| Radiation can damage multi-organ and multi-tissue, such as nervous system, endocrine system, hematopoietic system, particularly in bone marrow which is very sensitive to radioactive exposure. To alleviate the radiation injury, it is an urgent need to develop some non-toxic or low-toxic radiation protection agent. To date, there are many research findings regard to the protective effects of Angelica polysaccharide (APS) against radioactive exposure on mice. APS is the mostly extracted from Angelica, a Chinese herbal medicine which has been proven to increase blood cells refreshment and blood circulation. APS has been found to promote stromal cell excrete hematopoietic growth factor, and to mobilize hematopoietic stem and progenitor cells (HSPC) from bone marrow to peripheral blood. Moreover, some studies have shown that APS can speed up the peripheral blood restoration and strengthen immune surveillance capability on mice exposed by radiation. However, the anti-radiation mechanisms of APS still remain inconclusive.What is the mechanism that APS induces hematopoietic recovery? It is still unclear to promote peripheral blood through either accelerating mature blood cells migration from bone marrow to peripheral blood or advancing post-radiational HSPC relic to proliferation and differentiation? The regulation of blood cell generation is a complex network system, and the interaction between adhesion molecules and ligands is one of the most important processes. Both adhesion molecule receptors on hematopoietic cells and adhesion molecule ligands on stromal cells are damaged in the early period of acute radiation injury. Many adhesion molecules involve in the function of HSPC homing, generation and differentiation, especially CD44 and CD49d whose expression levels can regulate HSPC generation, differentiation and homing via the adhesion of HSPC and extracellular matrix. Meanwhile, it has been found that radiation can induce hematopoietic disturbance by blockage of G1 phase. Therefore, relieving the blockage of G1 phase is a very important way to rival radiation injury of bone marrow. Phases of cell cycle have different key check points. G1/S phase check point is the most important, and it can be regulated by cyclin and cyclin dependented kinase. CyclinD2 can regulate cell cycle transformation from G1 phase to S phase of hematopoietic cells in mice. We study the possible mechanisms of APS on hematopoietic recovery in acute radiation injury mice by expression changes of CD44, CD49d, the mRNA and protein of CyclinD2 in bone marrow mononuclear cell (BMNC). In this project we use BALB/c mice model of acute radiation injury for studying the effects of APS on adhesion molecule and cell cycle of BMNC. We want to provide new ideas for deeply exploring the underlying mechanisms of APS on hematopoietic recovery.Objective: To explore the effects of APS on adhesion molecule and cell cycle of BMNC in an acute radiation injuried mice model to deeply understand the molecular mechanisms of hematopoietic recovery.Methods:1. BALB/c mice were divided into 7 groups. Neither radioactive exposure nor treatment was given to the normal control group mice. 3 groups of mice were pretreated with Normal Saline (NS), 2 mg/kg APS and 8 mg/kg APS prior to radiation exposure. 3 groups of mice were post-treated with Normal Saline (NS), 2 mg/kg APS and 8 mg/kg APS after radiation exposure. Mice in above six groups were homogeneously radiated by X ray at 4.0 Gy and 1.25min.2. Quantity of peripheral blood cells and BMNC were counted.3. The expression levels of CD44 and CD49d in Sca-1+BMNC was detected by flow cytometry.4. Cell cycles of BMNC was detected by flow cytometry5. The effects of APS on the proliferation of BMNC was evaluated by MTT assay.6. The effects of APS on the mRNA and protein expression levels of BMNC CyclinD2 were quantified by RT-PCR and Western blot.Results:1. Mice model evaluation: After the model had been successfully established the count of peripheral blood cells and BMNC decreased, and mice showed symptoms, such as reducing drink, slowly eating, fur in a mess, shortness of glossiness, acting slowly, and drawing close with each other.2. Changes of peripheral blood cells and BMNC: The count of peripheral blood cells and BMNC decreased in all NS groups on day 7 and 14 after radiation, and it had significant difference with the normal control group(P<0.05).The counts of peripheral blood cells and BMNC in all APS groups descend less than that of NS groups at the same time. The counts of peripheral blood cells and BMNC in APS pretreatment groups recovered more quickly than APS treatment groups (P<0.05). 8mg/kg APS groups had no significant difference compared to 2mg/kg APS groups at the same time.3. Changes of the expression levels of CD44 and CD49d in Sca-1+BMNC: The expression levels of CD44 and CD49d in Sca-1+BMNC in all NS groups were lower than the normal control groups on day 7 and 14 after radiation (P<0.05),and the expression level of CD44 on day 14 is higher than that on the day 7(P<0.01). The expression levels of CD44 and CD49d on Sca-1+BMNC in APS treatment groups were obviously higher than the normal control group(P<0.05), but the expression level in APS pretreatment groups were lower than that in the normal control group on day 7(P<0.05). The expression of CD44 and CD49d on day 14 in all APS groups were lower than that on day 7, and they were all lower than the normal control group(P<0.05).4. Changes of the bone marrow cell proliferation: The cell viability and growth in bone marrow in APS groups were higher compared to NS groups via MTT assay(P<0.01), but both were lower than the normal control group(P<0.05).5. Changes of the BMNC cell cycle: G0/G1 phases were blocked and S phases were decreased in all NS groups on day 7 and 14 after radiation, and it had significant differences compared with the normal control group(P<0.05); S phases in all APS groups synthesize more actively on day 7 and 14, and the percentage of G0/G1 phases decreased as time lapses, but it still not returned back to normal level.6. Changes of the mRNA and protein expression levels of BMNC CyclinD2: The mRNA and protein levels of BMNC CyclinD2 in all NS groups were lower than the normal control groups on day 7 and 14 after radiation(P<0.05); The mRNA and protein levels of BMNC CyclinD2 in all APS groups were obviously higher than those in all NS groups at the same time(P<0.05).Conclusion: APS can promote hematopoietic recovery in a mouse model of acute radiation exposure. Pretreatment of APS showed better protective effects than post-treatment groups in the recovery of peripheral blood cells and BMNC. APS may increase the release speed of mature blood cell from bone marrow into peripheral blood circulation through regulating the adhesion molecular such as CD 44 and CD49d in Sca-1+BMNC. Meanwhile, it also stimulates cell cycle rate by up-regulating CyclinD2 level in BMNC on day 7 and 14. These discoveries provide new experimental evidences for further studying the effects of APS on hematopoietic function disturbance caused by acute radiation injury and providing a new potential medical agent Angelica against radiation exposure.
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