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Osthole ameliorates hepatic fibrosis and inhibits hepatic stellate cell activation.

Liu YW, Chiu YT, Fu SL, Huang YT - J. Biomed. Sci. (2015)

Bottom Line: Additionally, osthole reduced the expression of fibrosis-related genes significantly.Furthermore, osthole decreased TNF-α-triggered NF-κB activities significantly.In addition, osthole suppressed HSCs activation in vitro significantly.

View Article: PubMed Central - PubMed

Affiliation: Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, No. 155, Li-Nong Street, Sec. 2, Taipei, 11221, Taiwan. yaweiliu19850417@gmail.com.

ABSTRACT

Background: Hepatic fibrosis is a dynamic process which ultimately leads to cirrhosis in almost patients with chronic hepatic injury. However, progressive fibrosis is a reversible scarring response. Activation of hepatic stellate cells (HSCs) is the prevailing process during hepatic fibrosis. Osthole is an active component majorly contained in the fruit of Cnidium monnieri (L.) Cusson. This present study investigated the therapeutic effects of osthole on rat liver fibrosis and HSC activation.

Results: We established the thioacetamide (TAA)-model of Sprague-Dawley (SD) rats to induce hepatic fibrosis. Rats were divided into three groups: control, TAA, and TAA + osthole (10 mg/kg). In vivo, osthole significantly reduced liver injury by diminishing levels of plasma AST and ALT, improving histological architecture, decreasing collagen and α-SMA accumulation, and improving hepatic fibrosis scores. Additionally, osthole reduced the expression of fibrosis-related genes significantly. Osthole also suppressed the production of fibrosis-related cytokines and chemokines. Moreover, nuclear translocation of p65 was significantly suppressed in osthole-treated liver. Osthole also ameliorated TAA-induced injury through reducing cellular oxidation. Osthole showed inhibitory effects in inflammation-related genes and chemokines production as well. In vitro, we assessed osthole effects in activated HSCs (HSC-T6 and LX-2). Osthole attenuated TGF-β1-induced migration and invasion in HSCs. Furthermore, osthole decreased TNF-α-triggered NF-κB activities significantly. Besides, osthole alleviated TGF-β1- or ET-1-induced HSCs contractility.

Conclusions: Our study demonstrated that osthole improved TAA-caused liver injury, fibrogenesis and inflammation in rats. In addition, osthole suppressed HSCs activation in vitro significantly.

No MeSH data available.


Related in: MedlinePlus

Osthole downregulated HSC activation. a Wound healing assay in HSCs (both HSC-T6 and LX2 cells). Cells were treated by osthole (1, 3 and 10 μg/ml) after creating wounds. Graphs represent cell migration assessed at 0 h and 24 h after exposure to TGF-β1 (1 ng/ml), and quantification of wound closure was shown below, n = 3. b Cell invasion assay in HSCs. Cells were treated by osthole (1, 3 and 10 μg/ml) and then stimulated with TGF-β1 for 24 h. Graphs displaying the bottom side of the filter inserts with cells that migrated through the filter pores. Graphs of quantification graphs represent the analysis of the cell count. c NF-κB activity of HSCs which were transfected with CMV-βgal and the reporter plasmid containing NF-κB responsive region for 24 h. Cells were pre-treated with osthole (1, 3 and 10 μg/ml) for 1 h, followed by 6 h TNF-α (1 ng/ml) stimulation, then NF-κB activity was detected by luminescence. CMV-βgal was used as internal control to normalize the transfection efficiency. n = 3. d Osthole-inhibited ET-1- or TGF-β1-induced HSC contraction. Cultured HSCs were serum-starved 24 h prior to seeding onto collagen lattices. Cells were pre-treated with control or osthole in 10 μg/ml and subsequently treated with ET-1 (1 nmol/l) or TGF-β1(1 ng/ml) after 30 min. Graphs of quantification graphs represent the analysis of the area of collagen circle. n = 3. *p < 0.05; **p < 0.01, compared with other groups
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Fig4: Osthole downregulated HSC activation. a Wound healing assay in HSCs (both HSC-T6 and LX2 cells). Cells were treated by osthole (1, 3 and 10 μg/ml) after creating wounds. Graphs represent cell migration assessed at 0 h and 24 h after exposure to TGF-β1 (1 ng/ml), and quantification of wound closure was shown below, n = 3. b Cell invasion assay in HSCs. Cells were treated by osthole (1, 3 and 10 μg/ml) and then stimulated with TGF-β1 for 24 h. Graphs displaying the bottom side of the filter inserts with cells that migrated through the filter pores. Graphs of quantification graphs represent the analysis of the cell count. c NF-κB activity of HSCs which were transfected with CMV-βgal and the reporter plasmid containing NF-κB responsive region for 24 h. Cells were pre-treated with osthole (1, 3 and 10 μg/ml) for 1 h, followed by 6 h TNF-α (1 ng/ml) stimulation, then NF-κB activity was detected by luminescence. CMV-βgal was used as internal control to normalize the transfection efficiency. n = 3. d Osthole-inhibited ET-1- or TGF-β1-induced HSC contraction. Cultured HSCs were serum-starved 24 h prior to seeding onto collagen lattices. Cells were pre-treated with control or osthole in 10 μg/ml and subsequently treated with ET-1 (1 nmol/l) or TGF-β1(1 ng/ml) after 30 min. Graphs of quantification graphs represent the analysis of the area of collagen circle. n = 3. *p < 0.05; **p < 0.01, compared with other groups

Mentions: The in vivo results above suggested that osthole could suppress TAA-caused hepatic fibrogenesis and inflammation in rats. HSCs play an important role during hepatic fibrogenesis, and HSC activation is also crucially related to hepatic inflammation. Osthole (1, 3, and 10 μg/ml) showed no cytotoxicity to HSC-T6 and LX-2 cells after 24 h treatment (Additional file 5: Figure S4). We utilized HSC-T6 and LX-2 to determine whether osthole could inhibit HSC activation induced by chemotactic stimulation. Using wound-healing assays, the inhibitory effect of osthole on TGF-β1-induced HSC migration were revealed. After 24 h exposure to TGF-β1, HSCs (both HSC-T6 and LX-2) showed obvious migration to the cell-free zone. Moreover, osthole (10 μg/ml) inhibited TGF-β1-induced migration in HSCs significantly (Fig. 4a). We also performed trans-well invasion assay to assess the ability of cell invasion. Osthole (1-10 μg/ml) curbed HSC invasion in response to TGF-β1. The cell number of TGF-β1-stimulated HSCs was higher than that in the control group, whereas osthole treatment (3, 10 μg/ml) decreased TGF-β1-stimulated invasion significantly in both HSC cell lines (Fig. 4b). Furthermore, we used the luciferase assay to determine NF-κB activity, which is a hallmark of HSC activation. In both HSC cell lines, osthole (3, 10 μg/ml) led to a marked inhibition of TNF-α-induced NF-κB luciferase activity (Fig. 4c). HSC activation exhibits many features, the most prevailing of which includes tissue contraction mediated by contractile myofibroblasts. Therefore, we measured HSC contractility by ET-1 and TGF-β1 stimulation, both well known to induce HSC contraction in the liver. We treated HSCs plated on collagen lattices with osthole (10 μg/ml) and ET-1 or TGF-β1 for 48 h. Osthole significantly reduced both ET-1- and TGF-β1-promoted HSC contraction (Fig. 4d). We substantiated our hypothesis that osthole was capable of interrupting HSC activation in HSC-T6 and LX-2 cells.Fig. 4


Osthole ameliorates hepatic fibrosis and inhibits hepatic stellate cell activation.

Liu YW, Chiu YT, Fu SL, Huang YT - J. Biomed. Sci. (2015)

Osthole downregulated HSC activation. a Wound healing assay in HSCs (both HSC-T6 and LX2 cells). Cells were treated by osthole (1, 3 and 10 μg/ml) after creating wounds. Graphs represent cell migration assessed at 0 h and 24 h after exposure to TGF-β1 (1 ng/ml), and quantification of wound closure was shown below, n = 3. b Cell invasion assay in HSCs. Cells were treated by osthole (1, 3 and 10 μg/ml) and then stimulated with TGF-β1 for 24 h. Graphs displaying the bottom side of the filter inserts with cells that migrated through the filter pores. Graphs of quantification graphs represent the analysis of the cell count. c NF-κB activity of HSCs which were transfected with CMV-βgal and the reporter plasmid containing NF-κB responsive region for 24 h. Cells were pre-treated with osthole (1, 3 and 10 μg/ml) for 1 h, followed by 6 h TNF-α (1 ng/ml) stimulation, then NF-κB activity was detected by luminescence. CMV-βgal was used as internal control to normalize the transfection efficiency. n = 3. d Osthole-inhibited ET-1- or TGF-β1-induced HSC contraction. Cultured HSCs were serum-starved 24 h prior to seeding onto collagen lattices. Cells were pre-treated with control or osthole in 10 μg/ml and subsequently treated with ET-1 (1 nmol/l) or TGF-β1(1 ng/ml) after 30 min. Graphs of quantification graphs represent the analysis of the area of collagen circle. n = 3. *p < 0.05; **p < 0.01, compared with other groups
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4522080&req=5

Fig4: Osthole downregulated HSC activation. a Wound healing assay in HSCs (both HSC-T6 and LX2 cells). Cells were treated by osthole (1, 3 and 10 μg/ml) after creating wounds. Graphs represent cell migration assessed at 0 h and 24 h after exposure to TGF-β1 (1 ng/ml), and quantification of wound closure was shown below, n = 3. b Cell invasion assay in HSCs. Cells were treated by osthole (1, 3 and 10 μg/ml) and then stimulated with TGF-β1 for 24 h. Graphs displaying the bottom side of the filter inserts with cells that migrated through the filter pores. Graphs of quantification graphs represent the analysis of the cell count. c NF-κB activity of HSCs which were transfected with CMV-βgal and the reporter plasmid containing NF-κB responsive region for 24 h. Cells were pre-treated with osthole (1, 3 and 10 μg/ml) for 1 h, followed by 6 h TNF-α (1 ng/ml) stimulation, then NF-κB activity was detected by luminescence. CMV-βgal was used as internal control to normalize the transfection efficiency. n = 3. d Osthole-inhibited ET-1- or TGF-β1-induced HSC contraction. Cultured HSCs were serum-starved 24 h prior to seeding onto collagen lattices. Cells were pre-treated with control or osthole in 10 μg/ml and subsequently treated with ET-1 (1 nmol/l) or TGF-β1(1 ng/ml) after 30 min. Graphs of quantification graphs represent the analysis of the area of collagen circle. n = 3. *p < 0.05; **p < 0.01, compared with other groups
Mentions: The in vivo results above suggested that osthole could suppress TAA-caused hepatic fibrogenesis and inflammation in rats. HSCs play an important role during hepatic fibrogenesis, and HSC activation is also crucially related to hepatic inflammation. Osthole (1, 3, and 10 μg/ml) showed no cytotoxicity to HSC-T6 and LX-2 cells after 24 h treatment (Additional file 5: Figure S4). We utilized HSC-T6 and LX-2 to determine whether osthole could inhibit HSC activation induced by chemotactic stimulation. Using wound-healing assays, the inhibitory effect of osthole on TGF-β1-induced HSC migration were revealed. After 24 h exposure to TGF-β1, HSCs (both HSC-T6 and LX-2) showed obvious migration to the cell-free zone. Moreover, osthole (10 μg/ml) inhibited TGF-β1-induced migration in HSCs significantly (Fig. 4a). We also performed trans-well invasion assay to assess the ability of cell invasion. Osthole (1-10 μg/ml) curbed HSC invasion in response to TGF-β1. The cell number of TGF-β1-stimulated HSCs was higher than that in the control group, whereas osthole treatment (3, 10 μg/ml) decreased TGF-β1-stimulated invasion significantly in both HSC cell lines (Fig. 4b). Furthermore, we used the luciferase assay to determine NF-κB activity, which is a hallmark of HSC activation. In both HSC cell lines, osthole (3, 10 μg/ml) led to a marked inhibition of TNF-α-induced NF-κB luciferase activity (Fig. 4c). HSC activation exhibits many features, the most prevailing of which includes tissue contraction mediated by contractile myofibroblasts. Therefore, we measured HSC contractility by ET-1 and TGF-β1 stimulation, both well known to induce HSC contraction in the liver. We treated HSCs plated on collagen lattices with osthole (10 μg/ml) and ET-1 or TGF-β1 for 48 h. Osthole significantly reduced both ET-1- and TGF-β1-promoted HSC contraction (Fig. 4d). We substantiated our hypothesis that osthole was capable of interrupting HSC activation in HSC-T6 and LX-2 cells.Fig. 4

Bottom Line: Additionally, osthole reduced the expression of fibrosis-related genes significantly.Furthermore, osthole decreased TNF-α-triggered NF-κB activities significantly.In addition, osthole suppressed HSCs activation in vitro significantly.

View Article: PubMed Central - PubMed

Affiliation: Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, No. 155, Li-Nong Street, Sec. 2, Taipei, 11221, Taiwan. yaweiliu19850417@gmail.com.

ABSTRACT

Background: Hepatic fibrosis is a dynamic process which ultimately leads to cirrhosis in almost patients with chronic hepatic injury. However, progressive fibrosis is a reversible scarring response. Activation of hepatic stellate cells (HSCs) is the prevailing process during hepatic fibrosis. Osthole is an active component majorly contained in the fruit of Cnidium monnieri (L.) Cusson. This present study investigated the therapeutic effects of osthole on rat liver fibrosis and HSC activation.

Results: We established the thioacetamide (TAA)-model of Sprague-Dawley (SD) rats to induce hepatic fibrosis. Rats were divided into three groups: control, TAA, and TAA + osthole (10 mg/kg). In vivo, osthole significantly reduced liver injury by diminishing levels of plasma AST and ALT, improving histological architecture, decreasing collagen and α-SMA accumulation, and improving hepatic fibrosis scores. Additionally, osthole reduced the expression of fibrosis-related genes significantly. Osthole also suppressed the production of fibrosis-related cytokines and chemokines. Moreover, nuclear translocation of p65 was significantly suppressed in osthole-treated liver. Osthole also ameliorated TAA-induced injury through reducing cellular oxidation. Osthole showed inhibitory effects in inflammation-related genes and chemokines production as well. In vitro, we assessed osthole effects in activated HSCs (HSC-T6 and LX-2). Osthole attenuated TGF-β1-induced migration and invasion in HSCs. Furthermore, osthole decreased TNF-α-triggered NF-κB activities significantly. Besides, osthole alleviated TGF-β1- or ET-1-induced HSCs contractility.

Conclusions: Our study demonstrated that osthole improved TAA-caused liver injury, fibrogenesis and inflammation in rats. In addition, osthole suppressed HSCs activation in vitro significantly.

No MeSH data available.


Related in: MedlinePlus