Lipopolysaccharide infusion up-regulates hepcidin mRNA expression in equine liver

Abstract

Hepcidin has been found to be the key regulator of iron metabolism that leads to hypoferremia during inflammation. Recent work has shown that equine hepcidin is predominantly expressed in the liver of horses. In this study, hepcidin gene expression was determined in the liver and bone marrow of six healthy horses after iv infusion of Escherichia coli O55:B5 LPS. The IL-6 gene expression was also determined in liver and bone marrow samples. Clinical and laboratory evaluations were measured at multiple time points between 0 and 240 h post-LPS infusion (PI). Liver and bone marrow biopsies were taken immediately before (baseline) and at 6 and 18 h PI. In response to endotoxin infusion, all horses showed characteristic clinical signs of endotoxemia. Plasma iron concentration was decreased significantly from the pre-infusion level at 8 h PI. Hypoferremia peak was observed at 12 h and returned to normal levels at 30 h PI. Relative real-time RT-PCR analysis showed that liver hepcidin and IL-6 mRNA expression was up-regulated at 6 h PI. Bone marrow hepcidin relative expression was not influenced by LPS infusion. In another experiment, equine monocyte cultures were stimulated with LPS (1 µg/ml). Monocyte hepcidin and IL-6 gene expression was significantly induced after 2 h of LPS stimulus and returned to baseline levels thereafter. The present study describes that, in horses, LPS infusion up-regulates hepatic hepcidin mRNA expression resulting in early observed hypoferremia and suggests that hepcidin may act as an acute-phase protein in horses.

Freund's adjuvant-induced inflammation: clinical findings and its effect on hepcidin mRNA expression in horses

“…The edema that persisted at the injection site at the end of the experiment by Mills et al (1998) might have been due to the third injection of FCA on the fifth day of the experiment, despite the lower dosage used by these authors. The rapid increase in local temperature with three statistically significant peaks at 6 h, 30 h, and 60 h PI as well as the observed edema and localized pain not only represent the classical signs of inflammation but are similar to the results obtained using other protocols for intramuscular administration of FCA in horses (Auer et al 1989, Mills et al 1997a, 1997b, Oliveira-Filho et al 2012. Mills et al (1998) observed an elevated temperature in the horses 48 h after the first dose of FCA, which decreased to normal on the fourth day.…”

“…In the current study, the main objective was to verify the increased hepatic hepcidin mRNA expression and its effect on iron metabolism in horses by inducing a more focal inflammatory process using two intramuscular injections of FCA. The hepcidin expression level observed in the present study was more persistent compared with the endotoxemic experimental model induced with LPS (Oliveira-Filho et al 2012). This likely reflects the different duration of inflammation between the two models.…”

“…Recently, we verified that the increase in hepatic hepcidin mRNA expression was likely responsible for the hypoferremia observed in horses after LPS injection (Oliveira-Filho et al 2012). However, this increase was not persistent, and in a few hours, the hepcidin mRNA levels returned to normal.…”

“…Biopsy tissues were frozen in li-quid nitrogen and stored at -80 °C until processing. Total RNA purification, DNase treatment, and cDNA synthesis were performed using methodology that has been previously described (Oliveira--Filho et al 2012). The relative quantification of hepcidin mRNA in the liver samples was performed using the comparative Ct method (2 -ΔΔCt method) that was developed for relative real-time PCR data processing with a Real-Time PCR System (7300, Applied Biosystems TM , Carlsbad, USA) and the Real-Time PCR Master Mix (Power SYBR ® Green, Applied Biosystems TM ).…”

“…Recently, we cloned and sequenced equine hepcidin mRNA (Oliveira-Filho et al 2010a) and demonstrated a link between hepcidin and hypoferremia in horses that had been treated with lipopolysaccharide (LPS) (Oliveira-Filho et al 2012).…”