Downregulation of hepcidin by norcantharidin in macrophage

Abstract Norcantharidin (NCTD) is a demethylated analogue of cantharidin. It was recently demonstrated that NCTD reduces iron contents in the liver and spleen of mice in vivo, indicating that NCTD can affect iron metabolism via hepcidin. Here, we investigated the effects of NCTD on expression of iron storage protein ferritin-light chain (Ft-L), transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferroportin 1 (Fpn1), hepcidin, iron regulatory protein 1 (IRP1), IL-6, p-JAK2 and p-STAT3 in lipopolysaccharides (LPS)-treated RAW264.7 cells in vitro via Real-time PCR and Western blotting analysis. We demonstrate that NCTD down-regulates Ft-L, hepcidin, IL-6, pJAK2, pSTAT3 and up-regulates TfR1, DMT1, Fpn1 and IRP1 expression in LPS treated cells, showing that NCTD can inhibit hepcidin via the IL-6/JAK2/STAT3 signalling pathway and also increase TfR1, DMT1 and Fpn1 expression via down-regulating hepcidin and up-regulating IRP1. Our findings provide further evidence in vitro for the role of NCTD in iron metabolism. Graphical Abstract A hypothetical scheme for NCTD to affect the expression of iron transport, storage and regulatory proteins or mRNA in LPS-treated RAW264.7 cells. NCTD downregulates hepcidin by inhibiting the IL-6/JAK2/STAT3 signaling pathway and up-regulates Fpn1 via down-regulation of hepcidin. The upregulation of TfR1 and DMT1 expression is mainly induced by increased IRP1 expression. The positive effect of NCTD on Fpn1 may be more significant than its effect on TfR1 expression, leading to a reduced amount of iron intake relative to iron release and hence reduced Ft-L expression under inflammatory conditions. (Solid-line: a major (dominant) role, Dotted-line: a minor role).

A hypothetical scheme for NCTD to affect the expression of iron transport, storage and regulatory proteins or mRNA in LPS-treated RAW264.7 cells.NCTD downregulates hepcidin by inhibiting the IL-6/JAK2/STAT3 signaling pathway and up-regulates Fpn1 via down-regulation of hepcidin.The upregulation of TfR1 and DMT1 expression is mainly induced by increased IRP1 expression.The positive effect of NCTD on Fpn1 may be more significant than its effect on TfR1 expression, leading to a reduced amount of iron intake relative to iron release and hence reduced Ft-L expression under inflammatory conditions.(Solid-line: a major (dominant) role, Dotted-line: a minor role).

Results and discussion
We first investigated the effects of NCTD and/or LPS on the viability of RAW264.7 cells by incubating them with various concentrations of NCTD (0, 0.1, 1, 5, 10 and 20 μM) alone for 24-h or with NCTD (0, 0.1, 1, 5, 10, and 20 μM) alone for 18-h, and then LPS (1 μg/ml) for 6-h.The MTT assay demonstrated that viabilities in NCTD-treated cells (Figure 1C) or NCTD and LPS-co-treated cells were not significantly different from those of the controls, but treatment with 20 μM of NCTD plus LPS (LPS + 20μM) did induce a reduction in cell viability (Figure 1D).The 10 μM of NCTD was therefore used for all subsequent experiments.
Cell iron content depends mainly on the expression of iron uptake (TfR1/DMT1) and release (Fpn1) proteins (Qian et al. 1997;Ganz 2013), while Ft-L is closely associated with cellular iron storage (Harrison and Arosio 1996).To reveal whether NCTD affects the expression of iron transport and storage proteins, we measured TfR1, DMT1, Fpn1 and Ft-L expression by incubating the cells with 0 (Control) or 10 μM of NCTD (NCTD) for 24-h, or 0 (LPS) or 10 μM of NCTD for 18-h and then 1 μg/ml of LPS (NCTD + LPS) for 6-h.
of TfR1, and Fpn1 proteins in LPS-treated cells was significantly lower, and Ft-L higher than in the controls, showing that LPS down-regulated TfR1, DMT1, Fpn1 and up-regulated Ft-L protein expression.However, the levels of TfR1 (Figure 1E), DMT1 (Figure 1F) and Fpn1 (Figure 1G) proteins were significantly higher and Ft-L (Figure 1H) significantly lower in NCTD + LPS-co-treated cells (NCTD + LPS) when compared to those of LPS-treated cells.The fact that both TfR1 and Fpn1 expression was up-regulated while Ft-L was down-regulated by NCTD in LPS-treated cells may imply that the effect of NCTD on Fpn1 is more significant than its effect on TfR1 expression, leading to a lower amount of iron intake relative to iron release.This might be one of causes for the reduced Ft-L expression in the cells co-treated with NTCD and LPS.
Mammalian iron metabolism, including the expression of TfR1, DMT1, Fpn1 and Ft-L, is regulated systemically by hepcidin and cellularly by IRP (Hentze et al. 2010).To understand how NCTD affects the expression of iron transport and storage proteins, we subsequently investigated the effects of NCTD on the expression of hepcidin mRNA and IRP1 protein in RAW264.7 cells.As we reported previously (Wang et al. 2008;Ma et al., 2018), LPS induced a significant reduction in IRP1 protein expression (Figure 1I) and an increase in hepcidin mRNA (Figure 1J), and these LPS-induced changes could be significantly suppressed by NCTD in RAW264.7 cells.IRP1 protein contents were significantly higher and hepcidin mRNA lower in LPS + NCTD-co-treated cells when compared to LPS-treated cells.
The increased IRP1 protein and reduced hepcidin mRNA expression implied that NCTD affects the expression of iron transport (TfR1, DMT1, Fpn1) and storage (Ft-L) proteins by up-regulating IRP1 as well as down-regulating hepcidin expression in LPS-treated cells.Up-regulation of TfR1 and DMT1 expression is mainly due to increased IRP1 expression and may also be partly caused by the NCTD-induced down-regulation of hepcidin expression, as TfR1 (Muckenthaler et al. 2008;Du et al. 2011Du et al. , 2015) ) and DMT1 (Brasse-Lagnel et al. 2011;Jain et al. 2019) could be inhibited by hepcidin in different types of cells.Fpn1 expression was affected by hepcidin, as well as by IRP1.Increased IRP1 could induce a reduction in Fpn1, while reduced hepcidin led to an increase in Fpn1.The expression of Fpn1 increased, rather than decreased in NCTD + LPS-co-treated cells, implying that hepcidin may play a dominant role in the control of Fpn1 expression under inflammatory conditions.
Inflammation has been well-documented to affect iron metabolism via hepcidin.LPS regulates hepcidin expression (Wang et al. 2008) via the IL-6/JAK/STAT3 signaling pathway and then downregulates expression of TfR1 and Fpn1 in the brain (Li et al. 2016).To explore the mechanisms of the effect of NCTD on hepcidin, we investigated the effects of NCTD on the expression of IL-6 mRNA, pJAK2 and pSTAT3 proteins in LPS-treated cells.We found that the expression of IL-6 mRNA (Figure 1K), pSTAT3 (Figure 1L) and pJAK2 (Figure 1M) proteins in NCTD-treated cells was not different from that of the controls.However, LPS was shown to induce a significant increase in the expression of IL-6 mRNA (Figure 1K), pSTAT3 (Figure 1L) and pJAK2 (Figure 1M) proteins, while NCTD could significantly suppress these LPS-induced increases.In NCTD + LPS-co-treated cells, the expression of hepcidin mRNA as well as IL-6 mRNA, p-JAK2 and p-STAT3 was significantly reduced as compared with LPS-treated cells, indicating that NCTD downregulates hepcidin expression via its role in inhibiting the IL-6/JAK2/STAT3 pathway under inflammatory conditions.
In we demonstrated that NCTD has a significant effect on the expression of iron transport, storage and regulatory proteins in LPS-treated RAW264.7 cells (Graphical abstract).These findings also suggest that the ability of NCTD to reduce cell iron contents may be a novel mechanism associated with the anti-cancer effects of NCTD because iron is crucial to cell proliferation, growth and the development of many cancers.