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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

Anti-fibrotic effects of fluvastatin (Flu) on CDAA rats. (A) Representative result showed that Flu treatment reduced the protein expression of α-SMA in the liver tissues in CDAA rats at 4 and 8 weeks. **p < 0.01 vs. control rat; ##p < 0.01 vs. CDAA rat. (B) Quantitative PCR analyses for the expressions of Collagen I, α-SMA, and TIMP-1 transcripts in control rats, CDAA rats and CDAA rats receiving Flu (5 or 10 mg/kg/day). Densities of Collagen I, α-SMA, and TIMP-1 to G3PDH mRNA levels were analyzed by computerized densitometry and are expressed as the indicated ratios, respectively. Flu treatment attenuated the expressions of these pro-fibrogenic genes. The number of rats in each column was 8. *p <0.05 vs. control rat; #p < 0.05 vs. CDAA rat.
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Fig5: Anti-fibrotic effects of fluvastatin (Flu) on CDAA rats. (A) Representative result showed that Flu treatment reduced the protein expression of α-SMA in the liver tissues in CDAA rats at 4 and 8 weeks. **p < 0.01 vs. control rat; ##p < 0.01 vs. CDAA rat. (B) Quantitative PCR analyses for the expressions of Collagen I, α-SMA, and TIMP-1 transcripts in control rats, CDAA rats and CDAA rats receiving Flu (5 or 10 mg/kg/day). Densities of Collagen I, α-SMA, and TIMP-1 to G3PDH mRNA levels were analyzed by computerized densitometry and are expressed as the indicated ratios, respectively. Flu treatment attenuated the expressions of these pro-fibrogenic genes. The number of rats in each column was 8. *p <0.05 vs. control rat; #p < 0.05 vs. CDAA rat.

Mentions: Figure 5A shows that α-SMA protein expression was increased significantly in the livers of CDAA rats compared with that in the normal controls (p < 0.01). Treatment with either 5 mg/kg/day or 10 mg/kg/day of Flu for 4 and 8 weeks markedly reduced α-SMA protein expression (p < 0.01).Figure 5


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)

Anti-fibrotic effects of fluvastatin (Flu) on CDAA rats. (A) Representative result showed that Flu treatment reduced the protein expression of α-SMA in the liver tissues in CDAA rats at 4 and 8 weeks. **p < 0.01 vs. control rat; ##p < 0.01 vs. CDAA rat. (B) Quantitative PCR analyses for the expressions of Collagen I, α-SMA, and TIMP-1 transcripts in control rats, CDAA rats and CDAA rats receiving Flu (5 or 10 mg/kg/day). Densities of Collagen I, α-SMA, and TIMP-1 to G3PDH mRNA levels were analyzed by computerized densitometry and are expressed as the indicated ratios, respectively. Flu treatment attenuated the expressions of these pro-fibrogenic genes. The number of rats in each column was 8. *p <0.05 vs. control rat; #p < 0.05 vs. CDAA rat.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig5: Anti-fibrotic effects of fluvastatin (Flu) on CDAA rats. (A) Representative result showed that Flu treatment reduced the protein expression of α-SMA in the liver tissues in CDAA rats at 4 and 8 weeks. **p < 0.01 vs. control rat; ##p < 0.01 vs. CDAA rat. (B) Quantitative PCR analyses for the expressions of Collagen I, α-SMA, and TIMP-1 transcripts in control rats, CDAA rats and CDAA rats receiving Flu (5 or 10 mg/kg/day). Densities of Collagen I, α-SMA, and TIMP-1 to G3PDH mRNA levels were analyzed by computerized densitometry and are expressed as the indicated ratios, respectively. Flu treatment attenuated the expressions of these pro-fibrogenic genes. The number of rats in each column was 8. *p <0.05 vs. control rat; #p < 0.05 vs. CDAA rat.
Mentions: Figure 5A shows that α-SMA protein expression was increased significantly in the livers of CDAA rats compared with that in the normal controls (p < 0.01). Treatment with either 5 mg/kg/day or 10 mg/kg/day of Flu for 4 and 8 weeks markedly reduced α-SMA protein expression (p < 0.01).Figure 5

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