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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 NFκB p65 nuclear translocation, mRNA expression levels of pro-inflammatory genes in HepG2 cells and primary rat hepatocytes (PRHs). (A) Pre-treatment with Flu for 2 hr reduced the NFκB p65 nuclear translocation in PA-treated HepG2 cells and PRHs at 6 hr after treatment. *p < 0.05 vs. control; #p < 0.05 and ##p < 0.01 vs. PA treatment (n = 3). (B) Flu treatment inhibited the mRNA expressions of ICAM-1, IL-6 and TNF-α of PA-treated HepG2 cells while there were no significant differences in the expressions of ICAM-1, IL-6 and TNF-α between the control group and the group treated with Fluvastatin alone. **p < 0.01 vs. control; #p < 0.05 and ##p < 0.01 vs. PA treatment (n = 3). (C) Flu treatment decreased the mRNA expressions of ICAM-1, IL-6 and TNF-α of PA-treated PRHs, but no significant differences in the expressions of these markers between the control group and the group treated with Fluvastatin alone were noted. **p < 0.01 vs. control; #p < 0.05 and ##p < 0.01 vs. PA treatment (n = 3).
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Fig2: Effects of fluvastatin (Flu) on NFκB p65 nuclear translocation, mRNA expression levels of pro-inflammatory genes in HepG2 cells and primary rat hepatocytes (PRHs). (A) Pre-treatment with Flu for 2 hr reduced the NFκB p65 nuclear translocation in PA-treated HepG2 cells and PRHs at 6 hr after treatment. *p < 0.05 vs. control; #p < 0.05 and ##p < 0.01 vs. PA treatment (n = 3). (B) Flu treatment inhibited the mRNA expressions of ICAM-1, IL-6 and TNF-α of PA-treated HepG2 cells while there were no significant differences in the expressions of ICAM-1, IL-6 and TNF-α between the control group and the group treated with Fluvastatin alone. **p < 0.01 vs. control; #p < 0.05 and ##p < 0.01 vs. PA treatment (n = 3). (C) Flu treatment decreased the mRNA expressions of ICAM-1, IL-6 and TNF-α of PA-treated PRHs, but no significant differences in the expressions of these markers between the control group and the group treated with Fluvastatin alone were noted. **p < 0.01 vs. control; #p < 0.05 and ##p < 0.01 vs. PA treatment (n = 3).

Mentions: While PA (200 μM) stimulated NFκB p65 nuclear translocation in both HepG2 and PRHs (Figure 2A), Flu (1–20 μM) attenuated NFκB p65 nuclear translocation in both cell types (Figure 2A). In addition, Flu treatment inhibited the mRNA expression levels of pro-inflammatory gene transcripts (ICAM-1, IL-6, TNF-α) in both PA-treated HepG2 cells and PRHs (Figure 2B and C). Moreover, there were no significant differences in the expressions of pro-inflammatory gene transcripts between the control group and the group treated with Flu alone (Figure 2B and C).Figure 2


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 NFκB p65 nuclear translocation, mRNA expression levels of pro-inflammatory genes in HepG2 cells and primary rat hepatocytes (PRHs). (A) Pre-treatment with Flu for 2 hr reduced the NFκB p65 nuclear translocation in PA-treated HepG2 cells and PRHs at 6 hr after treatment. *p < 0.05 vs. control; #p < 0.05 and ##p < 0.01 vs. PA treatment (n = 3). (B) Flu treatment inhibited the mRNA expressions of ICAM-1, IL-6 and TNF-α of PA-treated HepG2 cells while there were no significant differences in the expressions of ICAM-1, IL-6 and TNF-α between the control group and the group treated with Fluvastatin alone. **p < 0.01 vs. control; #p < 0.05 and ##p < 0.01 vs. PA treatment (n = 3). (C) Flu treatment decreased the mRNA expressions of ICAM-1, IL-6 and TNF-α of PA-treated PRHs, but no significant differences in the expressions of these markers between the control group and the group treated with Fluvastatin alone were noted. **p < 0.01 vs. control; #p < 0.05 and ##p < 0.01 vs. PA treatment (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig2: Effects of fluvastatin (Flu) on NFκB p65 nuclear translocation, mRNA expression levels of pro-inflammatory genes in HepG2 cells and primary rat hepatocytes (PRHs). (A) Pre-treatment with Flu for 2 hr reduced the NFκB p65 nuclear translocation in PA-treated HepG2 cells and PRHs at 6 hr after treatment. *p < 0.05 vs. control; #p < 0.05 and ##p < 0.01 vs. PA treatment (n = 3). (B) Flu treatment inhibited the mRNA expressions of ICAM-1, IL-6 and TNF-α of PA-treated HepG2 cells while there were no significant differences in the expressions of ICAM-1, IL-6 and TNF-α between the control group and the group treated with Fluvastatin alone. **p < 0.01 vs. control; #p < 0.05 and ##p < 0.01 vs. PA treatment (n = 3). (C) Flu treatment decreased the mRNA expressions of ICAM-1, IL-6 and TNF-α of PA-treated PRHs, but no significant differences in the expressions of these markers between the control group and the group treated with Fluvastatin alone were noted. **p < 0.01 vs. control; #p < 0.05 and ##p < 0.01 vs. PA treatment (n = 3).
Mentions: While PA (200 μM) stimulated NFκB p65 nuclear translocation in both HepG2 and PRHs (Figure 2A), Flu (1–20 μM) attenuated NFκB p65 nuclear translocation in both cell types (Figure 2A). In addition, Flu treatment inhibited the mRNA expression levels of pro-inflammatory gene transcripts (ICAM-1, IL-6, TNF-α) in both PA-treated HepG2 cells and PRHs (Figure 2B and C). Moreover, there were no significant differences in the expressions of pro-inflammatory gene transcripts between the control group and the group treated with Flu alone (Figure 2B and C).Figure 2

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