Fluorofenidone protects liver against inflammation and fibrosis by blocking the activation of NF-κB pathway.

Despite the increasing understanding of the pathophysiology of hepatic fibrosis, the therapies to combat it remain inadequate. Fluorofenidone (AKF-PD) is a novel pyridone agent able to ameliorate hepatic fibrosis in an experimental hepatic fibrosis model induced by dimethylnitrosamine. However, the underlying mechanism remains to be further elucidated. In light of the critical role of the NF-κB pathway in inflammation and hepatic fibrosis, together with the preliminary finding that AKF-PD decreases the release of proinflammatory cytokines in the endotoxemia and unilateral ureteral occlusion model, the aim of this study was to explore whether AKF-PD exerts an antifibrotic effect in hepatic fibrosis by inhibiting inflammation and suppressing the activation of the NF-κB pathway in vivo and in vitro. To test this possibility, the effect of AKF-PD on hepatic fibrosis models induced by both carbon tetrachloride (CCL4 ) and porcine serum (PS) was investigated. Our results showed that AKF-PD treatment ameliorated hepatic injury and fibrosis in both models. Furthermore, the administration of AKF-PD induced a robust anti-inflammatory reaction revealed by the downregulation of the proinflammatory cytokines as well as the suppression of the infiltration of inflammatory cells in the fibrotic liver. The analysis of the mechanism of action demonstrated that the attenuation of the production of proinflammatory cytokines and chemokines mediated by AKF-PD in vivo and in vitro were accompanied by the suppression in the activation of the NF-κB signaling pathway. In conclusion, AKF-PD might be considered as an antifibrotic agent attenuating hepatic inflammation and fibrosis potentially through the suppression of the NF-κB pathway.

Correction to: A novel role of glutathione S-transferase A3 in inhibiting hepatic stellate cell activation and rat hepatic fibrosis.

An amendment to this paper has been published and can be accessed via the original article.

A novel role of glutathione S-transferase A3 in inhibiting hepatic stellate cell activation and rat hepatic fibrosis.

BACKGROUND AND AIMS: Glutathione S-transferase A3 (GSTA3) is known as an antioxidative protease, however, the crucial role of GSTA3 in liver fibrosis remains unclear. As a recently we developed water-soluble pyridone agent with antifibrotic features, fluorofenidone (AKF-PD) can attenuate liver fibrosis, present studies were designed to explore the role of GSTA3 in liver fibrosis and its modulation by AKF-PD in vivo and in vitro. METHODS: Rats liver fibrosis models were induced by dimethylnitrosamine (DMN) or carbon tetrachloride (CCl4). The two activated hepatic stellate cells (HSCs) lines, rat CFSC-2G and human LX2 were treated with AKF-PD respectively. The lipid peroxidation byproduct malondialdehyde (MDA) in rat serum was determined by ELISA. The accumulation of reactive oxygen species (ROS) was measured by dichlorodihydrofluorescein fluorescence analysis. The expression of α-smooth muscle actin (α-SMA), fibronectin (FN), and phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase (JNK) and glycogen synthase kinase 3 beta (GSK-3ß) were detected by western blotting (WB). RESULTS: GSTA3 was substantially reduced in the experimental fibrotic livers and transdifferentiated HSCs. AKF-PD alleviated rat hepatic fibrosis and potently inhibited HSCs activation correlated with restoring GSTA3. Moreover, GSTA3 overexpression prevented HSCs activation and fibrogenesis, while GSTA3 knockdown enhanced HSCs activation and fibrogenesis resulted from increasing accumulation of ROS and subsequent amplified MAPK signaling and GSK-3ß phosphorylation. CONCLUSIONS: We demonstrated firstly that GSTA3 inhibited HSCs activation and liver fibrosis through suppression of the MAPK and GSK-3ß signaling pathways. GSTA3 may represent a promising target for potential therapeutic intervention in liver fibrotic diseases.

Fluorofenidone alleviates liver fibrosis by inhibiting hepatic stellate cell autophagy via the TGF-ß1/Smad pathway: implications for liver cancer.

Objectives: Liver fibrosis is a key stage in the progression of various chronic liver diseases to cirrhosis and liver cancer, but at present, there is no effective treatment. This study investigated the therapeutic effect of the new antifibrotic drug fluorofenidone (AKF-PD) on liver fibrosis and its related mechanism, providing implications for liver cancer. Materials and Methods: The effects of AKF-PD on hepatic stellate cell (HSC) autophagy and extracellular matrix (ECM) expression were assessed in a carbon tetrachloride (CCl4)-induced rat liver fibrosis model. In vitro, HSC-T6 cells were transfected with Smad2 and Smad3 overexpression plasmids and treated with AKF-PD. The viability and number of autophagosomes in HSC-T6 cells were examined. The protein expression levels of Beclin-1, LC3 and P62 were examined by Western blotting. The Cancer Genome Atlas (TCGA) database was used for comprehensively analyzing the prognostic values of SMAD2 and SMAD3 in liver cancer. The correlation between SMAD2, SMAD3, and autophagy-related scores in liver cancer was explored. The drug prediction of autophagy-related scores in liver cancer was explored. Results: AKF-PD attenuated liver injury and ECM deposition in the CCl4-induced liver fibrosis model. In vitro, the viability and number of autophagosomes in HSCs were reduced significantly by AKF-PD treatment. Meanwhile, the protein expression of FN, α-SMA, collagen III, Beclin-1 and LC3 was increased, and P62 was reduced by the overexpression of Smad2 and Smad3; however, AKF-PD reversed these effects. SMAD2 and SMAD3 were hazardous factors in liver cancer. SMAD2 and SMAD3 correlated with autophagy-related scores in liver cancer. Autophagy-related scores could predict drug response in liver cancer. Conclusions: AKF-PD alleviates liver fibrosis by inhibiting HSC autophagy via the transforming growth factor (TGF)-ß1/Smadpathway. Our study provided some implications about how liver fibrosis was connected with liver cancer by SMAD2/SMAD3 and autophagy.

Fluorofenidone offers improved renoprotection at early interventions during the course of diabetic nephropathy in db/db mice via multiple pathways.

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.