RESUMO
The aim of the present research was to study the therapeutic impacts of fluorofenidone (AKF-PD) on pig serum (PS)-induced liver fibrosis in rats and the complex molecular mechanisms of its effects on hepatic stellate cells (HSCs). Wistar rats were randomly divided into normal control, PS and PS/AKF-PD treatment groups. The activated human HSC LX-2 cell line was also treated with AKF-PD. The expression of collagen I and III, and α-smooth muscle actin (α-SMA) was determined by immunohistochemical staining and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Western blotting and/or RT-qPCR analyses were used to determine the expression of transforming growth factor (TGF)-ß1, α-SMA, collagen I, mothers against decapentaplegic homolog (Smad)-3, extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK). AKF-PD attenuated the degree of hepatic fibrosis and liver injury in vivo, which was associated with the downregulation of collagen I and III, and α-SMA at the mRNA and protein levels. In vitro, AKF-PD treatment significantly reduced the TGF-ß1-induced activation of HSCs, as determined by the reduction in collagen I and α-SMA protein expression. The TGF-ß1-induced upregulation of the phosphorylation of Smad 3, ERK1/2, p38 and JNK was attenuated by AKF-PD treatment. These findings suggested that AKF-PD attenuated the progression of hepatic fibrosis by suppressing HSCs activation via the TGF-ß1/Smad and MAPK signaling pathways, and therefore that AKF-PD may be suitable for use as a novel therapeutic agent against liver fibrosis.
RESUMO
Diabetic nephropathy (DN) remains the leading cause of end-stage renal disease (ESRD), a situation that is in part attributable to the lack of effective treatments. Fluorofenidone is a newly developed reagent with anti-fibrotic activity. While fluorofenidone was previously demonstrated to possess renoprotection from DN pathogenesis in db/db mice, the protective process and its underlying mechanisms have not been well studied. To characterize fluorofenidone-derived renoprotection, we treated 5, 8, or 12-week old db/db mice with daily doses of placebo, fluorofenidone, or losartan until 24 weeks of age; the time at which diabetes and DN were fully developed in placebo-treated animals. In comparison to db/db mice receiving fluorofenidone at 12-weeks old, those treated at 5-weeks had less glomerular expansion and better preservation of renal functions, judged by serum creatinine levels, albumin to creatinine ratio, and urinary albumin excretion (mg/24 hours). These benefits of early treatment were associated with significant reductions of multiple DN-promoting events, such as decreased expression of TGF-ß1 and the p22phox subunit of NADPH oxidase as well as downregulated activation of protein kinase C-zeta (ζ), ERK and AKT. This improvement in renoprotection following early interventions is not a unique property of DN pathogenesis, as losartan does not apparently offer the same benefits and is not more renoprotective than fluorofenidone. Additionally, the enhanced renoprotection provided by fluorofenidone did not affect the diabetic process, as it did not alter serum levels of glycated serum proteins, glucose, triglyceride or cholesterol. Collectively, we provide evidence that fluorofenidone offers improved renoprotection at early stages of DN pathogenesis.