| Objective: To explore the antagonistic effect of low intensity microwave onγ-ray induced hematopoietic damage as well as the underlying mechanism, to provide basic data for the potential clinical application of low intensity microwave to prevent theγ-ray induced acute hematopoietic injury.Methods: In mice survival experiment, 140 mice were randomly divided into 5 groups:γ-ray irradiation alone group(8.0Gyγ-ray), positive control group (Amifostine+8.0Gyγ-ray) and three combined radiation groups (12μW/cm2, 120μW/cm2 and 1200μW/cm2 900MHz microwave+8.0Gyγ-ray). Each group consisted of 28 mice. Mice of combined radiation groups were exposed initially to 900MHz microwave radiation, 1hr/day for 14 days, and on day 15, they were subsequently exposed to 60Coγ-ray radiation. Mice survival rate, mean survival time and protective factors were determined.To study the antagonistic effect of low intensity microwave, mice were randomly divided into 4 groups: the control, microwave alone (120μW/cm2 microwave),γ-ray alone (8.0Gy 60Coγ-ray), and combined (120μW/cm2 microwave + 8.0Gy 60Coγ-ray) group. Mice of microwave alone group and the combined exposure group were exposed initially to 900MHz microwave radiation, 1hr/day for 14 days, on day 15, the combined andγ-ray alone groups were subsequently exposed to 8.0Gy 60Coγ-ray irradiation. The following analyzes were done on day 3, 6, 9, and 12 afterγ-ray irradiation respectively. (1) Number counting of peripheral blood cell and measurement of organ coefficient. (2) Pathological observations of bone marrow and spleen. (3) RT-PCR assays for expression of cell cycle related genes Cyclin-D1, Cyclin-E, CDK4, and CDK2 and apoptosis inhibiting gene bcl-2. (4) Colony forming unit-granulocyte and macrophage (CFU-GM) assays and colony forming unit-spleen (CFU-S) assays. (5) In vitro culturing and morphological observation of bone marrow stromal cells (BMSCs), and flow cytometer assay for expression of cell adhesion molecule VCAM-1. (6) Enzyme-linked immunosorbent assay (ELISA) for serum GM-CSF and IL-3.To investigate the underlying mechanism of antagonistic effect of low intensity microwave, qualitative and quantitative detection of intracellular Ca2+ level in mice bone marrow cells were determined by flow cytometer and confocal laser scanning microscopy, and expression of NF-κB protein were detected with western blotting assay.Results: Pre-treatment of 120μW/cm2 900MHz microwave could increase survival rate ofγray exposed mice from 17.86 %( normal control group) to 42.86%, and prolong survival time significantly, the protective factor of microwave was 1.47, which was stronger than amifostine.(1) Pre-exposure with 120μW/cm2 microwave obviously attenuated theγray-induced reduction of WBC number and organ (spleen, thymus) coefficient. (2) Compared with ionization alone group, bone marrow pathological alterations were less severe in combined group as evidenced by less hematopoietic cell reduction, higher hematopoietic tissue volume with less interstitial hemorrhage, and decreased edema. For spleen vissue, the lymphoid tissue in combined group was more compacted, apoptotic cells were relatively fewer, and proliferation of lymphocytes was observed in white pulp. (3) Compared to ionization alone group, the mRNA expressions of Bcl-2, Cyclin-D1, Cyclin-E, CDK4, and CDK2 were greatly up-regulated, and the number of CFU-GM was significantly increased in combined group. (4) The number of CFU-S of microwave alone group was significantly much than that of control group, and the number of CFU-S of combined group was greater than that ofγ-ray alone group. (5) The proliferation ability of BMSCs was inhibited significantly in ionization radiation alone group, while the number of BMSCs in combined group increased obviously and the cell adhesion ability restored quickly. The expression level of VCAM-1 in combined group was significantly higher than that of ionization alone group. (6) On day 9 and 12, the GM-CSF levels for combined group were significantly higher than that ofγ-rays group, and serum IL-3 levels for combined group were significantly increased on day 6 and 12.Research of antagonistic mechanisms for low intensity microwave: (1) The concentration of intracellular Ca2+ in combined group showed an upward trend 1h afterγ-rays, and increased significantly 24h afterγ-rays. (2) The level of NF-κB in combined group was higher than that of ionization alone group especially 24h afterγ-ray radiation. Conclusion: (1) pre-treatment with 120μW/cm2 900MHz microwave could increase survival rate ofγ-rays irradiated animals, prolong their survival time, increase peripheral white blood cells and enhance the restoration of hematopoietic function to a certain extent.(2) For bone marrow nucleated cells, mRNA expressions of bcl-2, Cyclin D1, Cyclin E, CDK2 and CDK4 were up-regulated greatly in BMNCs of mice pre-treated with low intensity microwaves. It could be assumed that the radioprotective effects might be the consequence of promoting BMNCs of marrow-depressed mice to get out of"G1-phase-block", suppressing apoptosis, speeding up the proliferation and differentiation of BMNCs, and promoting hematopoietic tissue regeneration.(3) For bone marrow microenvironment, pre-treatment of low intensity microwave could alleviate theγ-rays induced damage of BMNCs, as evidenced by enhanced proliferation ability and plating efficiencies, up-regulated expressions of VCAM-1 as well as increased serum GM-CSF and IL-3 level.(4) As a potential NF-κB inducing factor, the stimulating effects of low intensity microwave could be re-amplificated in the secondary radiation(γ-ray irradiation), Low intensity microwave exposure could induce NF-κB expression and trigger cascade reactions with a series of signaling molecules to antagonize theγ-ray induced damage. These reactions include: (1)Activation of downstream signaling molecules such as Cyclin-D1/CDK4, Cyclin-E/CDK2 could influence cell cycles; (2) Expressions of apoptosis inhibiting genes such as Bcl-2 could reduce apoptosis; (3) Up-regulation of downstream genes GM-CSF and IL-3 could stimulate hematopoietic proliferation; (4) Activation of downstream genes such as cell adhesion molecules VCAM-1 could promote HSCs/HPCs proliferation and enhance the reconstructions of hematopoietic system eventually.
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