Heme Oxygenase-1 Protects Bone Marrow Mesenchymal Stem Cells Against Iron Overload through Decreasing ROS and Promoting IL-10 Generation

Background:

Iron overload caused by frequent transfusions of suspended red blood cells which is of major concern in hematological disease. Heme oxygenase-1(HO-1) is well known as a crucial factor to cells against oxidative stress. In this study, significant upregulation of HO-1 was observed in bone marrow mesenchymal stem cells (BMMSCs) in the early phase of IO and predicted good prognosis in mice.

Aim: Given that the role of HO-1 in protecting (BMMSCs) against damage from IO was still unknown. In the study, we investigated the effects and mechanisms of iron overload-related ROS by inducing IL-10 by regulating HO-1.

METHODS:

Male C57BL/6Ly5.2 (Ly45.2) mice were housed under specific pathogen-free conditions and allowed to acclimate for 2 weeks before experiments. The mice were housed (5 individuals per cage) and used at a weight of approximately 20.0 g. Thirty male mice were recipient mice, and another five were competitive mice. The thirty-five mice were randomly divided into four groups: (a) a control group; (b) an IO group (25 mg/ml); (c) an IO + hemin group and (d) an IO + ZnPP group. The depositions of iron in the liver, spleen and BM were assessed using HE staining and BM iron staining. Then, the expression of HO-1 and IL-10 were examined by Western blotting and q-PCR. Cell proliferation was measured by CCK-8 assay. The quantity of Ferritin, Serum iron, Hepcidin and IL-10 in the blood serum from iron overload cell were detected by ELISA in mouse model and iron overload patients. Finally, the levels of apoptotic rate, intracellular ROS and labile iron pool (LIP) were analyzed by flow cytometer.

Result :

We found that HO-1 was upregulated in the excess iron culture of BMMSCs in the early phase. Meanwhile, the apoptosis rate of IO BMMSCs was increased by 3.0 times compared with that of CON group. On the contrary, the apoptotic rate of BMMSCs down-regulated by transcription of si-HO-1 virus increased by 7.12 times compared to the CON group. In addition, the apoptosis rate of HO-1 overexpressing BMMSCs was significantly decreased in BM-1 virus transfected with HO-1 virus. Next, we also observed LIP in IO BMMSCs transfected with HO-1 virus was significantly lower than IO BMMSCs in un-transfected group. It suggested that HO-1 up-regulated the BMMSCs was protected from IO damage by decreasing intracellular iron content in vitro.

To get an insight into the mechanism involved in cells protection from IO by HO-1 upregulation. We further explore production of ROS and secrection of IL10. As HO-1 was reported to have ability to reduce ROS, and IL-10 could promote excretion of intracellular iron. The results showed the ROS level in IO BMMSC was improved by 5.94 times compared with that of CON group, and the level of ROS in IO BMMSCs transcribed with si-HO-1 and HO-1 virus increased by 6.18 and 4.53 times, respectively. And IL-10 was increased significantly in the HO-1 up-regulated group.

Next, we used U0126 (an ERK pathway inhibitor) to co-culture with IO BMMSCs that transcribed HO-1 virus. We observed the secretion of ferritin and the expression of IL-10 were consistent with the BMMSCs concentration changed. In vivo, we found that the serum iron and ferritin levels in the peripheral blood of IO mice were reduced by 1.92 and 1.3 times through ELISA analysis. The levels of IL-10 and ferritin were detected by ELISA. The results of deposition detection in bone marrow were consistent with the in vitro model. Finally, from November 2015 to December 2016, 12 patients diagnosed iron overload were enrolled. Including 6 males and 6 females, aged 44 to 77 years old. By q-PCR, we also found that iron overload in BMMSCs with high expression of HO-1 and IL-10 was reduced in serum ferritin after deferoxamine treatment. Therefore, we hypothesized that HO-1 overexpression of iron overload patients with better prognosis.

Conclusions: we hypothesized that HO-1 overexpression of iron overload patients with better prognosis. The results of this study allow for further study of mechanisms of IO-related disease and provide new therapeutic targets for the treatment of IO patients.