Effects Of Angelica Polysaccharides On Bone Marrow Stromal Cell In Radiation Injured Mice

Bone marrow microenvironment provides a beneficial place for the development and maturation of hematopoietic cells, in which bone marrow stromal cells play an important role. In addition to direct role in hematopoietic stem/progenitor cells (HSPC),the adhesion molecules on the surface of BMSC may promote adhesion among HSPC, BMSC and the extracellular matrix of microenvironment, thereby promoting the proliferation and differentiation of hematopoietic cells.Vascular cell adhesion molecule-1(VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expressed mainly on the BMSC, the adhesion between BMSC and HSPC was mediated by them, and promote the proliferation and differentiation of hematopoietic cells. Moreover, in the role of several cytokines, the two molecules were also involved in hematopoietic stem / progenitor cell mobilization and homing. BMSC may also secrete some cytokines to regulate the haemopoiesis. Vascular endothelial growth factor(VEGF) is a glycoprotein that stromal cell secrete,which can stimulate endothelial cell proliferation, migration, increase vascular permeability and promote angiogenesis.The body may be damaged by radiation, especially in hematopoietic system.After radiation damage, the count of BMSC had decreased, most of them were arrested in G0/G1 phase, the proliferation ability of BMSC was impaired, and that lead to the capacity of BMSC forming CFU-F decreased significantly, the apoptosis ratio of BMSC can also be increased after radiation damage. In addition, radiation damage also affects the expression of BMSC adhesion molecules and inhibits the secretion of cytokines, inhibits the BMSC function of regulating haemopoiesis and lead to hematopoietic dysfunction.The research on radiation protection agent has been progressed for many years. The function was always decreased due to the serious toxicity effects of the chemical radioprotective agent.Consequently; it is indispensable to develop less toxic traditional Chinese medicine for clinical radiation protective agent. APS is the important component of Chinese herbal medicine. Some studies have shown that APS is radioprotective, which can speed up the peripheral blood and lymphocytes restoration, improve antioxidant capacity of the tissues. But the effect of APS on radiation injury in bone marrow hematopoietic microenvironment has not been reported. Base on this, from the changes of BMSC cell cycle, the count of CFU-F, apoptosis ratio, expression of CD54, CD106 and VEGF mRNA, we study the possible mechanisms of APS on hematopoietic recovery in radiation damage mice.In this study, BALB/c mice were irradiated using a linear accelerator on whole body. After the establishment of acute radiation injury animal model, we study the effects of APS on expression of adhesion molecules and vascular endothelial growth factor of BMSC in radiation injury mice. We want to provide new experimental basis for APS that used as a radioprotective agent in clinical.Objective: To study the effects of APS on proliferation,apoptosis ratio and expression of adhesion molecules and vascular endothelial growth factor of bone marrow stromal cell in radiation injured mice to explore the molecular mechanisms of hematopoietic recovery deeply.Methods:1. BALB/c mice were divided into 4 groups randomly: Normal group mice will not be made any processing. The other BALB/c mice were irradiated with a total dose of 4.0 Gy X rays for 1.33min. According to the different drugs and dosage, groups were divided into saline (NS) group, 2 mg/kg APS group and 8 mg/kg APS group after radiation exposure.2. We counted peripheral blood cells and BMNC with conventional methods.3. We cultured BMSC with conventional method and recorded the time of 80% adherent BMSC.4. We cultured BMSC with conventional method and counted the number of CFU-F in each group.5. Cell cycles of BMSC in radiation injury mice were detected by flow cytometry.6. Apoptosis ratio of BMSC in radiation injury mice was detected by flow cytometry.7. The expression levels of CD54 and CD106 on the surface of BMSC in radiation injury mice were detected by flow cytometry.8. The effects of APS on the VEGF mRNA expression levels of BMSC in radiation injury mice were detected by RT-PCR assay.Results:1. Establishment of mice model: After mice model had been successfully established, irradiated mice showed symptoms, such as drinking water and eating food were reduced; acting slowly, lethargy, fur in a mess, shortness of glossiness, occasional diarrhea, and moving close with each other.2. Changes of peripheral blood cells and BMNC counts: The counts of peripheral blood cells and BMNC decreased substantially in all NS groups on day 7,14,and 21 after radiation, and it had significant difference with the normal group(P<0.05).The counts of WBC, PLT and BMNC in all APS groups declined less than that of NS groups at each observed time point(P <0.05).The counts of WBC in 8 mg/kg APS groups recovered more rapidly than 2 mg/kg APS groups on day 14,and 21(P<0.05). The counts of BMNC in 8 mg/kg APS groups were more than 2 mg/kg APS groups at each observed time point(P<0.05).Only indicators in 8 mg / kg APS groups on day 21 returned to normal. The counts of RBC in APS groups descended more than that of NS groups on day 7 after radiation(P<0.05),but recovered more rapidly. The counts of RBC in 8 mg/kg APS groups on day 14 and 2 mg/kg APS groups on day 21 began increasing, and the one in 8 mg/kg APS groups returned to normal on day 21.3. Contrast on the time of 80% BMSC adherence:It cost much more time to adhere in NS groups compared with the normal level at each observed time Poin(tP<0.05).BMSC adhered more rapidly in APS groups than the one of NS groups at each observed time Point(P<0.05).The time of 80% adherence in 8 mg/kg APS and 2mg/kg APS groups had no significant difference at each observed time point. The capacity of adherence recovered normal level on day 21.4. Changes of CFU-F quantity: The counts of CFU-F reduced in all NS groups at each observed time point, and it had significant difference with the normal group(P<0.05),there was no significant difference between APS and NS group in the number of CFU-F on day 7. But the counts of CFU-F in APS groups increased significantly more than that of NS groups on day 14 and 21(P<0.05), there was no significant difference between 8 mg/kg APS and 2mg/kg APS group on day 7,14. The quantity in 8 mg/kg APS group were more than 2mg/kg APS group on day 21(P<0.05),but were similar to the normal control group.5. Changes of BMSC cell cycles: G0/G1 phases were arrested and S phases descended in all NS groups on day 7,14,21 after radiation, and it had significant differences with the normal group(P<0.05);The percentage of G0/G1 phases had no significant difference among each group on day 7. The percentage of G0/G1 phases in APS group was significantly lower than NS group on day 14,21(P<0.05). The percentage of S phases in 2mg/kg APS and NS groups had no significant difference at each observed time point, but S phases in 8mg/kg APS groups synthesize more actively than NS group(P<0.05).All phases recovered more quickly in 8mg/kg APS groups than 2mg/kg APS groups on day 14,21(P<0.05), and returned normal level on day 21.6. Changes of apoptosis ratio of BMSC: The apoptosis ratio in all NS groups were significantly higher compared with the normal control group on day 7,14,21(P<0.05); The apoptosis ratio in APS groups were lower than NS groups at each observed time point(P<0.05);but the apoptosis ratio in 2mg/kg APS groups and 8 mg/kg APS groups were similar at each observed time point, the apoptosis ratio in APS groups returned normal level on day 21.7. Changes of the expression levels of CD54 and CD106 on the surface of BMSC: The expression levels of CD54 and CD106 on BMSC in all NS groups were obviously lower than the normal groups at each observed time point (P<0.05); The expression levels of CD54 and CD106 in BMSC in APS groups were significantly higher than NS groups at each observed time point (P<0.05); The expression levels of CD54 in 2mg/kg APS groups and 8mg/kg APS groups had no significant difference at each observed time point, but the expression levels of CD106 in 8mg/kg APS groups were higher than 2mg/kg APS groups(P<0.05); The expression levels of CD54 and CD106 in BMSC in APS groups recovered on day 21.8. Changes of the expression levels of VEGF mRNA in BMSC: The expression levels of VEGF mRNA on BMSC in all NS groups were significantly lower than the normal control groups at each observed time point (P<0.05); The expression levels of VEGF mRNA in BMSC in APS were significantly higher than NS groups at each observed time point (P<0.05); The expression levels of VEGF mRNA in BMSC in 2mg/kg APS groups and 8mg/kg APS groups had no significant difference at each observed time point. The expression levels of VEGF mRNA in APS groups returned normal on day 21.Conclusion: APS can protect BMSC of the irradiated injury mice and then contribute to hematopoietic recovery. The mechanism may be APS promoted the BMSC proliferation,descend apoptosis ratio and increase adherence in cells by up-regulating the expression levels of CD54, CD106 of BMSC, and APS may promote new angiogenesis by stimulating the secretion of BMSC VEGF,which contribute to injured microenvironment recovery, then provide a good development environment for hematopoietic stem and progenitor cell proliferation and differentiation. Our results provide a new theoretical basis for the in-depth study of radiation protective effect of APS.