans ; Interferon-beta ; pharmacology

OBJECTIVETo investigate the effects of hydroxycamptothecin (HCPT) on proliferation and apoptosis of rat hepatic stellate cells (HSC).

METHODSRat HSC line (HSC-T6) and rat hepatocyte line (BRL-3A) were treated with different concentrations of HCPT (0, 0.008, 0.016, 0.031, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32 mg/L respectively) for 24 h. Cell proliferation was assessed by MTT colorimetric assay, apoptosis was detected with PI staging followed by flow cytometry, and by DNA ladder assay. The morphological change of apoptosis was observed under transmission electron microscopy (TEM).

RESULTSMTT assay indicated that HCPT significantly inhibited the proliferation of HSC-T6 and BRL-3A in a dose-dependent manner. 24 h after the treatment with different concentrations of HCPT (0.25, 0.5, 1 mg/L), the apoptosis rate (13.46%+/-2.42%, 26.25%+/-5.65%, 47.05%+/-8.76%, respectively) in HSC-T6 was significantly higher than that in control cells (4.89%+/-1.80%, F = 34.24, P less than 0.01). 24 h after 0.5 mg/L HCPT treatment, cell shrinkage, nucleoli disappearance, chromatin condensation were found under TEM, and DNA ladder was demonstrated by agarose gel electrophoresis.

CONCLUSIONHCPT could significantly inhibit proliferation and induce apoptosis of HSC-T6 in a dose-dependent manner.

Animals ; Apoptosis ; drug effects ; Camptothecin ; analogs & derivatives ; pharmacology ; Cell Line ; Cell Proliferation ; drug effects ; Hepatic Stellate Cells ; drug effects ; Rats

Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Hepatic Stellate Cells ; cytology ; drug effects ; Protease Inhibitors ; pharmacology ; Pyrazines ; pharmacology

Animals ; Apoptosis ; drug effects ; Cell Line ; Hepatic Stellate Cells ; cytology ; drug effects ; Rats ; gamma-Aminobutyric Acid ; pharmacology

OBJECTIVETo investigate the impact of the Artemisia annua plant-derived drug, artesunate, on proliferation of primary rat hepatic stellate cells (HSCs), and to analyze the underlying molecular mechanisms of its anti-fibrogenic effects involving the inhibition of transforming growth factor-beta 1 (TGF-b1) expression and secretion in liver.

METHODIsolated, cultured, and activated primary rat HSCs were divided into sixteen groups, including one untreated control group and fifteen artesunate-treated experimental groups with 125, 150, 175, 200 or 225 mumol/L for 24, 48 or 72 hours. The rate of cellular proliferation was measured using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. TGF-b1 mRNA expression was evaluated by reverse transcription-polymerase chain reaction and protein expression was evaluated by Western blotting. Enzyme-linked immunosorbent assay was used to evaluate secreted levels of TGF-b1 protein.

RESULTSArtesunate significantly inhibited proliferation of cultured HSCs in a dose- and time-dependent manner (all, P less than 0.01). After 24 hours of exposure, the inhibition ratios of the various artesunate concentrations were: 6.06%+/-1.44% (125 mumol/L), 21.47%+/-5.57% (150 mumol/L), 42.00%+/-7.36% (175 mumol/L), 67.12%+/-4.55% (200 mumol/L), and 79.83%+/-3.67% (225 mumol/L). Artesunate significantly inhibited the TGF-b1 mRNA expression in HSCs, and the higher the drug concentration, the higher the degree of inhibition (all, P less than 0.01). In addition, artesunate significantly inhibited the expression of intracellular and secreted TGF-b1 protein (all, P less than 0.01). In response to artesunate (mumol/L concentrations), the TGF-b1 levels were (164.24+/-6.88) pg/ml (0μmol/L), (102.68+/-4.45) pg/ml (150μmol/L), (86.54+/-5.56) pg/ml (175μmol/L), and (56.55+/-5.66) pg/ml (200μmol/L).

CONCLUSIONArtesunate exerts anti-fibrogenic effects on HSCs in vitro, possibly by reducing the expression, translation and secretion of TGF-b1.

Animals ; Artemisinins ; pharmacology ; Cells, Cultured ; Hepatic Stellate Cells ; drug effects ; secretion ; Rats ; Rats, Wistar ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo investigate the effects of cordyceps acid and cordycepin on the inflammatory phenotype and fibrogenic property of hepatic stellate cells (HSCs).

METHODSAn immortalized mouse HSC line (JS1) was stimulated with lippolysaccharide (LPS; 100 ng/ml) to induce an inflammatory response with or without co-administration of cordyceps acid or cordycepin in various concentrations (10, 50, or 200 mumol/L). Effects of the treatments on the chemokine monocyte chemotactic protein-1 (MCP-1) mRNA expression in the cells and the protein secretion in the cell culture supernatants were determined by reverse transcription and real-time quantitative PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. In addition, JS1 cells were treated with transforming growth factor-b1 (TGFb1; 10 ng/ml) to induce a fibrogenic response with or without co-administration of cordyceps acid or cordycepin in various concentrations (10, 50, or 200 mumol/L). Effects on the expression of fibrogenic proteins including collagen type I and a-smooth muscle actin (a-SMA), were investigated by Western blot.

RESULTSHigh-concentration (200 mumol/L) treatments of both cordyceps acid and cordycepin significantly inhibited the LPS-induced up-regulation of MCP-1 transcription and secretion (mRNA: 2.07 +/- 0.29 vs. 3.35 +/- 0.26, t = 15.90 and 1.15 +/- 0.23 vs. 4.17 +/- 0.61, t = 8.93; protein: 1.88 +/- 0.06 vs. 2.33 +/- 0.06, t = 10.39 and 1.47 +/- 0.25 vs. 1.97 +/- 0.04, t = 4.60; all P less than 0.05). All concentrations of cordyceps acid and cordycepin inhibited the TGFb1-induced up-regulation of collagen type I and a-SMA protein expression. However, the effects were more robust with the 200 mumol/L concentrations (P less than 0.05).

CONCLUSIONCordyceps acid and cordycepin ameliorate the LPS-induced inflammatory phenotype and TGFb1-induced fibrogenic response of cultured HSCs. These effects may contribute significantly to the drugs' therapeutic mechanisms to inhibit and resolve liver fibrosis.

Animals ; Cells, Cultured ; Chemokine CCL2 ; metabolism ; Cordyceps ; Hepatic Stellate Cells ; metabolism ; Transforming Growth Factor beta1 ; metabolism ; Up-Regulation ; drug effects

OBJECTIVETo investigate whether or not NADPH oxidase (NOX) participates in leptin-induced reactive oxygen species (ROS) production in hepatic stellate cells (HSC) and to explore the possible mechanism.

METHODSHSC-T6 cells (rat hepatic stellate cells line) were divided into nine groups: Group1: leptin (100 ng/ml) treated; Group2-6: leptin treated together with inhibitors that block different ROS-producing systems: diphenylene-iodonium (DPI) (20 micromol/L), Rotenone (20 micromol/L), Metyrapone (250 micromol/L), Allopurinol (100 micromol/L) and Indomethacin(100 micromol/L); Group7: leptin treated together with Janus kinase (JAK) inhibitor AG490 50 micromol/L; Group8: normal control group (treated DMEM with 0.1% DMSO); Group9: negative control group (untreated). Intracellular ROS levels were measured with dichlorodihydrofluorescein diacetate (DCFH-DA) dye assay by Fluorescence microscope and/or flow cytometry. NOX activity was analyzed by using spectrophotometer to calculate the absorbance of NADPH. The mRNA levels of Rac1 and p22Phox were evaluated by RT-PCR.

RESULTS(1) Leptin increased significantly the ROS production as compared to normal control group (92.91+/-4.19 vs.27.56+/-6.27, P<0.01) in HSC-T6 cells. Both the NADPH oxidase inhibitor DPI and AG490 (50 micromol/L) blocked the ROS production, inhibitors of other ROS producing systems had no significant effect on ROS production induced by lepin (P is more than 0.05). (2) Leptin treated HSC-T6 cells for 1 hour up-regulated the NOX activity significantly compared with that in normal control group [(1.90+/-0.22) pmol.min(-1).mg(-1) vs. (0.76+/-0.06) pmol.min(-1).mg(-1), P<0.05]. Furthermore, the NOX activity increased after being treated with leptin for 12 hours and 24 hours than being treated for 1 hour. Leptin-induced up-regulation of NOX activity was inhibited by pretreatment with DPI or AG490. (3) The RT-PCR results indicated that mRNA expressions of Rac1 and p22Phox in HSC-T6 cells with 12 hours of leptin stimulation increased significantly as compared with normal control group (0.41+/-0.13 vs 0.14+/-0.08, 0.45+/-0.12 vs 0.20+/-0.08, all P<0.05), while the DPI and AG490 had no effect on the mRNA expressions of Rac1 and p22Phox.

CONCLUSIONNOX is the main cellular source of the reactive oxygen species (ROS) generated by HSCs in response to leptin stimulation. The mechanism is probably that leptin can directly activate NOX through JAK signal transduction and hence induce the expression of NOX subunit to promote the activity of NOX which generates considerable ROS in HSC.

Animals ; Cells, Cultured ; Hepatic Stellate Cells ; drug effects ; metabolism ; Leptin ; pharmacology ; NADPH Oxidases ; genetics ; metabolism ; Rats ; Reactive Oxygen Species ; metabolism

OBJECTIVETo observe the contraction effect of endothelin-1 (ET-1) on human hepatic stellate cells (HSCs) and the inhibition of salianic-acid B (SA-B) on ET-1, to explore the acting link and the possible mechanism.

METHODSHSC were isolated from human normal liver tissue by enzyme digestion and Nycondenz density gradient centrifugation. The contraction of ET-1 on passage HSCs and the intervention of SA-B with three doses (low-, middle-, and high-) on the contraction were observed by collagen gel contraction. ET-1 and SA-B were directly added to the serum-free medium of HSCs, then calcium ion concentration was detected by laser scanning confocal microscope.

RESULTSCollagen gel contraction experiments showed that ET-1 could induce the contraction of HSC directly (P < 0.01). Three doses of SA-B significantly inhibited the contraction effects of ET-1 on HSCs (all P < 0.01). After adding the ET-1, HSCs morphology changed obviously with the number of cells decreased. However, SA-B inhibited the changes. Laser scanning confocal microscope experiments revealed that ET-1 stimulated the transiently rapid increase of intracellular calcium ion concentration, and the effects was obviously inhibited when SA-B was added.

CONCLUSIONSSA-B could inhibit the contraction of HSCs induced by ET-1, and its mechanism might be related to the lowing of free calcium ion concentration in HSCs. This anti-contraction effect of SA-B is perhaps one of the mechanisms of its anti-fibrosis and anti-portal hypertension effects.

Benzofurans ; pharmacology ; Cells, Cultured ; Endothelin-1 ; antagonists & inhibitors ; Hepatic Stellate Cells ; cytology ; Humans ; Isometric Contraction ; drug effects