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Fluvastatin attenuates hepatic steatosis-induced fibrogenesis in rats through inhibiting paracrine effect of hepatocyte on hepatic stellate cells.

Chong LW, Hsu YC, Lee TF, Lin Y, Chiu YT, Yang KC, Wu JC, Huang YT - BMC Gastroenterol (2015)

Bottom Line: Moreover, α-SMA protein expression was significantly decreased in HSC-T6 cultured with CM from PA-Flu-treated PRHs compared to those cultured with CM from PA-treated PRHs.Flu also reduced steatosis and fibrosis scores, α-SMA protein expression, mRNA expression of pro-inflammatory and pro-fibrogenic genes in livers of CDAA rats.We demonstrated PA-induced HSC activation through paracrine effect of hepatocyte in vitro that was significantly suppressed by pre-treating HSC with Flu.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Medicine, School of Medicine, National Yang Ming University, Taipei, Taiwan. leewonch@ms52.hinet.net.

ABSTRACT

Background: Non-alcoholic steatohepatitis (NASH) is associated with hepatic fibrogenesis. Despite well-known cholesterol-lowering action of statins, their mechanisms against NASH-mediated fibrogenesis remain unclear. This study aimed at investigating the in vitro and in vivo anti-fibrotic properties of fluvastatin (Flu).

Methods: Palmitate (PA)-induced changes in intracellular hydrogen peroxide levels in primary rat hepatocytes (PRHs) and human hepatoma cell line (HepG2) were quantified by dichlorofluorescein diacetate (DCF-DA) dye assay, whereas changes in expressions of NADPH oxidase gp91 (phox) subunit, α-smooth muscle actin (α-SMA), and NFκB p65 nuclear translocation were quantified with Western blotting. Quantitative real-time polymerase chain reaction (q-PCR) was used to investigate mRNA expressions of pro-inflammatory genes (ICAM-1, IL-6, TNF-α). Conditioned medium (CM) from PA-treated PRHs was applied to cultured rat hepatic stellate cell line, HSC-T6, with or without Flu-pretreatment for 2 h. Pro-fibrogenic gene expressions (COL1, TIMP-1, TGF-β1, α-SMA) and protein expression of α-SMA were analyzed. In vivo study using choline-deficient L-amino acid defined (CDAA) diet-induced rat NASH model was performed by randomly assigning Wistar rats (n = 28) to normal controls (n = 4), CDAA diet with vehicles, and CDAA diet with Flu (5 mg/kg or 10 mg/kg) (n = 8 each) through gavage for 4 or 8 weeks. Livers were harvested for histological, Western blot (α-SMA), and q-PCR analyses for expressions of pro-inflammatory (IL-6, iNOS, ICAM-1) and pro-fibrogenic (Col1, α-SMA, TIMP-1) genes.

Results: In vitro, Flu (1-20 μM) inhibited PA-induced free-radical production, gp91 (phox) expression, and NFκB p65 translocation in HepG2 and PRHs, while CM-induced α-SMA protein expression and pro-fibrogenic gene expressions in HSC-T6 were suppressed in Flu-pretreated cells compared to those without pretreatment. Moreover, α-SMA protein expression was significantly decreased in HSC-T6 cultured with CM from PA-Flu-treated PRHs compared to those cultured with CM from PA-treated PRHs. Flu also reduced steatosis and fibrosis scores, α-SMA protein expression, mRNA expression of pro-inflammatory and pro-fibrogenic genes in livers of CDAA rats.

Conclusions: We demonstrated PA-induced HSC activation through paracrine effect of hepatocyte in vitro that was significantly suppressed by pre-treating HSC with Flu. In vivo, Flu alleviated steatosis-induced HSC activation and hepatic fibrogenesis through mitigating inflammation and oxidative stress, suggesting possible therapeutic role of Flu against NASH.

No MeSH data available.


Related in: MedlinePlus

Effects of fluvastatin (Flu) on cell viability, ROS production and NADPH oxidase subunit gp91phoxexpression in HepG2 cells and primary rat hepatocytes (PRHs). (A) Effects of Flu on cell viability of HepG2 cells and PRHs at 24 hr after treatment (n = 3). (B) Flu significantly reduced the reactive oxygen species (ROS) production of PA-treated HepG2 cells and PRHs at 6 hr after treatments. *p < 0.05 and **p < 0.01 vs. control; #p < 0.05 and ##p < 0.01 vs. PA treatment (n = 3). (C) Flu significantly attenuated the ROS production of PA-treated HepG2 and PRHs at 12 hr after treatments. *p < 0.05 and **p < 0.01 vs. control; #p < 0.05 and ##p < 0.01 vs. PA treatment (n = 3). (D) Flu significantly decreased the expression of NADPH oxidase gp91phox in PA-treated HepG2 cells (left panel) and PRHs (right panel). **p < 0.01 vs. control; #p < 0.05 vs. PA treatment (n = 3).
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Fig1: Effects of fluvastatin (Flu) on cell viability, ROS production and NADPH oxidase subunit gp91phoxexpression in HepG2 cells and primary rat hepatocytes (PRHs). (A) Effects of Flu on cell viability of HepG2 cells and PRHs at 24 hr after treatment (n = 3). (B) Flu significantly reduced the reactive oxygen species (ROS) production of PA-treated HepG2 cells and PRHs at 6 hr after treatments. *p < 0.05 and **p < 0.01 vs. control; #p < 0.05 and ##p < 0.01 vs. PA treatment (n = 3). (C) Flu significantly attenuated the ROS production of PA-treated HepG2 and PRHs at 12 hr after treatments. *p < 0.05 and **p < 0.01 vs. control; #p < 0.05 and ##p < 0.01 vs. PA treatment (n = 3). (D) Flu significantly decreased the expression of NADPH oxidase gp91phox in PA-treated HepG2 cells (left panel) and PRHs (right panel). **p < 0.01 vs. control; #p < 0.05 vs. PA treatment (n = 3).

Mentions: To demonstrate the anti-fibrotic roles of Flu in steatosis-induced liver fibrosis, the beneficial effects of Flu on lipid-induced hepatocyte damage were first investigated. PRHs (isolated from Wistar rats) and HepG2 cells were cultured in the presence of PA, the most abundant free fatty acid in circulation. Firstly, we demonstrated that PA and Flu treatment did not alter the cell viability of PRHs and HepG2 cells after 24 hours of exposure (Figure 1A). PA treatment significantly enhanced ROS production in both HepG2 cells and PRHs at 6 hour and 12 hour detected by fluorescence DCF-DA dye (Figure 1B and C). PA (200 μM) significantly stimulated ROS production, and Flu (1–20 μM) concentration-dependently reduced PA-induced ROS production (Figure 1B and C), with higher concentrations achieving significant reduction in PRHs at 12 hour (Figure 1C). PA (200 μM) also increased gp91phox expression in HepG2 and PRHs (Figure 1D). By contrast, Flu (20 μM) reduced gp91phox expression in both cell types, indicating suppression of ROS generation after Flu treatment (Figure 1D).Figure 1


Fluvastatin attenuates hepatic steatosis-induced fibrogenesis in rats through inhibiting paracrine effect of hepatocyte on hepatic stellate cells.

Chong LW, Hsu YC, Lee TF, Lin Y, Chiu YT, Yang KC, Wu JC, Huang YT - BMC Gastroenterol (2015)

Effects of fluvastatin (Flu) on cell viability, ROS production and NADPH oxidase subunit gp91phoxexpression in HepG2 cells and primary rat hepatocytes (PRHs). (A) Effects of Flu on cell viability of HepG2 cells and PRHs at 24 hr after treatment (n = 3). (B) Flu significantly reduced the reactive oxygen species (ROS) production of PA-treated HepG2 cells and PRHs at 6 hr after treatments. *p < 0.05 and **p < 0.01 vs. control; #p < 0.05 and ##p < 0.01 vs. PA treatment (n = 3). (C) Flu significantly attenuated the ROS production of PA-treated HepG2 and PRHs at 12 hr after treatments. *p < 0.05 and **p < 0.01 vs. control; #p < 0.05 and ##p < 0.01 vs. PA treatment (n = 3). (D) Flu significantly decreased the expression of NADPH oxidase gp91phox in PA-treated HepG2 cells (left panel) and PRHs (right panel). **p < 0.01 vs. control; #p < 0.05 vs. PA treatment (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4336504&req=5

Fig1: Effects of fluvastatin (Flu) on cell viability, ROS production and NADPH oxidase subunit gp91phoxexpression in HepG2 cells and primary rat hepatocytes (PRHs). (A) Effects of Flu on cell viability of HepG2 cells and PRHs at 24 hr after treatment (n = 3). (B) Flu significantly reduced the reactive oxygen species (ROS) production of PA-treated HepG2 cells and PRHs at 6 hr after treatments. *p < 0.05 and **p < 0.01 vs. control; #p < 0.05 and ##p < 0.01 vs. PA treatment (n = 3). (C) Flu significantly attenuated the ROS production of PA-treated HepG2 and PRHs at 12 hr after treatments. *p < 0.05 and **p < 0.01 vs. control; #p < 0.05 and ##p < 0.01 vs. PA treatment (n = 3). (D) Flu significantly decreased the expression of NADPH oxidase gp91phox in PA-treated HepG2 cells (left panel) and PRHs (right panel). **p < 0.01 vs. control; #p < 0.05 vs. PA treatment (n = 3).
Mentions: To demonstrate the anti-fibrotic roles of Flu in steatosis-induced liver fibrosis, the beneficial effects of Flu on lipid-induced hepatocyte damage were first investigated. PRHs (isolated from Wistar rats) and HepG2 cells were cultured in the presence of PA, the most abundant free fatty acid in circulation. Firstly, we demonstrated that PA and Flu treatment did not alter the cell viability of PRHs and HepG2 cells after 24 hours of exposure (Figure 1A). PA treatment significantly enhanced ROS production in both HepG2 cells and PRHs at 6 hour and 12 hour detected by fluorescence DCF-DA dye (Figure 1B and C). PA (200 μM) significantly stimulated ROS production, and Flu (1–20 μM) concentration-dependently reduced PA-induced ROS production (Figure 1B and C), with higher concentrations achieving significant reduction in PRHs at 12 hour (Figure 1C). PA (200 μM) also increased gp91phox expression in HepG2 and PRHs (Figure 1D). By contrast, Flu (20 μM) reduced gp91phox expression in both cell types, indicating suppression of ROS generation after Flu treatment (Figure 1D).Figure 1

Bottom Line: Moreover, α-SMA protein expression was significantly decreased in HSC-T6 cultured with CM from PA-Flu-treated PRHs compared to those cultured with CM from PA-treated PRHs.Flu also reduced steatosis and fibrosis scores, α-SMA protein expression, mRNA expression of pro-inflammatory and pro-fibrogenic genes in livers of CDAA rats.We demonstrated PA-induced HSC activation through paracrine effect of hepatocyte in vitro that was significantly suppressed by pre-treating HSC with Flu.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Medicine, School of Medicine, National Yang Ming University, Taipei, Taiwan. leewonch@ms52.hinet.net.

ABSTRACT

Background: Non-alcoholic steatohepatitis (NASH) is associated with hepatic fibrogenesis. Despite well-known cholesterol-lowering action of statins, their mechanisms against NASH-mediated fibrogenesis remain unclear. This study aimed at investigating the in vitro and in vivo anti-fibrotic properties of fluvastatin (Flu).

Methods: Palmitate (PA)-induced changes in intracellular hydrogen peroxide levels in primary rat hepatocytes (PRHs) and human hepatoma cell line (HepG2) were quantified by dichlorofluorescein diacetate (DCF-DA) dye assay, whereas changes in expressions of NADPH oxidase gp91 (phox) subunit, α-smooth muscle actin (α-SMA), and NFκB p65 nuclear translocation were quantified with Western blotting. Quantitative real-time polymerase chain reaction (q-PCR) was used to investigate mRNA expressions of pro-inflammatory genes (ICAM-1, IL-6, TNF-α). Conditioned medium (CM) from PA-treated PRHs was applied to cultured rat hepatic stellate cell line, HSC-T6, with or without Flu-pretreatment for 2 h. Pro-fibrogenic gene expressions (COL1, TIMP-1, TGF-β1, α-SMA) and protein expression of α-SMA were analyzed. In vivo study using choline-deficient L-amino acid defined (CDAA) diet-induced rat NASH model was performed by randomly assigning Wistar rats (n = 28) to normal controls (n = 4), CDAA diet with vehicles, and CDAA diet with Flu (5 mg/kg or 10 mg/kg) (n = 8 each) through gavage for 4 or 8 weeks. Livers were harvested for histological, Western blot (α-SMA), and q-PCR analyses for expressions of pro-inflammatory (IL-6, iNOS, ICAM-1) and pro-fibrogenic (Col1, α-SMA, TIMP-1) genes.

Results: In vitro, Flu (1-20 μM) inhibited PA-induced free-radical production, gp91 (phox) expression, and NFκB p65 translocation in HepG2 and PRHs, while CM-induced α-SMA protein expression and pro-fibrogenic gene expressions in HSC-T6 were suppressed in Flu-pretreated cells compared to those without pretreatment. Moreover, α-SMA protein expression was significantly decreased in HSC-T6 cultured with CM from PA-Flu-treated PRHs compared to those cultured with CM from PA-treated PRHs. Flu also reduced steatosis and fibrosis scores, α-SMA protein expression, mRNA expression of pro-inflammatory and pro-fibrogenic genes in livers of CDAA rats.

Conclusions: We demonstrated PA-induced HSC activation through paracrine effect of hepatocyte in vitro that was significantly suppressed by pre-treating HSC with Flu. In vivo, Flu alleviated steatosis-induced HSC activation and hepatic fibrogenesis through mitigating inflammation and oxidative stress, suggesting possible therapeutic role of Flu against NASH.

No MeSH data available.


Related in: MedlinePlus