Norcantharidin down-regulates iron contents in the liver and spleen of lipopolysaccharide-treated mice

ABSTRACT Objective The inhibiting effect of Norcantharidin (NCTD) on IL-6 (interleukin-6) and STAT3 and the involvement of the IL-6/STAT3 pathway in hepcidin expression prompted us to speculate that NCTD could affect iron metabolism. 
Methods
 We examined the effects of NCTD on serum iron (SI) and transferrin (Tf) saturation, iron and ferritin light chain (FTL), transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferroportin 1 (Fpn1), iron regulatory protein 1 (IRP1) and hepcidin, as well as IL-6 and STAT3 in the liver, spleen and duodenum of mice treated with lipopolysaccharide (LPS) in vivo, using RT-PCR, Western blotting and immunofluorescence analysis. 
Results
 NCTD could increase SI and Tf saturation and reduce tissue iron and FTL content by affecting expression of cell-iron transport proteins TfR1, DMT1 and Fpn1. The impact of NCTD on TfR1, DMT1 and Fpn1 expression is mediated by up-regulating IRP1 and down-regulating hepcidin expression, while NCTD-induced down-regulation of hepcidin is mediated by the IL-6/STAT3 signalling pathway in LPS-treated mice. 
Conclusions
 NCTD affects iron metabolism by modifying the expression of IL-6/JAK2/STAT3/hepcidin and IRP1 and suggest that the ability of NCTD to reduce tissue iron contents may be a novel mechanism associated with the anti-cancer effects of NCTD.


Introduction
Cantharidin (CTD) is a naturally occurring compound isolated from 1500 species of medicinal insect blister beetle (Mylabris phalerata Pallas) [1]. The use of mylabris as a traditional medicine in China can be traced back more than 2000 years, and it is still used as a folk medicine today [2]. The most important of the medicinal uses of CTD is its anti-cancer activities [2,3]. However, the application of CTD is limited due to its toxicity to the gastrointestinal and urinary tracts. Norcantharidin (NCTD) is a demethylated analog of cantharidin (CTD) [2]. NCTD causes fewer nephrotoxic and inflammatory side effects than CTD [4], and like CTD has been demonstrated as a potential agent against certain cancers including human prostate cancer, stromal cancer, non-small lung cancer and hepatocellular carcinoma through inhibiting proliferation and metastasis of tumor cells [2,[5][6][7][8].
In the present in vivo study, we examined the effects of NCTD on serum iron (SI) and transferrin (Tf) saturation, iron contents and ferritin-light chain (FTL) expression in the liver, spleen and duodenum of LPS-treated mice. To find out the reasons for the changes in serum and tissue iron contents induced by NCTD, we then investigated the effects of NCTD on the expression of the major cell-iron importers transferrin receptor 1 (TfR1) and divalent metal transporter 1 (DMT1), and the celliron exporter ferroportin 1 (Fpn1). To clarify the mechanisms underlying the impact of NCTD on cell-iron-transporters under inflammatory conditions, we dissected the changes in the expression of IRP1 and hepcidin as well as IL-6, p-JAK2 and p-STAT3 in mice co-treated with NCTD and LPS. We demonstrated that NCTD could increase SI and Tf saturation and reduce iron contents in the liver, spleen and intestine in LPStreated mice by affecting both IRP and hepcidin expression, providing evidence for the effect of NCTD on iron metabolism.

Animals and treatments
Forty-five balb/c male mice (18-22 g) were used in the present study. The mice were housed under specific pathogen-free conditions at 22 ± 2°C with a relative humidity of 60-65% and maintained under a 12-h light/12-h dark cycle with ad libitum access to food and water as previously described. All animal care and experimental protocols were performed according to the Animal Management Rules of the Ministry of Health of China and approved by the Animal Ethics Committees of Nantong University (NSFC31271132). The mice were randomly divided into three groups: (1) Control group: intraperitoneal injection (i.p.j.) of 0.2 ml normal saline at 8am for 3 days and then 0.4 ml normal saline on the third day; (2) LPS group: intraperitoneal injection of 0.2 ml normal saline at 8am for 3 days and then LPS (1mg/ kg) in 0.4 ml normal saline at the third day; (3) NCTD + LPS group: intraperitoneal injection of NCTD (2 mg/kg) in 0.2 ml normal saline at 8am for 3 days and then LPS (1 mg/kg) in 0.4 ml normal saline at the third day. At 2pm on the third day (6-h after LPS treatment), animals were anesthetized with 1% (w/w) pentobarbital sodium (40 mg/kg body weight, intraperitoneally) and decapitated. The dose of the NCTD drug used was according to Liu et al. [19].

Sampling of blood and tissues
After anesthetization and decapitation, blood samples were collected into heparinized syringes for the determination of SI, UIBC and TF% saturation. Mice were then perfused with phosphate-buffered saline (PBS), the liver, spleen and duodenum were removed, excised, and rinsed in PBS, weighed and stored in a freezing chamber at −80°C for total RNA extraction, protein determination, and iron measurement [20,21].

Isolation of total RNA and quantitative real-time PCR
The extraction of total RNA and preparation of cDNA were performed using TRIzol reagent and the AevertAid First Strand cDNA Synthesis Kit respectively, in accordance with the instructions of the manufacturers. Real-time PCR was carried out by RT-PCR instrument (LC96, Roche, Switzerland) using Fast Start Universal SYBR Green Master and the Light Cycler96. The specific pairs of primers were: mouse β-actin, forward: 5 ′ -AAA TCG TGC GTG ACA TCA AAGA-3 ′ , reverse: 5 ′ -GCC ATC TCC TGC TCG AAG TC-3 ′ ; mouse hepcidin, forward: 5 ′ -AGA GCT GCA GCC TTT GCA C-3 ′ , reverse: 5 ′ -GAA GAT GCA GAT GGG GAA GT-3 ′ ; and IL-6, forward: Table 1). The CT values of each target gene were normalized to that of the β-actin mRNA. Relative gene expression was calculated by the 2 −ΔΔ CT method [25,26].

Tissue iron measurement
The total iron in the tissues (μg/g wet weight of tissue) was determined using a graphite furnace atomic absorption spectrophotometer as described [29]. In brief, tissues were homogenized in 20 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, followed by digestion in an equal volume of ultrapure nitric acid, and then measured with a graphite furnace atomic absorption spectrophotometer (Perkin-Elmer; Analyst 100).

Statistical analysis
Statistical analyses were performed using the Graphpad Prism 8.0. Data are presented as mean ± standard error of the mean. Differences between the means were analyzed using D'Agostino-Pearson omnibus normality test. For data not following normal distribution, Kruskal-Wallis test with Dunn's multiple comparisons test was performed. A p < .05 denoted statistical significance.

Results
Norcantharidin increased serum iron, serum transferrin saturation, and reduced iron content in the liver, spleen and duodenum in lipopolysaccharides-treated mice (F)) was lower in mice treated with NCTD + LPS when compared to mice treated with LPS only. There were no significant differences in UIBC among the control, LPS and NCTD/LPS groups (Figure 1(C)). The findings clearly showed that NCTD could up-regulate serum iron and transferrin saturation, but not UIBC, and down-regulated iron content in the liver, spleen and duodenum in LPS-treated mice.
We also examined the effects of NCTD on the expression of FTL in the liver, spleen and duodenum of LPS-treated mice. FTL was checked because FTL facilitates the storage of iron into the ferritin core [33][34][35], being more closely associated with cellular iron storage [36] and also because FTL is the predominant form of ferritin in the liver and spleen [37,38]. Western blot analysis showed that NCTD could significantly suppress the LPS-induced increase in expression of FTL (Figures 2, 3 and 4(D)) in the liver (Figure 2), spleen ( Figure  3) and duodenum (Figure 4) of mice. Also, the results were confirmed by immunofluorescence staining analysis ( Figures  2, 3 and 4(H)). These observations provide further evidence that NCTD could down-regulate iron contents in the liver, spleen and duodenum in LPS-treated mice.
Norcantharidin up-regulated expression of IRP1 in the liver, spleen and duodenum of lipopolysaccharides-treated mice To elucidate how NCTD up-regulates TfR1, DMT1 and Fpn1 expression and down-regulates FTL in LPS-treated mice, we subsequently investigated the effects of NCTD on IRP1 (iron regulatory protein 1) and hepcidin expression in mice because cellular expression of these proteins is regulated by IRPs (iron regulatory proteins) and systemically by hepcidin [39,40]. Western blot ( Figure 5(A-C)) and immunofluorescence staining ( Figure 5(D-F)) analysis both showed that IRP1 expression in the liver ( Figure 5(A,D)), spleen ( Figure 5(B,E)) and duodenum ( Figure 5(C,F)) was significantly higher in NCTD + LPS-treated mice than in LPS-treated mice, indicating that NCTD could significantly reverse the inhibition of LPS on IRP1 in all organs we examined. RT-PCR analysis demonstrated that expression of hepcidin mRNA in the liver ( Figure 5(G)), spleen ( Figure 5(H)) and duodenum ( Figure 5 (I)) was significantly lower in NCTD + LPS-treated mice than in LPS-treated mice, indicating that NCTD has the ability to significantly reverse the promoting role of LPS in the expression of hepcidin mRNA in these organs.
Norcantharidin down-regulated expression of IL-6, JAK2 and STAT3 in the liver, spleen and duodenum of lipopolysaccharides-treated mice It has been well-demonstrated that LPS up-regulates hepcidin expression via the IL-6/JAK/STAT3 signaling pathway [13,41]. To explore the possible mechanisms involved in the effect of NCTD on hepcidin, we next investigated the roles of NCTD in the expression of IL-6 mRNA, p-JAK2 and p-STAT3 proteins in LPS-treated mice. It was found that treatment with LPS only induced a significant increase in the expression of IL-6 mRNA ( Figure 6(A,D,G)), p-JAK2 ( Figure 6

Discussion
In the present study, we demonstrated that NCTD could significantly up-regulate serum iron level and Tf saturation (%), expression of TfR1, DMT1, Fpn1 and IRP1 proteins, and  down-regulate iron contents, expression of FTL, pJAK2 and pSTAT3 proteins and hepcidin and IL-6 mRNAs in the liver, spleen and duodenum of LPS-treated mice. These findings indicate that NCTD is capable of suppressing the effects of LPS or infection and inflammation on iron metabolism, and also provide in vivo evidence for the usefulness of NCTD for iron homeostasis under inflammatory conditions. The significant reduction in iron contents induced by NCTD in the liver, spleen and duodenum of LPS-treated mice may be due to the NCTD-induced up-regulation of Fpn1 expression because increased Fpn1 would increase the amount of iron exported from the cells. In most types of cells throughout the body, iron balance depends on the normal expression of in-and-out transporters of iron, TfR1, DMT1 and Fpn1 [16,[30][31][32]42]. By controlling their expression, the cell can determine the amount of iron acquired (via TfR1 and DMT1) and released (via Fpn1). In the present study, however, in addition to the increased Fpn1, we also found  that NCTD induces a significant increase in the expression of TfR1 and DMT1 in the organs of LPS-treated mice. The increased expression of these two iron importers should lead to increased iron import and contents in the cells or tissues. However, iron contents were found to be reduced in the organs we examined. This may indicate that the effect of NCTD on Fpn1 expression is more significant than its effect on TfR1 and DMT1. In other words, the increased amount of iron released from the cells via Fpn1 is more than that of iron imported into the cells via TfR1 and DMT1 under our experimental conditions (Figure 7).
It has been well-confirmed that TfR1, DMT1 and Fpn1 expression is mainly controlled by IRPs [43][44][45] as well as hepcidin [39,40,46,47]. The expression of TfR1, DMT1 and Fpn1 is coordinately regulated by the IRP/IRE (iron-responsive element) system, [39,40,48,49] positively for the 3 ′ untranslated region IRE motif in TfR1 and DMT1 and negatively for the 5 ′ untranslated region IRE motif in Fpn1 (and FTL), namely to up-regulate TfR1 and DMT1 and down-regulate Fpn1 (and FTL) expression. Hepcidin reduces the amount of Fpn1 on the membrane by directly binding with Fpn1, and the hepcidin/Fpn1 complex is then internalized and subsequently degraded [50]. It has also been reported that hepcidin is capable of directly inhibiting TfR1 in different types of cells [46][47][48] and down-regulating DMT1 expression in the intestine through proteasome internalization and degradation [51]. Therefore, the increased TfR1 and DMT1 caused by NCTD may be due to NCTD-induced up-regulation in IRP1 and down-regulation in hepcidin, while the increased Fpn1 is likely a result mainly of the down-regulation in hepcidin in the liver, spleen and duodenum of mice co-treated by LPS + NCTD.
Hepcidin is the master regulator in systemic iron metabolism, and hepcidin expression is regulated by a variety of factors, such as systemic iron levels [52] and inflammation [53]. Inflammation has been well-documented to affect iron metabolism via hepcidin. It has been demonstrated that LPS is able to up-regulate hepcidin expression via the IL-6/ JAK2/STAT3 signaling pathway [17]. In the mice co-treated with NCTD and LPS, the expression of IL-6 mRNA, p-JAK2 and p-STAT3 proteins and also hepcidin mRNA was found to be significantly reduced in the liver, spleen and duodenum, as compared with mice treated with LPS only. This clearly indicated that NCTD down-regulates the expression of hepcidin by inhibiting the IL-6/JAK2/STAT3 signaling pathway in mice under inflammatory conditions. It has been proved that NCTD, as an anti-tumor drug, can inhibit the growth and metastasis of tumor cells in vivo and in vitro [54,55]. Due to rapid proliferation and DNA synthesis, tumor cells demand more iron than normal cells. Iron plays an important role in matrix degradation and tumor metastasis by stimulating or stabilizing the activity of certain metalloproteinases [56]. Increased iron levels in the body have been considered as an important cofactor in the carcinogenesis of several cancers [57]. This notion was supported by most studies in which increased levels of iron have been shown to be associated with higher cancer risk [58][59][60]. In the present study, NCTD was found to have the ability to reduce tissue iron contents in LPS-treated mice. This may be a novel mechanism associated with the anti-cancer effects of NCTD. Further studies about this possibility are needed.
Anemia of inflammation (AI, or ACD anemia of chronic disease) is considered the second most prevalent type of anemia worldwide, after iron deficiency anemia (IDA) and the most frequent anemic entity observed in hospitalized or chronically ill patients [18,61,62]. In animal models of AI and in patients suffering from inflammatory diseases, increased hepcidin levels are associated with low Fpn1 expression on duodenal enterocytes and macrophages, along with impaired dietary iron absorption and retention of iron in macrophages   [63,64], thereby causing decreased serum iron, transferrin saturation, and iron delivery for erythropoiesis. Therefore, hepcidin measurement has been considered as a promising diagnostic tool for AI or ACD [64][65][66]. In the present study, we showed that NCTD could significantly suppress the LPS-induced increase in hepcidin expression and decrease in serum iron and transferrin saturation, indicating that NCTD may have a potential role in the treatment of AI as an ancillary drug.
In summary, we demonstrated that NCTD could increase serum iron and Tf saturation and reduce tissue iron and FTL contents by affecting the expression of cell-iron transport proteins TfR1, DMT1 and Fpn1. Our findings also revealed that the impact of NCTD on the expression of TfR1, DMT1 and Fpn1 is mediated by up-regulating IRP1 and down-regulating hepcidin expression, while down-regulation of NCTD on the expression of hepcidin is mediated by the IL-6/JAK2/ STAT3 signaling pathway in LPS-treated mice (Figure 7). Our findings provide further evidence in vivo to support the conclusion that NCTD modifies iron metabolism by affecting the expression of IL-6/JAK2/STAT3 /hepcidin and IRP1. The ability of NCTD to suppress the LPS-induced increase in hepcidin expression and decrease in serum iron and transferrin saturation implies that NCTD may have a potential role in the treatment of AI as an ancillary drug. The findings also suggest that reducing tissue iron contents may be a novel mechanism associated with the anti-cancer effects of NCTD. Further studies about this possibility are needed.