Down-regulation of transforming growth factor β1/activin receptor-like kinase 1 pathway gene expression by herbal compound 861 is related to deactivation of LX-2 cells

AIM： To investigate the effect of herbal compound 861 （Cpd861） on the transforming growth factor-β1 （TGFβ1）/ activin receptor-like kinase 1 （ALK1, type Ⅰ receptor） signaling-pathway-related gene expression in the LX-2 cell line, and the inhibitory mechanism of Cpd861 on the activation of LX-2 cells.
METHODS： LX-2 cells were treated with TGFβ1 （5 ng/mL） Cpd861 （0.1 mg/mL）, TGFβ1 （5 ng/mL） plus Cpd861 （5 ng/mL） for 24 h to investigate the effect of Cpd861 on the TGFβ1/ALK1 pathway. Real-time PCR was performed to examine the expression of α-SMA （α-smooth muscle actin）, ALK1, Id1 （inhibitor of differentiation 1）. Western blotting was carried out to measure the levels of α-SMA and phosphorylated Smad1, and immunocytochemical analysis for the expression of α-SMA.
RESULTS： In LX-2 cells, TGFβ1/ALK1-pathway-related gene expression could be stimulated by TGFβ1, which led to excessive activation of the cells. Cpd861 decreased the activation of LX-2 cells by reducing the expression of α-SMA mRNA and protein expression. This effect was related to inhibition of the above TGFβ1/ALK1-pathway- related expression of genes such as Id1 and ALK1, and phosphorylation of Smad1 in LX-2 cells, even with TGFβ1 co-treatment for 24 h.
CONCLUSION： Cpd861 can restrain the activation of LX-2 cells by inhibiting the TGFβ1/ALK1/Smad1 pathway.     

Effects of herbal compound 861 on human hepatic stellate cell proliferation and activation

AIM:To investigate the effects of herbal compound 861(Cpd 861)on cell proliferation in human hepatic stellatecells(LX-2)and human hepatocellular liver carcinoma cells(HepG2),and expression of α-smooth muscle actin(α-SMA)in LX-2 cells.METHODS:LX-2 and HepG2 cells were incubated withvarious concentrations of Cpd 861(0.1-0.003 mg/mL)for 1,2,3,5 and 7 d.Cell proliferation was analyzed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)assay.Effects of Cpd861on the expression of α-SMA mRNA in LX-2 cells weremeasured by real-time quantitative PCR method using SYBRGreen I technology.RESULTS:Cpd 861,at 0.1 mg/mL,significantly inhibitedLX-2 cell proliferation(15% decrease relative to control,P<0.05)after 3 d of incubation.The inhibitory effects seemedto increase with the treatment time(25% decrease after5 d of incubation and 35% decrease after 7 d of incubation,P<0.01).However,Cpd 861 did not affect HepG2 cellproliferation at the same concentration used for LX-2 cells.The expression levels of α-SMA mRNA decreased significantlywhen LX-2 cells were exposed to Cpd 861 for 48 h(59?crease relative to control,P<0.05)or 72 h(60% decreaserelative to control,P<0.01).CONCLUSION:Cpd 861 can significantly inhibit LX-2 cellproliferation in a dose-dependant manner,and reduce theexpression levels of α-SMA mRNA in LX-2 cells.Since hepaticcell proliferation and high level of α-SMA are associatedwith liver fibrosis,the results suggest that Clod 861 may beuseful in the treatment of this disease.     

Effects of herbal compound 861 on human hepatic stellate cell proliferation and activation

AIM: To investigate the effects of herbal compound 861 (Cpd 861) on cell proliferation in human hepatic stellate cells (LX-2) and human hepatocellular liver carcinoma cells (HepG2), and expression of α-smooth muscle actin (α-SMA) in LX-2 cells.METHODS: LX-2 and HepG2 cells were incubated with various concentrations of Cpd 861 (0.1-0.003 mg/mL)for 1, 2, 3, 5 and 7 d. Cell proliferation was analyzed by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Effects of Cpd861on the expression of cc-SMA mRNA in LX-2 cells weremeasured by real-time quantitative PCR method using SYBRGreen I technology.RESULTS: Cpd 861, at 0.1 mg/mL, significantly inhibitedLX-2 cell proliferation (15% decrease relative to control,P&lt;0.05) after 3 d of incubation. The inhibitory effects seemedto increase with the treatment time (25% decrease after5 d of incubation and 35% decrease after 7 d of incubation,P&lt;0.01). However, Cpd 861 did not affect HepG2 cellproliferation at the same concentration used for I_X-2 cells.The expression levels of ~-SMA mRNA decreased significantlywhen LX-2 cells were exposed to Cpd 861 for 48 h (59%decrease relative to control, P&lt;0.05) or 72 h (60% decreaserelative to control, P&lt;0.01).CONCLUSION: Cpd 861 can significantly inhibit LX-2 cellproliferation in a dose-dependant manner, and reduce theexpression levels of o~-SMA mRNA in LX-2 cells. Since hepaticcell proliferation and high level of ~-SMA are associatedwith liver fibrosis, the results suggest that Cpd 861 may beuseful in the treatment of this disease.     

Interaction between insulin-like growth factor binding protein-related protein 1 and trans-forming growth factor beta 1 in primary hepatic stellate cells

BACKGROUND:We previously showed that insulin-like growth factor binding protein-related protein 1(IGFBPrP1) is a novel mediator in liver fibrosis.Transforming growth factor beta 1(TGFβ1) is known as the strongest effector of liver fibrosis.Therefore,we aimed to investigate the detailed interaction between IGFBPrP1 and TGFβ1 in primary hepatic stellate cells(HSCs).METHODS:We overexpressed TGFβ1 or IGFBPrP1 and inhibited TGFβ1 expression in primary HSCs for 6,12,24,48,72,and 96 hours to investigate their interaction and observe the accompanying expressions of α-smooth muscle actin(α-SMA),collagen I,fibronectin,and phosphorylated-mothers against decapentaplegic homolog 2/3(p-Smad2/3).RESULTS:We found that the adenovirus vector encoding the TGFβ1 gene(Ad TGFβ1) induced IGFBPrP1 expression while that of α-SMA,collagen I,fibronectin,and TGFβ1 increased gradually.Concomitantly,Ad IGFBPrP1 upregulated TGFβ1,α-SMA,collagen I,fibronectin,and p-Smad2/3 in a time-dependent manner while IGFBPrP1 expression was decreased at 96 hours.Inhibition of TGFβ1 expression reduced the IGFBPrP1-stimulated expression of α-SMA,collagen I,fibronectin,and p-Smad2/3.CONCLUSIONS:These findings for the first time suggest the existence of a possible mutually regulation between IGFBPrP1 and TGFβ1,which likely accelerates liver fibrosis progression.Furthermore,IGFBPrP1 likely participates in liver fibrosis in a TGFβ1-depedent manner,and may act as an upstream regulatory factor of TGFβ1 in the Smad pathway.     

New role and molecular mechanism of Gadd45a in hepatic fibrosis

AIM: To investigate the role of Gadd45 a in hepatic fibrosis and the transforming growth factor(TGF)-β/Smad signaling pathway.METHODS: Wild-type male BALB/c mice were treated with CCl_4 to induce a model of chronic liver injury. Hepatic stellate cells(HSCs) were isolated from the liver of BALB/c mice and were treated with small interfering RNAs(si RNAs) targeting Gadd45 a or the pc DNA3.1-Gadd45 a recombinant plasmid. Cellular α-smooth muscle actin(α-SMA), β-actin, type Ⅰ collagen, phosphoSmad2, phospho-Smad3, Smad2, Smad3, and Smad4 were detected by Western blots. The m RNA levels of α-SMA, β-actin, and type Ⅰ collagen were determined by quantitative real-time(q RT)-PCR analyses. Reactive oxygen species production was monitored by flow cytometry using 2,7-dichlorodihydrofluorescein diacetate.Gadd45a, Gadd45 b, anti-Gadd45 g, type Ⅰ collagen, and SMA local expression in liver tissue were measured by histologic and immunohistochemical analyses. RESULTS: Significant downregulation of Gadd45 a, but not Gadd45 b or Gadd45 g, accompanied by activation of the TGF-β/Smad signaling pathways was detected in fibrotic liver tissues of mice and isolated HSCs with chronic liver injury induced by CCl_4 treatment. Overexpression of Gadd45 a reduced the expression of extracellular matrix proteins and α-SMA in HSCs, whereas transient knockdown of Gadd45 a with si RNA reversed this process. Gadd45 a inhibited the activity of a plasminogen activator inhibitor-1 promoter construct and(CAGA)_9 MLP-Luc, an artificial Smad3/4-specific reporter, as well as reduced the phosphorylation and nuclear translocation of Smad3. Gadd45 a showed protective effects by scavenging reactive oxygen species and upregulating antioxidant enzymes.CONCLUSION: Gadd45 a may counteract hepatic fibrosis by regulating the activation of HSCs via the inhibition of TGF-β/Smad signaling.     

miR-34a mediates oxaliplatin resistance of colorectal cancer cells by inhibiting macroautophagy via transforming growth factor-β/Smad4 pathway

AIM To investigate whether micro RNA(mi R)-34 a mediates oxaliplatin(OXA) resistance of colorectal cancer(CRC) cells by inhibiting macroautophagy via the transforming growth factor(TGF)-β/Smad4 pathway.METHODS miR-34 a expression levels were detected in CRC tissues and CRC cell lines by quantitative real-time polymerase chain reaction. Computational search, functional luciferase assay and western blotting were used to demonstrate the downstream target of miR-34 a in CRC cells. Cell viability was measured with Cell Counting Kit-8. Apoptosis and macroautophagy of CRC cells were analyzed by flow cytometry and transmission electron microscopy, and expression of beclin I and LC3-II was detected by western blotting.RESULTS Expression of miR-34 a was significantly reduced while expression of TGF-β and Smad4 was increased in CRC patients treated with OXA-based chemotherapy. OXA treatment also resulted in decreased mi R-34 a levels and increased TGF-β and Smad4 levels in both parental cells and the OXA-resistant CRC cells. Activation of macroautophagy contributed to OXA resistance in CRC cells. Expression levels of Smad4 and miR-34 a in CRC patients had a significant inverse correlation and overexpressing mi R-34 a inhibited macroautophagy activation by directly targeting Smad4 through the TGF-β/Smad4 pathway. OXA-induced downregulation of miR-34 a and increased drug resistance by activating macroautophagy in CRC cells.CONCLUSION miR-34 a mediates OXA resistance of CRC by inhibiting macroautophagy via the TGF-β/Smad4 pathway.     

Tetrandrine inhibits activation of rat hepatic stellate cells in vitro via transforming growth factor-β signaling

AIM: To investigate the effect of various concentrations of tetrandrine on activation of quiescent rat hepatic stellate cells (HSCs) and transforming growth factor-β(TGF-β) signaling in vitro. METHODS: HSCs were isolated from rats by in situ perfusion of liver and 18% Nycodenz gradient centrifugation, and primarily cultured on uncoated plastic plates for 24 h with DMEM containing 20% fetal bovine serum (FBS/ DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, the HSCs were cultured in 2% FBS/DMEM with tetrandrine (0.25, 0.5, 1, 2 mg/L, respectively). Cell morphological features were observed under an inverted microscope, smooth muscle-α-actin (α-SMA) was detected by immunocytochemistry and image analysis system, laminin (LN) and type Ⅲ procollagen (PCⅢ) in supernatants were determined by radioimmunoassay. TGF-β1 mRNA, Smad 7 mRNA and Smad 7 protein were analyzed with RT-PCR and Western blotting, respectively. RESULTS: Tetrandrine at the concentrations of 0.25-2 mg/L prevented morphological transformation of HSC from the quiescent state to the activated one, while α-SMA, LN and PCⅢ expressions were inhibited. As estimated by gray values, the expression of α-SMA in tetrandrine groups (0.25, 0.5, 1, 2 mg/L) was reduced from 21.3% to 42.2% (control: 0.67, tetrandrine groups: 0.82, 0.85, 0.96, or 0.96, respectively, which were statistically different from the control, P<0.01), and the difference was more significant in tetrandrine at 1 and 2 mg/L. The content of LN in supernatants was significantly decreased in tetrandrine groups to 58.5%, 69.1%, 65.8% or 60.0% that of the control respectively, and that of PCⅢ to 84.6%, 81.5%, 75.7% or 80.7% respectively (P<0.05 vs control), with no significant difference among tetrandrine groups. RT-PCR showed that TGF-β1 mRNA expression was reduced by tetrandrine treatments from 56.56% to 87.90% in comparison with the control, while Smad 7 mRNA was increased 1.4-4.8 times. The TGF-β1 mRNA and Smad 7 mRNA expression was in a significant negative correlation (r= -0.755, P<0.01), and both were significantly correlated with α-SMA protein expression (r= -0.938, P<0.01; r = 0.938,P<0.01, respectively). The up-regulation of Smad 7 protein by tetrandrine (1 mg/L) was confirmed by Western blotting as well. CONCLUSION: Tetrandrine has a direct inhibiting effect on the activation of rat HSCs in culture. It up-regulates the expression of Smad 7 which in turn blocks TGF-β1 expression and signaling.     

Tetrandrine inhibits activation of rat hepatic stellate cells in vitro via transforming growth factor-β signaling

AIM: To investigate the effect of various concentrations of tetrandrine on activation of quiescent rat hepatic stellate cells (HSCs) and transforming growth factor-β (TGF-β) signaling in vitro. METHODS: HSCs were isolated from rats by in situ perfusion of liver and 18% Nycodenz gradient centrifugugation, and primarily cultured on uncoated plastic plates for 24 h with DMEM containing 20% fetal bovine serum (FBS/DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, the HSCs were cultured in 2% FBS/DMEM with tetrandrine (0.25, 0.5, 1,2 mg/L, respectively). Cell morphological features were observed under an inverted microscope, smooth muscle-α-actin (α-SMA) was detected by immunocytochemistry and image analysis system, laminin (LN) and type Ⅲ procollagen (PCⅢ) in supernatants were determined by radioimmunoassay. TGF-β1 mRNA, Smad 7 mRNA and Smad 7 protein were analyzed with RT-PCR and Western blotting, respectively. RESULTS: Tetrandrine at the concentrations of 0.25-2 mg/L prevented morphological transformation of HSC from the quiescent state to the activated one, while α-SMA, LN and PCⅢ expressions were inhibited. As estimated by gray values, the expression of α-SMA in tetrandrine groups (0.25, 0.5, 1, 2 mg/L) was reduced from 21.3% to 42.2% (control: 0.67, tetrandrine groups: 0.82, 0.85, 0.96, or 0.96, respectively, which were statistically different from the control, P&lt;0.01), and the difference was more significant in tetrandrine at 1 and 2 mg/L. The content of LN in supernatants was significantly decreased in tetrandrine groups to 58.5%, 69.1%, 65.8% or 60.0% that of the control respectively, and that of PCII] to 84.6%, 81.5%, 75.7% or 80.7% respectively (P&lt;0.05 vs control), with no significant difference among tetrandrine groups. RTPCR showed that TGF-β1 mRNA expression was reduced by tetrandrine treatments from 56.56% to 87.90% in comparison with the control, while Smad 7 mRNA was increased 1.4-4.8 times. The TGF-β1 mRNA and Smad 7 mRNA expression was in a significant negative correlation (r = -0.755, P&lt;0.01), and both were significantly correlated with α-SMA protein expression (r = -0.938, P&lt;0.01; r = 0.938, P&lt;0.01, respectively). The up-regulation of Smad 7 protein by tetrandrine (1 mg/L) was confirmed by Western blotting as well. CONCLUSION: Tetrandrine has a direct inhibiting effect on the activation of rat HSCs in culture. It up-regulates the expression of Smad 7 which in turn blocks TGF-β1 expression and signaling.     

Upregulation of miR-29b contributes to the anti-fibrotic effect of macrophage migration inhibitory factor in diabetic myocardial fibrosis

Background Macrophage migration inhibitory factor(MIF) possesses proinflammatory function when secreted from the cells, and it also exhibits antioxidant properties based on its intrinsic oxidoreductase activity.However, the role of MIF in cardiac fibrosis is not well known. In the present study, the effect of MIF on fibrosis-associated gene expression and the underlying mechanism were examined. Methods The collagen content in mouse myocardium was detected by Masson staining. Expressions of MIF and fibrosis-associated Col1a1, Col3a1 and α-SMA in mouse myocardium or mouse cardiac fibroblasts were detected by quantitative real-time PCR and Western blot assay, respectively. Mature miR-29b expressions in mouse myocardium and cardiac fibroblasts were determined by real-time PCR. Smad3 activation in MIF-treated cardiac fibroblasts was also detected by Western blot assay. Results Compared with the db / m control mice, the collagen content was significantly increased in the myocardium of diabetic db / db mice. MIF was up-regulated, but miR-29b was down-regulated in the diabetic myocardium. Quantitative real-time PCR and Western blot assay showed that MIF could inhibit fibrosis-associated Col1a1, Col3a1 and α-SMA expressions in mouse cardiac fibroblasts.Smad3 activation was inhibited, but miR-29b was up-regulated in MIF-treated cardiac fibroblasts. Enforced expression of miR-29b significantly suppressed Col1a1, Col3a1, and α-SMA mRNA and 1protein expressions in cardiac fibroblasts. Conclusions MIF possesses the anti-fibrosis activity through inhibiting Smad3activation and through up-regulating miR-29b expression, and miR-29b can inhibit fibrosis-associated Col1a1,Col3a1 and α-SMA expressions in cardiac fibroblasts.     

Insulin-like growth factor binding protein-7 induces activation and transdifferentiation of hepatic stellate cells in vitro

AIM： To investigate the role of insulin-like growth factor binding protein-7 （IGFBP-7） in the activation and transdifferentiation of hepatic stellate cells （HSC） in vitro.
METHODS： Rat HSC-T6 cells were cultured in separate dishes and treated with various concentration of transforming growth factor （TGF）-β1, IGFBP-7 or anti- IGFBP-7 antibody for 24 h. The supernatant or a cytoplasm suspension was obtained from cultured HSC, followed by transfer of cells to form cell-coated dishes. Immunocytochemistry and Western blotting were used to analyze the expression of IGFBP-7 induced by TGF-β1 and the level of fibronectin, collagen Ⅰ and α-smooth muscle actin （SMA）. The pro-apoptotic effect of anti- IGFBP-7 antibody was determined by flow cytometry.
RESULTS： Immunocytochemistry and Western blotting revealed that the expression of IGFBP-7 in TGF-β1 treated HSC was significantly up-regulated compared to that in the control group. In addition, fibronectin, collagen Ⅰ and α-SMA also showed enhanced expression in accordance with the transdifferentiation process in a dose-dependent manner to some extent. Moreover, flow cytometry suggested that anti-IGFBP-7 antibody induced apoptosis of activated HSC, which is responsible for the development of liver fibrosis, and may represent a novel pathway and target for therapeutic intervention.
CONCLUSION： IGFBP-7 showed increased expression in activated HSC and played an important role in the activation and transdifferentiation process of HSC. Anti- IGFBP-7 antibody may ameliorate liver fibrogenesis.     

Dynamic changes of α-AR,β1-AR and β2-AR expression during hepatic fibrogenesis

Objective To investigate the dynamic changes of α-AR,β1-AR and β2-AR expression in hepatic fibrosis.Methotis Rat hepatic fibrosis model was established by bile duct ligation(BDL).HE and Masson staining were used to determine hepatic fibrosis levels.Immunohistochemistry was applied to detect α-sooth muscle actin(α-SMA),a marker of hepatic stellate cell(HSC)activation;Western blot and realtime RT-PCR were used to measure the dynamic changes of α-AR,β1-AR,β2-AR expression on protein and mRNA levels.respectively,during the development of hepatic fibrosis.Results(1)HE and Masson trichrome staining showed that the liver fibrosis modeIs were established successfully.(2)At 1,2,3,4 wk after BDL,α-SMA positive area density of the model group(10.58%±1.75%,24.1 4%±2.02%,29.74%±2.59%,34.28%±2.01%)was significantly higher than that of the sham operation group (4.12%±1.51%),P＜0.01.(3)The expression of α-AR,β1-AR,β2-AR protein and mRNA was increased with the development of the hepatic fibrosis(P＜0.05).(4)α-SMA expression Was positively associated with α-AR,β1-AR,β2-AR,r values were 0.564,0.753 and 0.606,respectively.Conclusions The expression of α-SMA is increased dramatically during the fibrosis,and is positively associated with the expression of α-AR,β1-AR and β2-AR.     

Histone deacetylase inhibitor suberoylanilide hydroxamic acid alleviates liver fibrosis by suppressing the transforming growth factor-β1 signal pathway

Background: Histone deacetylases(HDACs) inhibitors are new anti-fibrotic drugs that inhibit the activity of hepatic stellate cells. The present study focused on the anti-fibrotic function of HDAC inhibitor suberoylanilide hydroxamic acid(SAHA) by suppressing transforming growth factor-β1(TGF-β1) signaling. Methods: Male Sprague-Dawley rats were used to induce liver fibrosis with carbon tetrachloride(CCl 4) and LX2 cell(human hepatic stellate cell line) was stimulated by TGF-β1. Both animals and cells were treated with SAHA. The Smad7 and connective tissue growth factor(CTGF) mRNA levels were detected by real-time polymerase chain reaction(PCR). Western blotting was used to examine the protein levels of CTGF, Histone H3(H3), Smad7, Smad2/3, Acetyl-Histone H3(AH3), HDAC2, α-smooth muscle actin( α-SMA), HDAC6, p-Smad2/3 and HDAC8. In addition, the TGF-β1 and liver enzyme levels from rat serum were detected. Histopathological changes were examined by hematoxylin and eosin(HE), Sirius red and Masson trichrome staining. The α-SMA expression was detected by immumohistochemical staining. Results: Compared with control group, the TGF-β1 and liver enzyme levels from rat serum, together with the mRNA levels of CTGF and protein levels of CTGF, HDAC2, α-SMA, HDAC6, p-Smad2/3 and HDAC8 were elevated in fibrotic rats( P 0.01). But the Smad7 mRNA and AH3 protein levels were notably suppressed in the fibrotic rats( P 0.01). Pathological examination showed the typical changes of liver fibrosis in the fibrotic rats. After the treatment with SAHA, the levels of liver enzymes, TGF-β1, CTGF, HDAC2, α-SMA, HDAC6, p-Smad2/3 and HDAC8 were reduced( P 0.01) and Smad7 and AH3 protein contents were elevated in liver fibrotic rats( P 0.01). Moreover, immumohistochemistry showed that SAHA significantly suppressed the α-SMA protein content in fibrotic liver( P 0.01). Conclusion: The HDAC inhibitor SAHA alleviated liver fibrosis by suppressing the TGF-β1 signaling.     

Antifibrogenic effects of vitamin D derivatives on mouse pancreatic stellate cells

AIM To study the molecular effects of three different D-vitamins, vitamin D2, vitamin D3 and calcipotriol, in pancreatic stellate cells(PSCs).METHODS Quiescent PSCs were isolated from mouse pancreas and activated in vitro by seeding on plastic surfaces. The cells were exposed to D-vitamins as primary cultures(early-activated PSCs) and upon re-culturing(fullyactivated cells). Exhibition of vitamin A-containing lipid droplets was visualized by oil-red staining. Expression of α-smooth muscle actin(α-SMA), a marker of PSC activation, was monitored by immunofluorescence and immunoblot analysis. The rate of DNA synthesis was quantified by 5-bromo-2'-deoxyuridine(Brd U) incorporation assays. Real-time PCR was employed to monitor gene expression, and protein levels of interleukin-6(IL-6) were measured by ELISA. Uptake of proline was determined using 18 F-proline.RESULTS Sustained culture of originally quiescent PSCs inducedcell proliferation, loss of lipid droplets and exhibition of stress fibers, indicating cell activation. When added to PSCs in primary culture, all three D-vitamins diminished expression of α-SMA(to 32%-39% of the level of control cells; P 0.05) and increased the storage of lipids(scores from 1.97-2.15 on a scale from 0-3; controls: 1.49; P 0.05). No such effects were observed when D-vitamins were added to fully-activated cells, while incorporation of Brd U remained unaffected under both experimental conditions. Treatment of re-cultured PSCs with D-vitamins was associated with lower expression of IL-6(-42% to-49%; P 0.05; also confirmed at the protein level) and increased expression of the vitamin D receptor gene(209%-321% vs controls; P 0.05). There was no effect of D-vitamins on the expression of transforming growth factor-β1 and collagen type 1(chain α1). The lowest uptake of proline, a main component of collagen, was observed in calcipotriol-treated PSCs.CONCLUSION The three D-vitamins inhibit, with similar efficiencies, activation of PSCs in vitro, but cannot reverse the phenotype once the cells are fully activated.