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Acute lung injury (ALI) is a severe disease that presents serious damage and excessive inflammation in lungs with high mortality without effective pharmacological therapy. Fluorofenidone (AKFPD) is a novel pyridone agent that has anti-fibrosis, anti-inflammation, and other pharmacological activities, while the effect of fluorofenidone on ALI is unclarified. Here, we elucidated the protective effects and underlying mechanism of fluorofenidone on lipopolysaccharide (LPS)-induced ALI. In this study, fluorofenidone alleviated lung tissue structure injury and reduced mortality, decreased the pulmonary inflammatory cell accumulation and level of inflammatory cytokines IL-1β, IL-6, and TNF-α in the bronchoalveolar lavage fluid, and attenuated pulmonary apoptosis in LPS-induced ALI mice. Moreover, fluorofenidone could block LPS-activated phosphorylation of ERK, JNK, and P38 and further inhibited the phosphorylation of IκB and P65. These results suggested that fluorofenidone can significantly contrast LPS-induced ALI through suppressing the activation of the MAPK/NF-κB signaling pathway, which indicates that fluorofenidone could be considered as a novel therapeutic candidate for ALI.

Purpose: The present study aimed to investigate the inhibitory effect of fluorofenidone against transforming growth factor β2-induced proliferation and epithelial-mesenchymal transition in human lens epithelial cell line FHL 124 and its potential mechanism. Methods: We evaluated the effect of fluorofenidone on proliferation and epithelial-mesenchymal transition of human lens epithelial cell line FHL 124 in vitro. After treatment with 0, 0.1, 0.2, 0.4, 0.6, and 1.0 mg/mL fluorofenidone, cell proliferation was measured via MTT assay. Cell viability was evaluated by lactate dehydrogenase activity from damaged cells. FHL 124 cells were treated with different transforming growth factor β2 concentrations (0-10 ng/mL) for 24 h and the expression of CTGF, α-SMA, COL-I, E-cadherin, and Fn were detected via quantitative polymerase chain reaction and Western blot analysis. After treatment with 0, 0.2, and 0.4 mg/ml fluorofenidone, the expressions of transforming growth factor β2 and SMADs were detected with real-time polymerase chain reaction and Western blot analysis. Expressions of CTGF, α-SMA, COL-I, and Fn were analyzed by immunocytochemistry assay. Results: The viability of FHL 124 cells was not inhibited when the fluorofenidone concentration was ≤0.4 mg/mL after the 24h treatment. Cytotoxicity was not detected via lactate dehydrogenase assay after the 24h and 36h treatment with 0.2 and 0.4 mg/mL fluorofenidone. Transforming growth factor β2 increased mRNA and protein expression of CTGF, α-SMA, COL-I, and Fn. However, fluorofenidone significantly suppressed expression of SMADs, CTGF, α-SMA, COL-I, and Fn in the absence or presence of transforming growth factor β2 stimulation. Conclusions: Fluorofenidone significantly inhibited expression of SMADs, CTGF, α-SMA, COL-I, and Fn in FHL 124 cells. Due to noncompliance in infants, fluorofenidone may become a novel therapeutic drug against posterior capsular opacification in infants.

Cisplatin (CP) is one of the most effective chemotherapeutics in the treatment of human cancers. However, the beneficial effects of CP are limited by the toxic effects, especially nephrotoxicity. Fluorofenidone (AKFPD) is a promising multifunctional antifibrosis pyridinone drug discovered by our group. But there is no evidence of its protective effects against acute kidney injury (AKI). Therefore, we investigated the protective effects of AKFPD on CP-induced AKI

Background/Aims: Cyclosporine A (CsA) is an immunosuppressant drug that is used during organ transplants. However, its utility is limited by its nephrotoxic potential. This study aimed to investigate whether fluorofenidone (AKF-PD) could provide protection against CsA-induced nephrotoxicity. Methods: Eighty-five male Sprague-Dawley rats were divided into 5 groups: drug solvent, CsA, CsA with AKF-PD (250, 500 mg/kg/day), and CsA with pirfenidone (PFD, 250 mg/kg/day). Tubulointerstitial injury index, extracellular matrix (ECM) deposition, expression of type I and IV collagen, transforming growth factor (TGF)-β1, platelet-derived growth factor (PDGF), Fas ligand (FASL), cleaved-caspase-3, cleaved-poly(ADP-ribose) polymerase (PARP)-1, and the number of transferase-mediated nick end-labeling (TUNEL)-positive renal tubule cells were determined. In addition, levels of TGF-β1, FASL, cleaved-caspase-3, cleaved-PARP-1, and number of annexin V-positive cells were determined in rat proximal tubular epithelial cells (NRK-52E) treated with CsA (20 μmol/L), AKF-PD (400 μg/mL), PFD (400 μg/mL), and GW788388 (5 μmol/L). Results: AKF-PD (250, 500 mg/kg/day) significantly reduced tubulointerstitial injury, ECM deposition, expression of type I and IV collagen, TGF-β1, PDGF, FASL, cleaved-caspase-3, cleaved-PARP-1, and number of TUNEL-positive renal tubule cells in the CsA-treated kidneys. In addition, AKF-PD (400 μg/mL) significantly decreased TGF-β1, FASL, cleaved-caspase-3, and PARP-1 expression in NRK-52E cells and further reduced the number of annexin V-positive cells. Conclusion: AKF-PD protect kidney from fibrosis and apoptosis in CsA-induced kidney injury.

Paraquat (PQ) is a widely used herbicide that is severely toxic to humans and animals. Pulmonary fibrosis is a disorder that can result from PQ poisoning. Fluorofenidone (AKF‑PD) is a novel small molecule pyridone drug with a widespread and clear anti‑organ fibrosis effect; however, its mechanism of action on PQ poisoning‑induced pulmonary fibrosis is not clear. The purpose of the present study was to investigate the protective effect and underlying mechanism of AKF‑PD on PQ poisoning‑induced pulmonary fibrosis. Human alveolar epithelial cells (HPAEpiC) and Sprague‑Dawley rats were treated with AKF‑PD in the presence or absence of PQ. Hematoxylin‑eosin and Masson staining were used to observe the morphological changes in lung tissue. Cell Counting Kit‑8 and lactate dehydrogenase assays were used to evaluate the viability of HPAEpiC cells. ELISA was used to detect inflammatory factors and the collagen content. Finally, the effects of AKF‑PD on pulmonary fibrosis, as well as the underlying mechanisms, were evaluated via western blotting, reverse transcription‑quantitative PCR and immunofluorescence analysis. AKF‑PD effectively alleviated PQ‑induced pulmonary fibrosis and reduced the expression of oxidative stress and inflammatory factors. Moreover, AKF‑PD treatment effectively inhibited the PI3K/Akt/mTOR signaling pathway and upregulated autophagy. Overall, these findings suggested that AKF‑PD can alleviate PQ‑induced inflammation and pulmonary fibrosis by inhibiting the PI3K/Akt/mTOR signaling pathway and by upregulating autophagy.

Cell death and sterile inflammation are major mechanisms of renal fibrosis, which eventually develop into end-stage renal disease. “Necroptosis” is a type of caspase-independent regulated cell death, and sterile inflammatory response caused by tissue injury is strongly related to necrosis. Fluorofenidone (AKF-PD) is a novel compound shown to ameliorate renal fibrosis and associated inflammation. We investigated whether AKF-PD could alleviate renal fibrosis by inhibiting necroptosis. Unilateral ureteral obstruction (UUO) was used to induce renal tubulointerstitial fibrosis in C57BL/6J mice. AKF-PD (500 mg/kg) or necrostatin-1 (Nec-1; 1.65 mg/kg) was administered simultaneously for 3 and 7 days. Obstructed kidneys and serum were harvested after euthanasia. AKF-PD and Nec-1 ameliorated renal tubular damage, inflammatory-cell infiltration, and collagen deposition, and the expression of proinflammatory factors (interlukin-1β, tumor necrosis factor [TNF]-α) and chemokines (monocyte chemoattractant protein-1) decreased. AKF-PD or Nec-1 treatment protected renal tubular epithelial cells from necrosis and reduced the release of lactate dehydrogenase in serum. Simultaneously, production of receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like protein (MLKL) was also reduced 3 and 7 days after UUO. AKF-PD and Nec-1 significantly decreased the percentage of cell necrosis, inhibiting the phosphorylation of MLKL and RIPK3 in TNF-α- and Z-VAD–stimulated human proximal tubular epithelial (HK-2) cells. In conclusion, AKF-PD and Nec-1 have effective anti-inflammatory and antifibrotic activity in UUO-induced renal tubulointerstitial fibrosis, potentially mediated by the RIPK3/MLKL pathway.

Aim We explored whether Fluorofenidone reduced interleukin-1β (IL-1β) production by interacting with NLRP3 inflammasome in unilateral ureteral obstruction (UUO). Methods Ureteral obstruction rats were treated with Fluorofenidone (500 mg/kg per day) for 3, 7 days. Morphologic analysis and leukocytes infiltration were assessed in ligated kidneys. Furthermore, plasmids of NLRP3, ASC, pro-Caspase-1, pro-IL-1β were co-transfected into 293 T cells, and then treated with Fluorofenidone (2 mM). The expression of NLRP3, ASC, pro-caspase-1, cleavage caspase-1, pro-IL-1β and cleavage IL-1β were measured by Western blot or real-time PCR in vivo and in vitro. Moreover the interaction of NLRP3 inflammasome-assembly was detected by co-immunoprecipitation and confocal immunofluorescence. Results Fluorofenidone treatment significantly attenuated renal fibrosis and leukocytes infiltration in UUO model. Fluorofenidone had no effect on the expression of pro-IL-1β. Interestingly, Fluorofenidone inhibited the activation of NLRP3 inflammasome, downregulated Caspase-1 levels and thereby decreased the cleavage of pro-IL-1β into IL-1β in vivo and in vitro. Fluorofenidone treatment distinctively weakened the interaction between NLRP3 and ASC, as well as ASC and pro-Caspase-1 in vivo. However, Fluorofenidone treatment only significantly weakened the interaction between ASC and pro-Caspase-1 in co-transfected 293 T cells. Conclusion Fluorofenidone serves as a novel anti-inflammatory agent that attenuates IL-1β production in UUO model by interacting with NLRP3 inflammasome.

Background: Lung carcinoma is the leading cause of malignant tumor related mortality in China in recent decades, and the development of new and effective therapies for patients with advanced lung carcinoma is needed. We recently found that fluorofenidone (FD), a newly developed pyridine compound, reduced the activation of Stat3 (Signal transducer and activator of transcription 3) in fibroblasts. Stat3 plays a crucial role in the development of lung cancer and may represent a new therapeutic target. In this study, we examined the effect of FD on human lung adenocarcinoma cells in vivo and in vitro. Methods: The effect of FD on the growth of lung cancer cells was measured with a CCK-8 assay, colony formation assay and xenograft tumor model. A flow cytometry analysis was performed to study cell cycle arrest and apoptosis. Western blotting and immunohistochemistry were used to observe the expression of Stat3. Changes in the expression of RNA induced by FD were assessed using gene chip and real-time RT-PCR assays. Results: In vitro, FD inhibited the growth of lung adenocarcinoma A549 and SPC-A1 cells in a dose-dependent manner. After treatment with FD, the A549 and SPC-A1 cells were arrested in the G1 phase, and apoptosis was induced. In vivo, this compound significantly inhibited the growth of tumors that were subcutaneously implanted in mice. Moreover, FD decreased Stat3 activity in lung cancer cells and xenograft tumor tissue, and microarray chip results showed that FD altered the gene expression profile of lung cancer cells. Specifically, NUPR1, which plays a significant role in cancer development, was down-regulated by FD in lung cancer cells. Conclusion: Our study supports the clinical evaluation of FD as a potential lung adenocarcinoma therapy.

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-3β) 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-3β phosphorylation. Conclusions We demonstrated firstly that GSTA3 inhibited HSCs activation and liver fibrosis through suppression of the MAPK and GSK-3β signaling pathways. GSTA3 may represent a promising target for potential therapeutic intervention in liver fibrotic diseases.

This study aimed to investigate the mechanism of fluorofenidone (AKF-PD) in treating renal interstitial fibrosis in rats with unilateral urinary obstruction (UUO). Thirty-two male Sprague-Dawley rats were randomly divided into sham, UUO, UUO + enalapril, and UUO + AKF-PD groups. All rats, except sham, underwent left urethral obstruction surgery to establish the animal model. Rats were sacrificed 14 days after surgery, and serum was collected for renal function examination. Kidneys were collected to observe pathological changes. Immunohistochemistry was performed to assess collagen I (Col I) protein expression, and terminal deoxynucleotidyl transferase-mediated nick end-labeling staining to observe the apoptosis of renal tubular epithelial cells. The expression of Fas-associated death domain (FADD), apoptotic protease activating factor-1 (Apaf-1), and C/EBP homologous protein (CHOP) proteins was evaluated by immunohistochemistry and western blot analysis. AKF-PD showed no significant effect on renal function in UUO rats. The pathological changes were alleviated significantly after enalapril or AKF-PD treatment, but with no significant differences between the two groups. Col I protein was overexpressed in the UUO group, which was inhibited by both enalapril and AKF-PD. The number of apoptotic renal tubular epithelial cells was much higher in the UUO group, and AKF-PD significantly inhibited epithelial cells apoptosis. The expression of FADD, Apaf-1, and CHOP proteins was significantly upregulated in the UUO group and downregulated by enalapril and AKF-PD. In conclusion, AKF-PD improved renal interstitial fibrosis by inhibiting apoptosis of renal tubular epithelial cells in rats with UUO.

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)-β1, 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-β1-induced activation of HSCs, as determined by the reduction in collagen I and α-SMA protein expression. The TGF-β1-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-β1/Smad and MAPK signaling pathways, and therefore that AKF-PD may be suitable for use as a novel therapeutic agent against liver fibrosis.

Interleukin-1 beta (IL-1β) is a potent pro-inflammatory and pro-fibrotic cytokine that plays an important role in renal fibrosis. Fluorofenidone (AKF-PD) is a novel pyridone agent that exerts a strong renal anti-fibrotic effect. We previously found that administration of AKF-PD could significantly attenuate IL-1β production in vitro and in vivo. However, the underlying mechanism is not fully understood. Here we show that AKF-PD has no effect on the expression of pro-IL-1β in activated mouse macrophages in vitro. Instead, AKF-PD inhibits the inflammasome, lowering caspase-1 levels and thereby decreasing cleavage of pro-IL-1β into IL-1β. AKF-PD was found to block inflammasome activity induced by various signals, including ATP, alum crystals, and Salmonella typhimurium. These results provide a novel mechanistic insight into how AKF-PD exerts its anti-inflammatory and anti-fibrotic activities, and suggest that AKF-PD might block IL-1β production via suppression of inflammasomes in renal fibrosis. In addition, the results suggest that AKF-PD may be of therapeutic potential in other inflammasome-related diseases.

Lung-targeting fluorofenidone (AKF) loaded PLGA microspheres (AKF-MS) for the treatment of paraquat (PQ)-induced acute lung injury in rats, were constructed by a solvent evaporation method. The microspheres' morphology, size distribution, drug loading ratio, encapsulation efficiency, in vitro release characteristics and tissue distributions in rats were systematically studied. Scanning electron microscopy shows the microspheres are spherical and well dispersed. The average particle size is 18.1 μm with 90% of the microspheres being in the range of 7 to 30 μm. The encapsulation efficiency (EE%) and drug loading ratio (DL%) are 80.2 ± 2.5% and 8.2 ± 1.9%, respectively. The in vitro drug release behavior of AKF-MS follows the Korsmeyer–Peppas model: Q = 11.141t0.292 (R2 = 0.9797). The tissue distribution shows that the drug concentration in lung tissue for the AKF-MS/18.1 μm suspension is significantly higher than that for the AKF solution and AKF-MS/3.9 μm, and the drug-targeting index for lung is 6.4 and 4.6-fold higher than that of AKF solution and AKF-MS/3.9 μm, respectively. In addition, AKF-MS/18.1 μm significantly reduced the circulating levels of TNF-α and IL-1β. Histopathological studies confirm that the AKF-MS treatment significantly reduced edema and neutrophil infiltration, as well as the lung interval damage. Taken together, the results of the present study demonstrate that AKF-MS/18.1 μm improved the treatment efficacy of AKF against PQ-induced acute lung injury, compared to other forms of AKF (AKF solution and AKF-MS/3.9 μm).

Idiopathic pulmonary fibrosis is a progressive, fatal lung disease with poor survival. The advances made in deciphering this disease have led to the approval of different antifibrotic molecules, such as pirfenidone and nintedanib. An increasing number of studies with particles (liposomes, nanoparticles [NPs], microspheres, nanopolymersomes, and nanoliposomes) modified with different functional groups have demonstrated improvement in lung-targeted drug delivery. In the present study, we prepared, characterized, and evaluated spermidine (Spd)-modified poly(lactic-co-glycolic acid) (PLGA) NPs as carriers for fluorofenidone (AKF) to improve the antifibrotic efficacy of this drug in the lung. Spd-AKF-PLGA NPs were prepared and functionalized by modified solvent evaporation with Spd and polyethylene glycol (PEG)-PLGA groups. The size of Spd-AKF-PLGA NPs was 172.5±4.3 nm. AKF release from NPs was shown to fit the Higuchi model. A549 cellular uptake of an Spd–coumarin (Cou)-6-PLGA NP group was found to be almost twice as high as that of the Cou-6-PLGA NP group. Free Spd and difluoromethylornithine (DFMO) were preincubated in A549 cells to prove uptake of Spd-Cou-6-PLGA NPs via a polyamine-transport system. As a result, the uptake of Spd-Cou-6-PLGA NPs significantly decreased with increased Spd concentrations in incubation. At higher Spd concentrations of 50 and 500 µM, uptake of Spd-Cou-6-PLGA NPs reduced 0.34- and 0.49-fold from that without Spd pretreatment. After pretreatment with DFMO for 36 hours, cellular uptake of Spd-Cou-6-PLGA NPs reached 1.26-fold compared to the untreated DFMO group. In a biodistribution study, the drug-targeting index of Spd-AKF-PLGA NPs in the lung was 3.62- and 4.66-fold that of AKF-PLGA NPs and AKF solution, respectively. This suggested that Spd-AKF-PLGA NPs accumulated effectively in the lung. Lung-histopathology changes and collagen deposition were observed by H&E staining and Masson staining in an efficacy study. In the Spd-AKF-PLGA NP group, damage was further improved compared to the AKF-PLGA NP group and AKF-solution group. The results indicated that Spd-AKF-PLGA NPs are able to be effective nanocarriers for anti–pulmonary fibrosis therapy.

The aim of this study was to investigate the protective effect of fluorofenidone (5‐methyl‐1‐[3‐fluorophenyl]‐2‐[1H]‐pyridone, AKF‐PD) on ultraviolet (UV)‐A‐induced senescence in human dermal fibroblasts (HDF) and examine the mechanisms involved. HDF were treated with AKF‐PD. Senescence‐associated (SA)‐β‐galactosidase level, cell viability and expression of p16 were evaluated. In addition, UV‐A‐irradiated HDF were treated with AKF‐PD, rapamycin and MHY1485; SA‐β‐galactosidase staining, 3‐(4 5‐dimethylthiazol‐2‐yl)‐2 5‐diphenyltetrazolium bromide assay and western blot for SIRT1 were performed; and phosphorylated mammalian target of rapamycin (p‐mTOR) expression and reactive oxygen species (ROS) levels were measured. Intracellular ROS was detected by the 2′,7′‐dichlorofluroescein diacetate probe. Our results showed that AKF‐PD substantially attenuated the changes of p16 expression, SA‐β‐galactosidase staining and cellular proliferation induced by UV‐A irradiation in HDF. AKF‐PD rescued the increased mTOR phosphorylation and reduced SIRT1 expression induced by UV‐A irradiation in HDF. AKF‐PD and rapamycin together had a synergistic effect on p‐mTOR reduction and SIRT1 increase. mTOR activator MHY1485 partly blocked the above effects. Moreover, intracellular ROS level induced by UV‐A irradiation could partly decrease by AKF‐PD, and MHY1485 could reduce this effect. Our results indicated that AKF‐PD could alleviate HDF senescence induced by UV‐A‐irradiation by inhibiting the p‐mTOR and increasing SIRT1. Moreover, AKF‐PD may be a potential treatment material for skin.

Interleukin (IL)‐1β plays an important role in the pathogenesis of idiopathic pulmonary fibrosis. The production of IL‐1β is dependent upon caspase‐1‐containing multiprotein complexes called inflammasomes and IL‐1R1/MyD88/NF‐κB pathway. In this study, we explored whether a potential anti‐fibrotic agent fluorofenidone (FD) exerts its anti‐inflammatory and anti‐fibrotic effects through suppressing activation of NACHT, LRR and PYD domains‐containing protein 3 (NALP3) inflammasome and the IL‐1β/IL‐1R1/MyD88/NF‐κB pathway in vivo and in vitro. Male C57BL/6J mice were intratracheally injected with Bleomycin (BLM) or saline. Fluorofenidone was administered throughout the course of the experiment. Lung tissue sections were stained with haemotoxylin and eosin and Masson's trichrome. Cytokines were measured by ELISA, and α‐smooth muscle actin (α‐SMA), fibronectin, collagen I, caspase‐1, IL‐1R1, MyD88 were measured by Western blot and/or RT‐PCR. The human actue monocytic leukaemia cell line (THP‐1) were incubated with monosodium urate (MSU), with or without FD pre‐treatment. The expression of caspase‐1, IL‐1β, NALP3, apoptosis‐associated speck‐like protein containing (ASC) and pro‐caspase‐1 were measured by Western blot, the reactive oxygen species (ROS) generation was detected using the Flow Cytometry, and the interaction of NALP3 inflammasome‐associated molecules were measured by Co‐immunoprecipitation. RLE‐6TN (rat lung epithelial‐T‐antigen negative) cells were incubated with IL‐1β, with or without FD pre‐treatment. The expression of nuclear protein p65 was measured by Western blot. Results showed that FD markedly reduced the expressions of IL‐1β, IL‐6, monocyte chemotactic protein‐1 (MCP‐1), myeloperoxidase (MPO), α‐SMA, fibronectin, collagen I, caspase‐1, IL‐1R1 and MyD88 in mice lung tissues. And FD inhibited MSU‐induced the accumulation of ROS, blocked the interaction of NALP3 inflammasome‐associated molecules, decreased the level of caspase‐1 and IL‐1β in THP‐1 cells. Besides, FD inhibited IL‐1β‐induced the expression of nuclear protein p65. This study demonstrated that FD, attenuates BLM‐induced pulmonary inflammation and fibrosis in mice via inhibiting the activation of NALP3 inflammasome and the IL‐1β/IL‐1R1/MyD88/ NF‐κB pathway.

Background/Aims: Fluorofenidone [1-(3-fluorophenyl)-5-methyl-2-(1H)-pyridone, AKF-PD], a novel pyridone agent, showed potent antifibrotic properties. The aim of the present study was to investigate the effects of AKF-PD on diabetic nephropathy and kidney fibrosis, and to obtain an insight into its mechanisms of action. Methods: We administered AKF-PD to diabetic db/db mice for 12 weeks. Moreover, we performed in vitro cultures using murine mesangial cells exposed to high ambient glucose concentrations. Results: AKF-PD reduced renal hypertrophy, mesangial matrix expansion and albuminuria in the db/db mice. The upregulated expression of α1(I)- and α1(IV)-collagen and fibronectin mRNAs, transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA), and tissue inhibitors of metalloproteinase 1 (TIMP-1) mRNAs and proteins was inhibited by AKF-PD treatment in the renal cortex of db/db mice. The maximal effective dose of AKF-PD was about 500 mg/kg body weight. AKF-PD inhibited the upregulated expression of α1(I)- and α1(IV)-collagens, TGF-β1, TIMP-1 and α-SMA induced by high glucose concentrations in cultured mesangial cells. Conclusions: Our data indicate that AKF-PD diminishes the abnormal accumulation of mesangial matrix through the inhibition of upregulated expression of TGF-β target genes in kidneys of db/db mice, resulting in attenuation of renal fibrosis and amelioration of renal dysfunction despite persistent hyperglycemia. Therefore, AKF-PD, a potent antifibrotic agent, holds great promise in the treatment of diabetic nephropathy.

Background/Aims: We evaluated the therapeutic effects of fluorofenidone (AKF-PD), a novel pyridone agent, targeting oxidative stress and fibrosis in obstructive nephropathy. Methods: AKF-PD was used to treat renal interstitial fibrosis in unilateral ureteral obstruction (UUO) obstructive nephropathy in rats. The expression of NOX2 (gp91phox), fibronectin and extracellular signal regulated kinase (ERK) were detected by western blot. A level of Malondialdehyde (MDA), an oxidative stress marker, was measured by ELISA. In addition, ROS and the expressions of NOX2, collagen I (a1), fibronectin and p-ERK were measured in angiotensin (Ang) II-stimulated rat proximal tubular epithelial cells (NRK-52E) in culture. Results: In NRK-52E cells, AKF-PD reduced AngII induced expressions of ROS, NOX2, fibronectin, collagen I (a1) and p-ERK. In UUO kidney cortex, AKF-PD attenuated the degree of renal interstitial fibrosis, which was associated with reduced the expressions of collagen I (a1) and fibronectin. Furthermore, AKF-PD downregulated the expressions of NOX2, MDA and p-ERK. Conclusion: AKF-PD treatment inhibits the progression of renal interstitial fibrosis by suppressing oxidative stress and ERK/MAPK signaling pathway.

Pyridone compounds, such as pirfenidone (PFD) and fluorofenidone (AKF-PD), are multi-target anti-fibrotic agents. Using PFD and AKF-PD as the leading compounds, two series of novel (5-substituent)-2(1H)-pyridone compounds were synthesized with the purpose of maintaining multi-targeting property and overcoming the drawbacks of fast metabolism. These derivatives demonstrated good proliferation inhibiting activity against NIH3T3 cells by MTT assay with AKF-PD as the positive control. Compound 5b exhibited a high potent of anti-fibrosis with a IC(50) of 0.08 mmol/L about 34 times of AKF-PD. The SAR of pyridone derivatives as anti-fibrosis agents was also discussed.

Fluorofenidone (AKF-PD) is a novel pyridone agent. The purpose of this study is to investigate the inhibitory effects of AKF-PD on liver fibrosis in rats and the involved molecular mechanism related to hepatic stellate cells (HSCs). Rats treated with dimethylnitrosamine or CCl4were randomly divided into normal, model, AKF-PD treatment, and pirfenidone (PFD) treatment groups. The isolated primary rat HSCs were treated with AKF-PD and PFD respectively. Cell proliferation and cell cycle distribution were analyzed by bromodeoxyuridine and flow cytometry, respectively. The expression of collagen I and α-smooth muscle actin (α-SMA) were determined by Western blot, immunohistochemical staining, and real-time RT-PCR. The expression of cyclin D1, cyclin E, and p27kip1and phosphorylation of MEK, ERK, Akt, and 70-kDa ribosomal S6 kinase (p70S6K) were detected by Western blot. AKF-PD significantly inhibited PDGF-BB-induced HSC proliferation and activation by attenuating the expression of collagen I and α-SMA, causing G0/G1 phase cell cycle arrest, reducing expression of cyclin D1and cyclin E, and promoting expression of p27kip1. AKF-PD also downregulated PDGF-BB-induced MEK, ERK, Akt, and p70S6K phosphorylation in HSCs. In rat liver fibrosis, AKF-PD alleviated hepatic fibrosis by decreasing necroinflammatory score and semiquantitative score, and reducing expression of collagen I and α-SMA. AKF-PD attenuated the progression of hepatic fibrosis by suppressing HSCs proliferation and activation via the ERK/MAPK and PI3K/Akt signaling pathways. AKF-PD may be used as a potential novel therapeutic agent against liver fibrosis.

Pirfenidone, a pyridone compound, is an effective and novel antifibrotic agent. In this article, we describe the design, synthesis and activity evaluation of novel antifibrotic agents, 1-(substituted aryl)-5-trifluoromethyl-2(1H) pyridones modified with carbohydrate. Most of the title compounds exhibited comparable or better inhibitory activity than fluorofenidone. Notably, compound 19a demonstrated the highest cell-based inhibitory activity against NIH 3T3 (IC(50) = 0.17 mM).