| The hematopoietic system as well as the hematocytes is known to be sensitive to radiation, and low doses of radiation can induce damage. One of major syndromes of hematopoietic system by high dose total-body exposure to ionizing radiation is bone marrow aplasia. It is general agreed that radiation death in the midlethal dose range is due to impairment of bone marrow hematopoietic function such as leucopenia, erthropenia and thrombocytopenia which will ultimately develope predispose to whole body infection, hemorrhage, immune suppression and even death. Thus, the degree of hematopoietic dysfunction and recovery speed of hematopoiesis play crucial roles in determining the severity, duration, and therapeutic efficiency of radiation diseases.Radioprotectors are that are administered before exposure to ionizing radiation in order to reduce its damaging effects, including radiation induced lethality. Many synthetic or natural agents have been investigated in the past several years for their efficacy to protect against radiation damage. Among the numerous radioprotectors, estrogens are among the most effective classes. Some evidences indicated that both estradiol, which belongs to the natural estrogens, and the synthetic estrogens like diethylstilbestrol (DES) could exert protective effect on radiation sickness in experimental animals, including increasing the survival, elongating the mean survival time, and accelerating the recovery of the peripheral blood. Moreover, estrogens also promoted recovery of cancer radiotherapy- or chemotherapy- induced leucopenia in the clinic. However, their inherent toxicities, especially on reproductive systems, resulted in the applications of them in the clinic limited and warranted further search of a safer and effective radioprotector. To reduce toxicities, a strategy of combining radioprotective molecules working through different modes of action has also been attempted.Genistein (GEN) (4', 5', 7'-trihydroxy-isoflavone), a naturally occurring isoflavone found in soybeans, has structural similarity to 17β-estradiol but rather weak estrogenic activities (10-2 - to 10-3 -fold) compared to 17β-estradiol. As one of the most important phytoestrogens, GEN has no toxicity on human health. Many studies have demonstratedthat GEN possessed potential properties to act as both an estrogen and anti-estrogen, inhibit the activities of tyrosine kinases and DNA topoisomerases I / II, and improve immune system. Consequently, GEN has gained increasing attention because of its association with beneficial effects for patients with breast cancer, prostate cancer, cardiovascular disease, high cholesterol levels, and osteoporosis. Recently, newly promising physiological properties of GEN were obtained. In female ovariectomized mice, GEN could regulate B-lymphopoisis and prevent bone loss caused by estrogen deficiency without exhibiting estrogenic actions in the uterus. In surgically postmenopausal macaques, GEN could not induce proliferation or any other clinical changes in endometrial and mammary tissue. In addition, stimulating T-lymphocyte to secret IL-2 and IL-3 was found in GEN treated mice. All results indicated that GEN might exhibit selective estrogenic actions on bone marrow and hematopoietic system but nonendocrine effects on reproductive systems. Moreover, GEN was an effective antioxidant which could eliminate the free radicals and boost the antioxidant enzymes activities, resulted in increasing survival of irradiated mice and alleviating ultraviolet-B radiation-induced skin diseases. However, the effect of genistein on protection from radiation-induced hematopoietic system injury has yet to be examined.Based on both domestic and overseas researches, the aim of the experiments presented here was to assess in vivo radioprotection in irradiated mice and to study the possible mechanisms of radioprotective properties of GEN. During the investigations, adult male Kunming mice whole-body exposed to a sublethal dose of 6.0 Gy 60Co gamma-rays only, or a single oral administration of GEN at a dose of 160 mg/kg b.w. 24h before irradiation. The 30-day survival rate, some indices related to hematogenesis, and difference of radioprotection actions between GEN and DES were examined following irradiation. Meanwhile the distribution of cell cycle, the cell cycle-specific expression of protein and mRNA of bcl-2, cyclins, CDKs, and the changes of CDKs activities in bone marrow nucleated cells (BMNCs) were observed. In addition, the expression of hematopoietic factor receptors, the hematopoiesis- stimulating effects of serum, the adhesion capabilities of bone marrow stromal cells (BMSCs) were studied as well as the expression of some factors associated with cell adhesion. In brief, the effects of GEN on hematopoiesis and hematopoietic microenvironment (HME) were investigated to explore the radioprotective mechanisms of GEN.The main results and conclusions were summarized as follow:1. GEN administration rendered approximately 53.33% survival on day 30 post-irradiation (about 36.66% higher than that of irradiated group) and lengthened the mean survival time (about 3.53 days longer than that of irradiated group). It showed that administered to mice 24h prior to irradiation, GEN possessed highly protective efficacy on prevention of mortality in sublethally irradiated mice and its protective factor was 1.60, although it has no significant effects on the survival of non-irradiated mice. Moreover, protecting abilitys of GEN was powerful than those of DES (45.56% survival in mice protected by DES).2. Granulocyte and lymphocyte counts in the peripheral blood as well as BMNCs were changed rapidly and returned to normal level in normal mice within 6 days after GEN given. After irradiation, in comparison to mice of only irradiated group, the damages on bone marrow tissue and spleen were alleviated, and the declined level of circulating granulocyte, lymphocyte, platelet and BMNCs counts, as well as hemoglobin content, were lessened in mice protected by GEN. Stimulating recovery of hematograms and BMNCs and the increase of numbers of CFU-S and CFU-GM were also detected in GEN pre-treated group. Above-mentioned effects of GEN were similar to those in DES-pretreated mice and the activities of stimulating recovery of granulocytes, lymphocytes and BMNCs were powerful than those of DES. These findings revealed that GEN has radioprotective action on hematopoietic system against radiation-induced damage, which perhaps attributed to strengthen the radioresistant and the viability of hemapoietic stem cells (HSCs)/ hematopoietic progenitor cells (HPCs), and reduce the direct damage of rays on blood cell, resulting in the increase of HSCs/HPCs numbers in irradiated mice as well as potentiation of the abilities of proliferation and differentiation of HSCs/HPCs to promote the rehabilitation of hematopoiesis.3. The cell cycle of BMNCs changed significantly in short period after GEN treated to mice. First, the proliferation suppression of BMNCs was observed and most cells were in Go/Gi phase on day 1 (from 60.47% up to 67.25%). Then, that in S + G2/M phase increased greatly, mainly that in S phase on day 2 (from 28.70% up to 34.55%). Finally, cell proportion declined to normal level on day 4. Results suggested that the properties of GEN-induced the redistribution of cell cycle of BMNCs could contribute to enhance the radiotolerance of BMNCs, and to promote the survival, proliferation and differentiation of hematopoietic cells in irradiated mice.All these were beneficial effects for stimulating hematopoietic system injury to restoration. Moreover, we inferred that those also maybe an underlying mechanism for GEN inducing the changes in the peripheral blood of normal mice.4. Compared to mice of only radiated group, the number of BMNCs in Go/Gi phase decreased obviously following irradiation, protein and mRNA expressions of bcl-2, Cyclin Dl, Cyclin E, CDK2, CDK4, and the activities of Cyclin E-CDKs were up-regulated greatly in BMNCs of mice pretreated with GEN 24h before irradiation. Regarding the sequence of changes induced by GEN in the post-irradiation, it could be assumed that the radioprotective action of GEN might be consequence of accelerating BMNCs of marrow-depressed mice to get out of "Gl-phase-block", suppressing apoptosis, entering into cell cycle and radically speeding up the proliferation and differentiation of BMNCs to uphold the hematopoietic tissue regeneration. Briefly, changing the cell cycle and enhancing the abilities of proliferation and differentiation of BMNCs played an important role in amelioration of bone marrow hematopoietic tissue after radiation damage.5. In mice pre-treated with GEN 24h before irradiation, apart from the damage of BMSCs alleviated, the capabilities of survival and proliferation, CFU-F formation and plating efficiencies of BMSCs were strengthened in comparison with those of only irradiated mice. FCM analysis displayed that the up-regulated expressions of VCAM-1, Fibronectin (FN) and Laminin (LN) were found in mice protected by GEN after irradiation. Above-mentioned changes resulted in the reinforcing of the cell adhesion between BMSCs and blood cells or intercellular adhesions of blood cells. Those maybe correlate with GEN changing the cell cycle of BMSCs and hematopoietic cells, inducing proliferation and differentiation, and promoting reconstitution of hematopoietic system after radiation insult.6. The hematopoiesis-stimulating activities of serum enhanced distinctly in all irradiated groups. With GEN administration 24h before irradiation, the hematopoiesis-stimulating activities of serum became more powerful resulting in the increase of numbers of CFU-GM and CFU-E, compared to those in only irradiated mice, and stimulating activities on CFU-GM were more powerful than that on CFU-E. Furthermore, the hematopoietic-suppressing activities of serum were decreased in GEN pre-treated mice, and no further significant differences between CFU-GM and CFU-E were found. In addition, the declined level of the expressions of c-kit and IL-3Ra on BMNCs were decreased inmice protected by GEN, and number of positive BMNCs was much more than those of only radiated mice on day 9 after irradiation. These experimental evidences demonstrated that changing hematopoiesis-stimulating activities of serum and inducing the expression of hemopoietic growth factors were important mechanisms of GEN in protecting against radiation-induced hematopoietic syndrome and accelerating restoration of hematopoiesis.In brief, GEN was an effective radioprotector. This present study suggested that the protective properties associated with the pre-treatment of GEN were largely a consequence of the reducing of radiation-induced damage of hematopoietic cells and bone marrow stromal cells directly or indirectly, promoting cell adhesion between BMSCs and blood cells, induction of haemopoietic colony-stimulating activities and potentially the activation and/or enhancement of haemopoietic cytokine cascades, and ameliorating hematopoietic microenvironment in the sublethaly irradiated animals. These changes might ultimately impact the cell cycle profile of haemopoietic cells and bone marrow stromal cells, and therefore their ability to withstand and/or recover from radiation insult and enhance survival.
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