Objective: To investigate the effects of deferoxamine on macrophage polarization and wound healing in mice with deep tissue injury (DTI) and its mechanism. Methods: The experimental research methods were adopted. Fifty-four male C57BL/6J mice of 6-8 weeks old were divided into DTI control group, 2 mg/mL deferoxamine group, and 20 mg/mL deferoxamine group according to random number table, with 18 mice in each group. DTI was established on the back of mice by magnet compression method. From post injury day (PID) 1, mice were injected subcutaneously with 100 µL normal saline or the corresponding mass concentration of deferoxamine solution every other day at the wound edge until the samples were collected. Another 6 mice without any treatment were selected as normal control group. Six mice in each of the three DTI groups were collected on PID 3, 7, and 14 to observe the wound changes and calculate the wound healing rate. Normal skin tissue of mice in normal control group was collected on PID 3 in other groups (the same below) and wound tissue of mice in the other three groups on PID 7 and 14 was collected for hematoxylin-eosin (HE) staining to observe the tissue morphology. Normal skin tissue of mice in normal control group and wound tissue of mice in the other three groups on PID 7 were collected, and the percentages of CD206 and CD11c positive area were observed and measured by immunohistochemical staining, and the mRNA and protein expressions of CD206, CD11c, and inducible nitric oxide synthase (iNOS) were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. Normal skin tissue of mice in normal control group and wound tissue of mice in DTI control group and 20 mg/mL deferoxamine group were collected on PID 3, 7, and 14, and the protein expressions of signal transducer and activator of transcription 3 (STAT3) and interleukin-10 (IL-10) were detected by Western blotting. The sample number in each group at each time point in the above experiments. The RAW264.7 cells were divided into 50 μmol/L deferoxamine group, 100 μmol/L deferoxamine group, 200 μmol/L deferoxamine group, and blank control group, which were treated correspondingly, with 3 wells in each group. The positive cell percentages of CD206 and CD86 after 48 h of culture were detected by flow cytometry. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and least significant difference test. Results: On PID 7, the wound healing rates of mice in 2 mg/mL and 20 mg/mL deferoamine groups were (17.7±3.7)% and (21.5±5.0)%, respectively, which were significantly higher than (5.1±2.3)% in DTI control group (P<0.01). On PID 14, the wound healing rates of mice in 2 mg/mL and 20 mg/mL deferoamine groups were (51.1±3.8)% and (57.4±4.4)%, respectively, which were significantly higher than (25.2±3.8)% in DTI control group (P<0.01). HE staining showed that the normal skin tissue layer of mice in normal control group was clear, the epidermis thickness was uniform, and skin appendages such as hair follicles and sweat glands were visible in the dermis. On PID 7, inflammation in wound tissue was obvious, the epidermis was incomplete, and blood vessels and skin appendages were rare in mice in DTI control group; inflammatory cells in wound tissue were reduced in mice in 2 mg/mL and 20 mg/mL deferoxamine groups, and a few of blood vessels and skin appendages could be seen. On PID 14, inflammation was significantly alleviated and blood vessels and skin appendages were increased in wound tissue of mice in 2 mg/mL and 20 mg/mL deferoxamine groups compared with those in DTI control group. On PID 7, the percentages of CD206 positive area in wound tissue of mice in 2 mg/mL and 20 mg/mL deferoxamine groups were significantly higher than that in DTI control group (P<0.01), the percentage of CD206 positive area in wound tissue of mice in DTI control group was significantly lower than that in normal skin tissue of mice in normal control group (P<0.01), the percentage of CD206 positive area in wound tissue of mice in 20 mg/mL deferoxamine group was significantly higher than that in normal skin tissue of mice in normal control group (P<0.01). The percentages of CD11c positive area in wound tissue of mice in 2 mg/mL and 20 mg/mL deferoxamine groups were significantly lower than those in DTI control group and normal skin tissue in normal control group (P<0.05 or P<0.01), and the percentage of CD11c positive area in normal skin tissue of mice in normal control group was significantly higher than that in DTI control group (P<0.05). On PID 7, the CD206 mRNA expressions in the wound tissue of mice in 2 mg/mL and 20 mg/mL deferoxamine groups were significantly higher than that in DTI control group (P<0.01), but significantly lower than that in normal skin tissue in normal control group (P<0.01); the CD206 mRNA expression in wound tissue of mice in DTI control group was significantly lower than that in normal skin tissue in normal control group (P<0.01). The mRNA expressions of CD11c and iNOS in wound tissue of mice in 2 mg/mL and 20 mg/mL deferoamine groups were significantly lower than those in DTI control group (P<0.01). The mRNA expressions of CD11c in the wound tissue of mice in DTI control group, 2 mg/mL and 20 mg/mL deferoamine groups were significantly higher than that in normal skin tissue in normal control group (P<0.01). Compared with that in normal skin tissue in normal control group, the mRNA expressions of iNOS in wound tissue of mice in 2 mg/mL and 20 mg/mL deferoamine groups were significantly decreased (P<0.01), and the mRNA expression of iNOS in wound tissue of mice in DTI control group was significantly increased (P<0.01). On PID 7, the protein expressions of CD206 in the wound tissue of mice in 2 mg/mL and 20 mg/mL deferoamine groups were significantly higher than those in DTI control group and normal skin tissue in normal control group (P<0.01), and the protein expression of CD206 in wound tissue of mice in DTI control group was significantly lower than that in normal skin tissue in normal control group (P<0.01). The protein expressions of CD11c and iNOS in wound tissue of mice in 2 mg/mL and 20 mg/mL deferoamine groups were significantly lower than those in DTI control group (P<0.01). The protein expressions of CD11c and iNOS in wound tissue of mice in DTI control group were significantly higher than those in normal skin tissue in normal control group (P<0.01). The CD11c protein expressions in wound tissue of mice in 2 mg/mL and 20 mg/mL deferoamine groups were significantly higher than those in normal skin tissue in normal control group (P<0.05 or P<0.01). The protein expression of iNOS in wound tissue of mice in 2 mg/mL deferoamine group was significantly lower than that in 20 mg/mL deferoamine group and normal skin tissue in normal control group (P<0.05). On PID 3, 7, and 14, the protein expressions of STAT3 and IL-10 in wound tissue of mice in 20 mg/mL deferoxamine group were significantly higher than those in DTI control group (P<0.05 or P<0.01), and the protein expressions of STAT3 were significantly higher than those in normal skin tissue in normal control group (P<0.05 or P<0.01). On PID 7 and 14, the protein expressions of IL-10 in wound tissue of mice in 20 mg/mL deferoxamine group were significantly higher than those in normal skin tissue in normal control group (P<0.01). On PID 3, 7, and 14, the protein expressions of IL-10 in wound tissue of mice in DTI control group were significantly lower than those in normal skin tissue in normal control group (P<0.05 or P<0.01). After 48 h of culture, compared with those in blank control group, the CD206 positive cell percentages in 100 μmol/L and 200 μmol/L deferoamine groups were significantly increased (P<0.01), while the CD86 positive cell percentages in 100 μmol/L and 200 μmol/L deferoamine groups were significantly decreased (P<0.01). Conclusions: Deferoxamine can promote the polarization of macrophages toward the anti-inflammatory M2 phenotype and improve wound healing by enhancing the STAT3/IL-10 signaling pathway in DTI mice.
Animals ; Deferoxamine/pharmacology* ; Inflammation ; Interleukin-10 ; Macrophages ; Male ; Mice ; Mice, Inbred C57BL ; Wound Healing

OBJECTIVETo investigate the effect of the iron chelator deferoxamine (DFA) in suppressing microglia activation and protecting against secondary neural injury in a rat model of intracerebral hemorrhage (ICH).

METHODSSD rats were randomly divided into sham-operated group, ICH group and DFA treatment group. ICH model was established by infusion of type IV collagenase into the right basal ganglia, and starting from 1 h after the operation, the rats received intraperitoneal DFA injections every 12 h for 7 days. The iron content in the perihematoma brain tissue was determined at different time points after DFA administration, and OX42 immunohistochemistry was used to observe the changes in the microglia. The contents of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the brain tissue were detected by ELISA. The neural death and neurological deficiency were measured using Nissl staining and neurological scores, respectively.

RESULTSThe iron content in the brain tissues around the hematoma was significantly increased 3 days after ICH and maintained a high level till 28 days, accompanied by a marked increase of microglial cells as compared to the sham-operated group. DFA injection caused significantly decreased iron content in the brain tissue, reduced number of microglial cells, and lowered levels of IL-1β and TNF-α. Neuronal loss around the hematoma was obviously reversed after DFA injections, which resulted in improved neurological deficiency.

CONCLUSIONDFA can suppress microglia activation by removing iron overload from the perihematoma brain tissue, thus reducing secondary neuronal death and neurological deficiency in rats with ICH.

Animals ; Cerebral Hemorrhage ; metabolism ; pathology ; Deferoxamine ; pharmacology ; Interleukin-1beta ; metabolism ; Iron ; metabolism ; Male ; Microglia ; drug effects ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo study the molecular mechanism of apoptosis of leukemic cells (K562 cells) induced by iron chelating agent deferoxamine (DFO).

METHODSThe exponentially growing K562 cells were used (1×10(6)/mL) in this study. The K562 cells were treated with different concentrations of DFO (10, 50 and 100 mmol/L), DFO+FeCl3 (10 μmol/L each) or normal saline (blank control). The cellular labile iron pool was measured with a fluorimetric assay using the metalsensitive probe calcein-AM. The viable count and cell viability were determined by typanblue assay. Cell apoptosis was determined by morphological study and flow cytometry assay. Caspase-3 activity in K562 cells was detected by colorimetry.

RESULTSAfter DFO treatment, the cellular labile iron pool and the viability of K562 cells were reduced and the cell apoptosis increased in a time- and dose-dependent manner compared with the blank control group. The apoptosis rate of K562 cells in the DFO+FeCl3 treatment group was not significantly different from that in the blank control group. The caspase-3 activity in K562 cells increased significantly 24 hrs after 50 and 100 μmmol DFO treatment when compared with the blank control group (P<0.01). There was a negative correlation between cellular labile iron pool and caspase-3 activity of K562 cells (r=-0.894, P<0.05).

CONCLUSIONSDFO induces apoptosis of leukemic cells possibly through decreasing cellular labile iron pool and increasing caspase-3 activity of the cells.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Deferoxamine ; pharmacology ; Flow Cytometry ; Humans ; Iron Chelating Agents ; pharmacology ; K562 Cells

OBJECTIVETo investigate the effect of deferoxamine (DFO) and DFO in combination with arsenic trioxide (ATO) on inhibition of HL-60 cells xenograft tumor growth in nude mice and its mechanism.

METHODSXenograft tumor model of HL-60 cell line in nude mice was established by inoculating HL-60 cells subcutaneously into nude mice. The tumor-bearing mice were randomly divided into four groups: 50 mg/kg DFO group (group I), 3 mg/kg ATO group (group II), combination group (50 mg/kg DFO + 1.5 mg/kg ATO (group III) and normal saline control group. The drugs were administered intraperitoneally from the day of inoculation (once a day for 10 days). The inhibitory effects on the tumor growth were compared. NF-κBp65 expression levels of the tumors were detected by immunohistochemistry (24h after the last administration).

RESULTS(1) Tumors growth could be observed in all of the nude mice on day 7 to day 8 after inoculation, 0.5 - 1.0 cm in diameter, and then grew rapidly; (2) Tumor weight of control group, group I, group II and group III were (2.62 ± 0.54) g, (2.55 ± 0.82) g, (2.34 ± 0.79) g and (1.95 ± 0.39) g respectively, and the growth inhibition rates in group I, group II and group III were 2.67%, 10.69% and 25.57% respectively. Both DFO alone and in combination with ATO could inhibit the growth of transplanted tumors, and the combination group exhibited more effects, with no vital organ damages in the tumor-bearing mice. (3) There was significant difference in mean value of NF-κBp65 expression among the three experimental groups (P < 0.05), with a descending order of control group > group II, > group I > group III.

CONCLUSION(1) Both DFO and ATO have antitumor activities on tumor-bearing mice, and their combination has an obvious and significant effect. (2) DFO combined with ATO, is well tolerated with no significant adverse effects in the nude mice. (3) Both DFO and ATO can downregulate NF-κBp65 expression of transplanted tumors, especially for their combination.

Animals ; Antineoplastic Agents ; pharmacology ; therapeutic use ; Arsenicals ; pharmacology ; therapeutic use ; Deferoxamine ; pharmacology ; therapeutic use ; Female ; HL-60 Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Oxides ; pharmacology ; therapeutic use ; Transcription Factor RelA ; metabolism ; Xenograft Model Antitumor Assays

Iron is an essential element for cell growing including tumor cells. This study was purposed to explore the effect of desferrioxamine (DFO) on cell line K562/A02 and its mechanism. K562/A02 cells were cultured with different concentrations of DFO. The inhibitory effects of adriamycin (ADM) used alone or combined with DFO on the proliferation of K562/A02 was evaluated by MTT assay. The apoptosis rate of K562/A02 cells after treatment with 0, 12.5, 25 and 50 µmol/L DFO alone or in combination with 1 mg/L ADM were analyzed by flow cytometry. ADM accumulation in K562/A02 cells after treatment with different concentrations of 0, 12.5, 25 and 50 µmol/L DFO were also analyzed by flow cytometry. The levels of BAX/BCL-2 and MDR1 mRNA were determined by RT-PCR, and then the protein level of P-glycoprotein (P-gp) was detected by Western blot. The results showed that the IC(50) of ADM for K562 and K562/A02 cells were (1.46 ± 0.07) mg/L and (40.98 ± 3.05) mg/L respectively. The resistance of K562/A02 cells to ADM was 28.06 times as that of K562 cells. After treatment of K562/A02 cell with DFO of 12.5, 25 and 50 µmol/L for 48 hours, the resistance of K562/A02 cells to ADM were increased by 24.95, 16.11 and 9.99 times respectively. When K562/A02 cells were incubated with different concentrations of DFO of 12.5, 25, 50 µmol/L for 48 hours, the apoptosis rat were (3.50 ± 0.30)%, (7.27 ± 0.32)% and (12.53 ± 1.21)% respectively. After co-culture with DFO and ADM for 48 hours, apoptosis rate were (6.13 ± 0.29)%, (9.57 ± 0.40)% and (18.97 ± 1.10)% respectively. The above apoptosis rates was much higher than that of control group (p < 0.05) and they were dose-dependent. In comparison between DFO + ADM group and DFO group, there was no significant difference (p > 0.05). Expression rate of BAX/BCL-2 increased. The levels of MDR1 mRNA reduced. Furthermore, expression of P-gp also decreased in K562/A02 cells. It is concluded that iron increase can promote K562/A02 cells growth and inhibit their apoptosis. Otherwise, iron-deprivation can induce K562/A02 cells apoptosis. DFO disturbs the iron metabolism and inhibits DNA synthesis of K562/A02 cells. This action of DFO may enhance the susceptibility of K562/A02 cells to apoptosis induced by chemotherapeutic drugs. The iron-deprivation may play a role in the treatment of leukemia with combination of DFO with other anticancer agents.
ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Apoptosis ; Deferoxamine ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Humans ; Iron ; metabolism ; K562 Cells ; drug effects ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism

The standard iron-chelator deferoxamine is known to prevent the growth of coagulase-negative staphylococci (CoNS) which are major pathogens in iron-overloaded patients. However, we found that deferoxamine rather promotes the growth of coagulase-positive Staphylococcus aureus. Accordingly, we tested whether deferiprone, a new clinically-available iron-chelator, can prevent the growth of S. aureus strains as well as CoNS. Deferiprone did not at least promote the growth of all S. aureus strains (n=26) and CoNS (n=27) at relatively low doses; moreover, it could significantly inhibit the growth of all staphylococci on non-transferrin-bound-iron and the growth of all CoNS on transferrin-bound iron at relatively high doses. At the same doses, it did not at least promote the growth of all S. aureus strains on transferrin-bound-iron. These findings indicate that deferiprone can be useful to prevent staphylococcal infections, as well as to improve iron overload, in iron-overloaded patients.
Deferoxamine/pharmacology ; Humans ; Iron/metabolism ; Iron Chelating Agents/*pharmacology ; Iron Overload/metabolism ; Microbial Sensitivity Tests ; Pyridones/*pharmacology ; Staphylococcus/*drug effects/growth & development ; Staphylococcus aureus/drug effects/growth & development ; Transferrin/metabolism

Apoptosis can be caused by hypoxia, a major factor during ischemic injury, in cardiomyocytes. However, the regulatory mechanisms underlying hypoxia-induced cardiomyocyte apoptosis have not yet been fully understood. E2F6, an identified E2F family member, has been demonstrated to repress DNA damage-induced apoptosis in our recent study. However, it is unclear whether E2F6 is involved in hypoxia-induced apoptosis. In this study, we determined the expression property of E2F6 during hypoxia-induced apoptosis in H9c2 cells, a rat ventricular myoblast cell line. The results showed that physical hypoxia and chemical hypoxia-mimetic agents desferrioxamine (DFO) and cobalt chloride (CoCl(2)) induced apoptosis in H9c2 cells. Physical hypoxia- and CoCl(2)-induced apoptosis was accompanied with a downregulation of endogenous E2F6 mRNA expression, but not protein expression. DFO treatment resulted in a significant downregulation of both mRNA and protein expressions of endogenous E2F6. These results suggest that E2F6 may be involved in DFO-induced apoptosis, while it is less sensitive in physical hypoxia- and CoCl(2)-induced apoptosis in H9c2 cells. In addition, the apoptosis induced by DFO may share different pathways from that induced by physical hypoxia and CoCl(2).
Animals ; Apoptosis ; Cell Hypoxia ; Cell Line ; Cobalt ; pharmacology ; Deferoxamine ; pharmacology ; Down-Regulation ; E2F6 Transcription Factor ; metabolism ; Myocytes, Cardiac ; cytology ; metabolism ; Rats

OBJECTIVETo investigate whether desferoxamine (DFO) preconditioning can induce tolerance against cerebral ischemia and its effect on the expression of hypoxia inducible factor 1alpha (HIF-1alpha) and erythropoietin (EPO) in vivo and in vitro.

METHODSRat model of cerebral ischemia was established by middle cerebral artery occlusion with or without DFO administration. Infarct size was examined by TTC staining, and the neurological severity score was evaluated according to published method. Cortical neurons were cultured under ischemia stress which was mimicked by oxygen-glucose deprivation (OGD), and the neuron damage was assessed by MTT assay. Immunofluorescent staining was employed to detect the expressions of HIF-1alpha and EPO.

RESULTSThe protective effect induced by DFO (decreasing the infarction volume and ameliorating the neurological function) appeared at 2 d after administration of DFO (post-DFO), lasted until 7 d and disappeared at 14 d (P < 0.05); the most effective action was observed at 3 d post-DFO. DFO induced tolerance of cultured neurons against OGD: neuronal viability was increased 23%, 34%, 40%, 48% and 56% at 8 h, 12 h, 24 h, 36 h, and 48 h, respectively, post-DFO (P < 0.05). Immunofluorescent staining found that HIF-1alpha and EPO were upregulated in the neurons of rat brain at 3 d and 7 d post-DFO; increase of HIF-1alpha and EPO appeared in cultured cortex neurons at 36 h and 48 h post-DFO.

CONCLUSIONDFO induced tolerance against focal cerebral ischemia in rats, and exerted protective effect on OGD cultured cortical neurons. DFO significant induced the expression of HIF-1alpha and EPO both in vivo and in vitro. DFO preconditioning can protect against cerebral ischemia, which may be associated with the synthesis of HIF-1alpha and EPO.

Animals ; Brain Ischemia ; drug therapy ; metabolism ; physiopathology ; Cells, Cultured ; Cerebral Infarction ; drug therapy ; metabolism ; physiopathology ; Deferoxamine ; pharmacology ; therapeutic use ; Disease Models, Animal ; Erythropoietin ; metabolism ; Fluorescent Antibody Technique ; Hypoxia-Inducible Factor 1, alpha Subunit ; drug effects ; metabolism ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; physiopathology ; Infarction, Middle Cerebral Artery ; drug therapy ; metabolism ; physiopathology ; Iron ; metabolism ; Ischemic Preconditioning ; methods ; Nerve Degeneration ; drug therapy ; metabolism ; physiopathology ; Neurons ; drug effects ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley ; Siderophores ; pharmacology ; therapeutic use ; Time Factors ; Treatment Outcome ; Up-Regulation ; drug effects ; physiology

This study was purposed to observe the changes of caspase-3 activity during apoptosis of HL-60 cells induced by an iron chelator, DFO (deferoxamine), and to explore the mechanism underlying apoptosis in HL-60 cells. The HL-60 cells treated with DFO were examined by light microscopy, flow cytometry (FCM) and DNA agarose gel electrophoresis; the activity of caspase-3 was determined by cellular immunohistochemistry; the transcription of the apoptotic gene of bax was detected by hybridization in situ. The results showed that the typical morphological character of apoptosis cells, DNA ladder and FCM assay confirmed that DFO could induce the apoptosis in HL-60 cells. The apoptotic rate increased in dose-and time-dependent manner. When cells had been cultivated with 100 micromol DFO for 12 hours, a few caspase-3 positive cells were found. In the process of time, the rate of caspase-3 positive cells was progressively higher than that in control (P < 0.05), while the level of bax transcription was also higher than that in the control. It is concluded the activation of caspase-3 and gene bax may be involved in the apoptosis of HL-60 cells induced by DFO.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Deferoxamine ; pharmacology ; HL-60 Cells ; Humans ; bcl-2-Associated X Protein ; biosynthesis ; genetics

To explore a rapid and easy method to detect labile iron of pool (LIP) in cells, HL-60 and K562 cells were cultured at a concentration 1 x 10(6)/ml in RPMI 1640 containing 10% heat-inactivated fetal bovine serum. The iron deprivation was induced by adding desferrioxamine (DFO) 10 - 100 micromol/L for 0 - 48 hours. The intracellular LIP was measured by probe calcein-AM. Calcein fluorescence was monitored in 1420 multilabel counter. The results indicated that when HL-60 and K562 cells were incubated with different concentrations of DFO, the calcein fluorescence intensity was higher than that of control group at 12, 24 and 48 hours (P < 0.05). Fluorescence value of representing LIP in DFO groups was lower than that in the control group. In conclusion, DFO can decrease LIP in leukemia cells. The approach used in this study may provide a simple and reliable method for detection of intracellular iron homeostasis.
Cation Transport Proteins ; antagonists & inhibitors ; biosynthesis ; metabolism ; Deferoxamine ; pharmacology ; Fluoresceins ; Fluorescent Dyes ; HL-60 Cells ; Humans ; Iron ; metabolism ; Iron Chelating Agents ; analysis ; metabolism ; Iron-Regulatory Proteins ; metabolism ; K562 Cells