RESUMEN
An amendment to this paper has been published and can be accessed via the original article.
RESUMEN
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.
Asunto(s)
Glutatión Transferasa/metabolismo , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/enzimología , Animales , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células Estrelladas Hepáticas/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/patología , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Piridonas/farmacología , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
AIM: We explored whether Fluorofenidone reduced interleukin-1ß (IL-1ß) 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-1ß 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-1ß and cleavage IL-1ß 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-1ß. Interestingly, Fluorofenidone inhibited the activation of NLRP3 inflammasome, downregulated Caspase-1 levels and thereby decreased the cleavage of pro-IL-1ß into IL-1ß 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-1ß production in UUO model by interacting with NLRP3 inflammasome.
Asunto(s)
Antiinflamatorios/farmacología , Inflamasomas/efectos de los fármacos , Interleucina-1beta/metabolismo , Riñón/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Nefritis/prevención & control , Piridonas/farmacología , Obstrucción Ureteral/tratamiento farmacológico , Animales , Proteínas Adaptadoras de Señalización CARD/metabolismo , Caspasa 1/metabolismo , Quimiotaxis de Leucocito/efectos de los fármacos , Modelos Animales de Enfermedad , Regulación hacia Abajo , Fibrosis , Células HEK293 , Humanos , Inflamasomas/genética , Inflamasomas/inmunología , Inflamasomas/metabolismo , Interleucina-1beta/genética , Riñón/inmunología , Riñón/metabolismo , Riñón/patología , Masculino , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Nefritis/inmunología , Nefritis/metabolismo , Nefritis/patología , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Obstrucción Ureteral/inmunología , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patologíaRESUMEN
Sterile inflammation is initiated by damage-associated molecular patterns (DAMPs) and a key contributor to acute liver injury (ALI). However, the current knowledge on those DAMPs that activate hepatic inflammation under ALI remains incomplete. We report here that circulating peroxiredoxin-1 (Prdx1) is a novel DAMP for ALI. Intraperitoneal injection of acetaminophen (APAP) elicited a progressive course of ALI in mice, which was developed from 12 to 24â¯h post injection along with liver inflammation evident by macrophage infiltration and upregulations of cytokines (IL-1ß, IL-6 and TNF-α); these alterations were concurrently occurred with a robust and progressive production of serum Prdx1. Similar observations were also obtained in carbon tetrachloride (CCl4)-induced ALI in mice. Removal of the source of serum Prdx1 protected mice deficient in Prdx1 from APAP and CCl4-induced liver injury, and decreased macrophage infiltration, IL-1ß, IL-6 and TNF-α production. As a result, Prdx1-/- mice were strongly protected from APAP-induced death that was likely progressed from ALI. Additionally, intravenous re-introduction of recombinant Prdx1 (rPrdx1) in Prdx1-/- mice reversed or reduced all the above events, demonstrating an important contribution of circulating Prdx1 to ALI. rPrdx1 potently induced in primary macrophages the expression of pro-IL-1ß, IL-6, TNF-α, and IL-1ß through the NF-κB signaling as well as the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome signaling, evident by caspase-1 activation. Furthermore, a significant elevation of serum Prdx1 was demonstrated in patients (nâ¯=â¯15) with ALI; the elevation is associated with ALI severity. Collectively, we provide the first demonstration for serum Prdx1 contributing to ALI.
Asunto(s)
Alarminas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Inflamación/metabolismo , Hígado/patología , Macrófagos/inmunología , Peroxirredoxinas/metabolismo , Acetaminofén , Adulto , Animales , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , FN-kappa B/metabolismo , Peroxirredoxinas/genética , Transducción de SeñalRESUMEN
Mefunidone is a new pyridone agent that attenuates renal tubulointerstitial fibrosis. However, the signaling pathways involved in the effect of mefunidone on renal tubulointerstitial fibrosis have not been well explained. Inflammatory response initiates and promotes renal tubulointerstitial fibrosis, and the inhibitor of nuclear factor kappa-B kinase beta (IKKß) is a master regulator of inflammation. This study is determined to clarify the influence of mefunidone on renal inflammation and the phosphorylation of IKKß. Experimental renal tubulointerstitial fibrosis was induced by unilateral ureteral obstruction (UUO) for 3, 7 and 14days in sprague dawley rat. Treatment with mefunidone was conducted simultaneously. Obstructed kidneys were harvested for the assessment. Our results showed that treatment with mefunidone ameliorated renal inflammatory injury, renal tubular lesions and interstitial fibrosis. Further studies indicated that treatment with mefunidone mitigated the expressions of tumor necrosis factorα (TNFα) and interleukin-1ß (IL-1ß) in the kidney. The phosphorylation of IKKß and inhibitor of kappa-B (IκB) and the expression of NOD-like receptor family, pyrin domain containing 3 (NALP3) were also reduced in vivo after treatment with mefunidone. In vitro, peritoneal macrophages were incubated with necrotic cells or lipopolysaccharide in the presence or absence of mefunidone. Mefunidone markedly decreased necrotic cell or LPS induced IL-1ß production and LPS induced TNFα production in primary peritoneal macrophages. Furthermore, mefunidone significantly inhibited the phosphorylation of IKKß/IκB and nuclear transition of NF-κB p65 in peritoneal macrophages stimulated by necrotic cell or lipopolysaccharide. In conclusion, mefunidone serves as a novel anti-inflammatory agent that attenuates UUO-induced renal interstitial inflammation and fibrosis, possibly through suppressing IKKß phosphorylation.