[Establishment of macrophage model of iron overload in vitro and the injury induced by oxidative stress on macrophage with iron overload].

Abstract

OBJECTIVE:

To establish macrophage iron overload model in vitro by co-culture macrophages with iron, and to explore the effect of iron overload on cell reactive oxygen species (ROS) and the impact of ROS on macrophages.

METHOD:

Iron overload group were treated with different concentrations (0, 5, 10, 20, 40, 80 μmol/L respectively) of ferric ammonium citrate (FAC). The control group was the group of macrophages without FAC treatment. We detected the number and state of cells, metabolic activity, the change of phagocytosis, the levels of ROS and reactive nitrogen, and changes of related oxidative stress signaling pathways in different groups. Changes in the above indexes were detected after application of deferasirox (DFX) to remove iron and the antioxidant N -acetylcysteine (NAC) to clear excess oxidative stress.

RESULTS:

(1)The levels of labile iron pool (LIP) in macrophages co-cultivated with iron was increased with the increase of iron concentration in a dose-dependent manner. The LIP levels was the highest in the macrophages treated with 80 μmol/L. (2)The increase of FAC concentration, the metabolic activity of macrophages in the 5 FAC-treated groups decreased to 51.58%, 40.98%, 16.23%, 3.46%, and 0.05% of the activity level of the control group (all P< 0.05). The group with the metabolic activity decreased to 16.23% (20 μmol/L) was selected as the iron overload group for the following experiments. (3)Compared with the control group, the number of macrophages in the iron overload group reduced to 32.80% (P<0.05), and the state of cells changed from adherence to partial suspension. The phagocytosis of macrophages in the iron overload group reduced to 20.40% of the control group (P<0.05). (4)Our further experiment showed that the levels of ROS and the activity nitrogen in the iron overload group increased by 7.71-and 1.45-fold compared with the control group (both P<0.05). The RT-PCR showed up-regulated mRNA expression of genes related with ROS production, i. e. nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX 4) gene related with ROS production and inducible nitric oxide synthase (iNOS) gene related with reactive nitrogen production, down-regulated mRNA expression of glutathione peroxidase 1 (GPX1) gene which participated in ROS clearance. Moreover, mRNA expression of phosphatidylinositol-3-kinase (PI3K) gene involved in oxidative stress signaling pathway in the iron overload group was up-regulated, while fork head protein O3 (FOXO3) which regulated oxidative stress through negative feedback showed a down-regulation level of mRNA expression compared with the control group. (5)After iron chelation and antioxidant treatment, the above-mentioned damage in the iron overload group were partially reversed.

CONCLUSIONS:

The damages of iron overload on macrophages may be mediated by inducing oxidative stress and activating oxidative stress signaling pathways. Our established model provides a method to explore the mechanism of iron overload on macrophage, and may shed some new light on possible therapeutic target in treating iron overload patients.

• Authors: X L Cao, M F Zhao, D G Li, Y Xing, Y C Zhang, J Chen, X Y He, R Cui, J X Meng, X Xiao, J Mu, Y Y Jiang, R M Wu
• Journal: Zhonghua yi xue za zhi (Zhonghua Yi Xue Za Zhi) Vol. 96 Issue 2 Pg. 129-33 (Jan 12 2016) ISSN: 0376-2491 [Print] China
• PMID: 26792697 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antioxidants
• Ferric Compounds
• Quaternary Ammonium Compounds
• Reactive Oxygen Species
• Iron
• Phosphatidylinositol 3-Kinases
• ferric ammonium citrate
• Acetylcysteine

Topics

• Acetylcysteine
• Antioxidants
• Down-Regulation
• Ferric Compounds
• Humans
• Iron
• Iron Overload
• Macrophages
• Oxidative Stress
• Phosphatidylinositol 3-Kinases
• Quaternary Ammonium Compounds
• Reactive Oxygen Species
• Signal Transduction