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

Lee-Won Chong, Yi-Chao Hsu, Ting-Fang Lee, Yun Lin, Yung-Tsung Chiu, Kuo-Ching Yang, Jaw-Ching Wu, Yi-Tsau Huang, Lee-Won Chong, Yi-Chao Hsu, Ting-Fang Lee, Yun Lin, Yung-Tsung Chiu, Kuo-Ching Yang, Jaw-Ching Wu, Yi-Tsau Huang

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.

Figures

Figure 1
Figure 1
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).
Figure 2
Figure 2
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).
Figure 3
Figure 3
Effects of fluvastatin (Flu) on α-SMA protein expression and pro-fibrogenic gene transcripts in conditioned medium (CM) or TGF-β1-treated HSC-T6 cells. (A) HSC-T6 cells incubated in the conditioned medium (CM) from PA-treated PRHs showed significantly increased α-SMA protein expression, while α-SMA protein expression was not up-regulated in HSC-T6 cells treated directly with PA. TGF-β1 (1 ng/mL) was used as a positive control *p < 0.05 vs. control. (B) mRNA expressions of TIMP-1 and α-SMA were increased in HSC-T6 cells incubated with CM, whereas there were no significant differences in the expressions of pro-fibrogenic genes when HSC-T6 cells were treated with PA directly. TGF-β1 (1 ng/mL) was used as a positive control. **p < 0.01 vs. control. (C) Treatment with Flu significantly reduced CM- and TGF-β1-induced α-SMA protein expression. Besides, α-SMA protein expression was also suppressed in HSC-T6 cells incubated with Flu (5 μM) alone. *p < 0.05 vs. control; **p < 0.01 vs. control; ##p < 0.01 vs. CM treatment; &&p < 0.01 vs. TGF-β1 treatment. (D) Flu treatment attenuated CM-induced mRNA expressions of Col1, TGF-β1 and α-SMA, and TGF-β1-induced mRNA expressions of Col1 and α-SMA. **p < 0.01 vs. control; #p < 0.05 vs. CM treatment; ##p < 0.01 vs. CM treatment; &p < 0.05 vs. TGF-β1 treatment. (E & F) α-SMA protein and mRNA expressions were significantly decreased in HSC-T6 cells incubated with CM collected from PA-Flu-treated PRHs compared to those incubated with CM collected from PA-treated PRHs. Furthermore, α-SMA protein and mRNA expressions were also reduced in HSC-T6 cells when treated with CM collected from Flu-treated PRHs compared to those incubated with CM without Flu treatment. **p < 0.01 vs. control; #p < 0.05 vs. CM (PA 200 μM) treatment; &p < 0.05 vs. CM without Flu treatment. n=3 for all experiments.
Figure 4
Figure 4
Histological examination of liver sections. Representative liver sections were obtained from rats of normal controls (n = 4), rats fed with CDAA diet with vehicles, and rats fed with CDAA diet with Flu (5 mg/kg or 10 mg/kg) (n = 8 each). Sections were stained with (A) Hematoxylin-eosin or (B) Sirius red. Scale bar = 100 μm.
Figure 5
Figure 5
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.
Figure 6
Figure 6
Anti-inflammatory effects of fluvastatin (Flu) on CDAA rats. Quantitative PCR analyses for the expressions of IL6, iNOS and ICAM-1 transcripts in control rats, CDAA rats and CDAA rats receiving Flu (5 or 10 mg/kg/day). Densities of IL6, iNOS and ICAM-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-inflammatory genes. The number of rats in each column was 8. *p <0.05 and **p <0.01 vs. the control rat; #p < 0.05 and ##p < 0.01 vs. CDAA rat.

References

    1. Angulo P. Nonalcoholic fatty liver disease. N Engl J Med. 2002;346(16):1221–31. doi: 10.1056/NEJMra011775.
    1. Malaguarnera M, Di Rosa M, Nicoletti F, Malaguarnera L. Molecular mechanisms involved in NAFLD progression. J Mol Med (Berl) 2009;87(7):679–95. doi: 10.1007/s00109-009-0464-1.
    1. Leamy AK, Egnatchik RA, Young JD. Molecular mechanisms and the role of saturated fatty acids in the progression of non-alcoholic fatty liver disease. Prog Lipid Res. 2013;52(1):165–74. doi: 10.1016/j.plipres.2012.10.004.
    1. Chavez-Tapia NC, Rosso N, Tiribelli C. Effect of intracellular lipid accumulation in a new model of non-alcoholic fatty liver disease. BMC Gastroenterol. 2012;12:20. doi: 10.1186/1471-230X-12-20.
    1. Sumida Y, Niki E, Naito Y, Yoshikawa T. Involvement of free radicals and oxidative stress in NAFLD/NASH. Free Radic Res. 2013;47(11):869–80. doi: 10.3109/10715762.2013.837577.
    1. Paik YH, Kim J, Aoyama T, De Minicis S, Bataller R, Brenner DA. Role of NADPH oxidases in liver fibrosis. Antioxid Redox Signal. 2014;20(17):2854–72. doi: 10.1089/ars.2013.5619.
    1. Cao S, Zhang X, Edwards JP, Mosser DM. NF-kappaB1 (p50) homodimers differentially regulate pro- and anti-inflammatory cytokines in macrophages. J Biol Chem. 2006;281(36):26041–50. doi: 10.1074/jbc.M602222200.
    1. Pal S, Bhattacharjee A, Ali A, Mandal NC, Mandal SC, Pal M. Chronic inflammation and cancer: potential chemoprevention through nuclear factor kappa B and p53 mutual antagonism. J Inflamm (Lond) 2014;11:23. doi: 10.1186/1476-9255-11-23.
    1. Salamone F, Galvano F, Cappello F, Mangiameli A, Barbagallo I, Li Volti G. Silibinin modulates lipid homeostasis and inhibits nuclear factor kappa B activation in experimental nonalcoholic steatohepatitis. Transl Res. 2012;159(6):477–86. doi: 10.1016/j.trsl.2011.12.003.
    1. Baigent C, Keech A, Kearney PM, Blackwell L, Buck G, Pollicino C, et al. Efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins. Lancet. 2005;366(9493):1267–78. doi: 10.1016/S0140-6736(05)67394-1.
    1. Roglans N, Sanguino E, Peris C, Alegret M, Vazquez M, Adzet T, et al. Atorvastatin treatment induced peroxisome proliferator-activated receptor alpha expression and decreased plasma nonesterified fatty acids and liver triglyceride in fructose-fed rats. J Pharmacol Exp Ther. 2002;302(1):232–9. doi: 10.1124/jpet.302.1.232.
    1. Egawa T, Toda K, Nemoto Y, Ono M, Akisaw N, Saibara T, et al. Pitavastatin ameliorates severe hepatic steatosis in aromatase-deficient (Ar−/−) mice. Lipids. 2003;38(5):519–23. doi: 10.1007/s11745-003-1093-x.
    1. Gomez-Dominguez E, Gisbert JP, Moreno-Monteagudo JA, Garcia-Buey L, Moreno-Otero R. A pilot study of atorvastatin treatment in dyslipemid, non-alcoholic fatty liver patients. Aliment Pharmacol Ther. 2006;23(11):1643–7. doi: 10.1111/j.1365-2036.2006.02926.x.
    1. Hyogo H, Tazuma S, Arihiro K, Iwamoto K, Nabeshima Y, Inoue M, et al. Efficacy of atorvastatin for the treatment of nonalcoholic steatohepatitis with dyslipidemia. Metabolism. 2008;57(12):1711–8. doi: 10.1016/j.metabol.2008.07.030.
    1. Lin YL, Wu CH, Luo MH, Huang YJ, Wang CN, Shiao MS, et al. In vitro protective effects of salvianolic acid B on primary hepatocytes and hepatic stellate cells. J Ethnopharmacol. 2006;105(1–2):215–22. doi: 10.1016/j.jep.2005.10.021.
    1. Parkes JG, Templeton DM. Effects of retinol and hepatocyte-conditioned medium on cultured rat hepatic stellate cells. Ann Clin Lab Sci. 2003;33(3):295–305.
    1. Vogel S, Piantedosi R, Frank J, Lalazar A, Rockey DC, Friedman SL, et al. An immortalized rat liver stellate cell line (HSC-T6): a new cell model for the study of retinoid metabolism in vitro. J Lipid Res. 2000;41(6):882–93.
    1. Chong LW, Hsu YC, Chiu YT, Yang KC, Huang YT. Anti-fibrotic effects of thalidomide on hepatic stellate cells and dimethylnitrosamine-intoxicated rats. J Biomed Sci. 2006;13(3):403–18. doi: 10.1007/s11373-006-9079-5.
    1. Chong LW, Hsu YC, Chiu YT, Yang KC, Huang YT. Antifibrotic effects of triptolide on hepatic stellate cells and dimethylnitrosamine-intoxicated rats. Phytother Res. 2011;25(7):990–9. doi: 10.1002/ptr.3381.
    1. Wobser H, Dorn C, Weiss TS, Amann T, Bollheimer C, Buttner R, et al. Lipid accumulation in hepatocytes induces fibrogenic activation of hepatic stellate cells. Cell Res. 2009;19(8):996–1005. doi: 10.1038/cr.2009.73.
    1. Kirsch R, Clarkson V, Shephard EG, Marais DA, Jaffer MA, Woodburne VE, et al. Rodent nutritional model of non-alcoholic steatohepatitis: species, strain and sex difference studies. J Gastroenterol Hepatol. 2003;18(11):1272–82. doi: 10.1046/j.1440-1746.2003.03198.x.
    1. Takeuchi-Yorimoto A, Noto T, Yamada A, Miyamae Y, Oishi Y, Matsumoto M. Persistent fibrosis in the liver of choline-deficient and iron-supplemented l-amino acid-defined diet-induced nonalcoholic steatohepatitis rat due to continuing oxidative stress after choline supplementation. Toxicol Appl Pharmacol. 2013;268(3):264–77. doi: 10.1016/j.taap.2013.01.027.
    1. Tsai MK, Lin YL, Huang YT. Effects of salvianolic acids on oxidative stress and hepatic fibrosis in rats. Toxicol Appl Pharmacol. 2010;242(2):155–64. doi: 10.1016/j.taap.2009.10.002.
    1. Lopez-De Leon A, Rojkind M. A simple micromethod for collagen and total protein determination in formalin-fixed paraffin-embedded sections. J Histochem Cytochem. 1985;33(8):737–43. doi: 10.1177/33.8.2410480.
    1. Brunt EM. Pathology of nonalcoholic fatty liver disease. Nat Rev Gastroenterol Hepatol. 2010;7(4):195–203. doi: 10.1038/nrgastro.2010.21.
    1. Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005;41(6):1313–21. doi: 10.1002/hep.20701.
    1. De Minicis S, Bataller R, Brenner DA. NADPH oxidase in the liver: defensive, offensive, or fibrogenic? Gastroenterology. 2006;131(1):272–5. doi: 10.1053/j.gastro.2006.05.048.
    1. De Minicis S, Seki E, Paik YH, Osterreicher CH, Kodama Y, Kluwe J, et al. Role and cellular source of nicotinamide adenine dinucleotide phosphate oxidase in hepatic fibrosis. Hepatology. 2010;52(4):1420–30. doi: 10.1002/hep.23804.
    1. Aram G, Potter JJ, Liu X, Wang L, Torbenson MS, Mezey E. Deficiency of nicotinamide adenine dinucleotide phosphate, reduced form oxidase enhances hepatocellular injury but attenuates fibrosis after chronic carbon tetrachloride administration. Hepatology. 2009;49(3):911–9. doi: 10.1002/hep.22708.
    1. Bataller R, Schwabe RF, Choi YH, Yang L, Paik YH, Lindquist J, et al. NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis. J Clin Invest. 2003;112(9):1383–94. doi: 10.1172/JCI18212.
    1. Tobar N, Villar V, Santibanez JF. ROS-NFkappaB mediates TGF-beta1-induced expression of urokinase-type plasminogen activator, matrix metalloproteinase-9 and cell invasion. Mol Cell Biochem. 2010;340(1–2):195–202. doi: 10.1007/s11010-010-0418-5.
    1. Mantawy EM, Tadros MG, Awad AS, Hassan DA, El-Demerdash E. Insights antifibrotic mechanism of methyl palmitate: impact on nuclear factor kappa B and proinflammatory cytokines. Toxicol Appl Pharmacol. 2012;258(1):134–44. doi: 10.1016/j.taap.2011.10.016.
    1. Abe Y, Izumi T, Urabe A, Nagai M, Taniguchi I, Ikewaki K, et al. Pravastatin prevents myocardium from ischemia-induced fibrosis by protecting vascular endothelial cells exposed to oxidative stress. Cardiovasc Drugs Ther. 2006;20(4):273–80. doi: 10.1007/s10557-006-9525-7.
    1. Gianella A, Nobili E, Abbate M, Zoja C, Gelosa P, Mussoni L, et al. Rosuvastatin treatment prevents progressive kidney inflammation and fibrosis in stroke-prone rats. Am J Pathol. 2007;170(4):1165–77. doi: 10.2353/ajpath.2007.060882.
    1. Ikeuchi H, Kuroiwa T, Yamashita S, Hiramatsu N, Maeshima A, Kaneko Y, et al. Fluvastatin reduces renal fibroblast proliferation and production of type III collagen: therapeutic implications for tubulointerstitial fibrosis. Nephron Exp Nephrol. 2004;97(4):e115–22. doi: 10.1159/000079176.
    1. Mukai Y, Wang CY, Rikitake Y, Liao JK. Phosphatidylinositol 3-kinase/protein kinase Akt negatively regulates plasminogen activator inhibitor type 1 expression in vascular endothelial cells. Am J Physiol Heart Circ Physiol. 2007;292(4):H1937–42. doi: 10.1152/ajpheart.00868.2006.
    1. Wang W, Zhao C, Zhou J, Zhen Z, Wang Y, Shen C. Simvastatin ameliorates liver fibrosis via mediating nitric oxide synthase in rats with non-alcoholic steatohepatitis-related liver fibrosis. PLoS One. 2013;8(10):e76538. doi: 10.1371/journal.pone.0076538.
    1. Miyaki T, Nojiri S, Shinkai N, Kusakabe A, Matsuura K, Iio E, et al. Pitavastatin inhibits hepatic steatosis and fibrosis in non-alcoholic steatohepatitis model rats. Hepatol Res. 2011;41(4):375–385. doi: 10.1111/j.1872-034X.2010.00769.x.

Source: PubMed

Patrocinadores y Colaboradores

Condiciones médicas

Intervenciones de drogas

3
Suscribir