| Ionizing radiation can induce the organism to produce a lot of free radicals. Free radicals may cause structural changes and loss of biological activity of biological macromolecules such as DNA,RNA, resulting in the occurrence of oxidative damage. Superoxide dismutase(SOD) is a kind of metal enzyme which is widely exists in plants and microbes, can catalyze the superoxide anion radical to disproportionation reaction, scavenge free radicals, thereby protect the damaged cells, so it plays major role in the oxidant and antioxidant balance. Mmaleic dialdehyde (MDA) is product of lipid peroxide degradation. Usually we can determine the degree of lipid peroxidation by detecting the content of MDA, which indirect react to the degree of cell injury.Apoptosis (AP) refers to be a process in which the cells take acceptance of some signal or be stimulated by some factors and die out by the interactions of some apoptosis related gene. It is a complexly, involved many factors biological process. Signaling pathways of cell apoptosis induced by ionizing radiation:signaling pathway of dependent on the DNA damages, signaling pathway of dependent on the cytoplasmic ionized damage, dependent on membrane damage and signaling pathway of SAPK/JNK. P53, Bc1-2, Caspase-3play an important role in apoptosis process which is induced by ionizing radiation. Ionizing radiation can induce cell apoptosis by P53that is dependent on the way to induce death receptor(Fas) and its ligand (FasL) expression. Bc1-2is one of cell apoptosis protein which is taken most attention in the study of apoptosis related gene and it can inhibit cell apoptosis induced by radiation. During apoptosis process, Caspase-3is one of the key executive elements and can cut many of the key proteins in the apoptotic process. It is cut in the inactivity full-length fragment Caspase-3(35KD) between Asp28and Ser29, Asp175and Ser176then generates activity of17KD subunit and12KD subunit. Its activation should be regarded as an important index of cell apoptosis.Bone marrow is highly sensitive to ionizing radiation. Bone marrow mononuclear cells(BMNC) are mainly consisted of lymphocytes and monocytes. After radiation injury, the number of BMNC greatly reduce and activity of SOD decrease while the content of MDA increase. On the other hand, the percentage of Go/Gi phase and apoptosis rate elevate as well as the expression of apoptosis related protein Bcl-2, P53, Caspase-3change; resulting in apoptosis increase.Angelica polysaccharide (APS) is one of main activity in angelic which is Chinese traditional hemopoietic medicine. Research shows:APS can be impact on the hematopoietic system, antiradiation, antioxidant, antiviral, antitumor. At present, it is not very clear that the mechanisms how APS resistant radiation. This experiment is on the basis of the acute radiation injury animal models with C57BL/6mice, then we study the effect of APS on oxidative damage and apoptosis of BMNC in injuryed mice, aiming to explore the molecular mechanism of APS in radiation and provide theoretical basis for the development of radio-protective agent.Objective:To study the effect of APS on BMNC oxidative damage, proliferation, apoptosis rate and expression of apoptosis related proteins in acute radiation injuryed mice to deeply understand the molecular mechanism of APS radiation.Methods:1.C57BL/6mice were randomly divided into10groups.The normal group (no treatment),normal saline(NS) group,2mg/kg APS group and8mg/kg APS group. According to time,the posterior3groups were divided into ld,3d,7d groups and the total were9groups.Mice in above9groups were homogeneously radiated by X-ray at4.0Gy about1.25min.The absorbed rate were3.76Gy/min.The length from focus to rats were100cm and the area of it is about25x25cm2.Then the mice were given different dosages of APS(2mg/kg APS and8mg/kg APS) or normal saline(NS) for7days continuously by intraperitoneal injection after radiation(injecting once a day after weight and then according to the weight of mice were calculated the corresponding injection volume). These mice were killed and their BMNC were extracted after1,3, and7days of injection.2.The activity of SOD and content of MDA of BMNCs were detected by the method of colorimetric assay.3.The cell cycle of BMNC were detected by flow cytometry(FCM).4.The cell apoptosis rate of BMNC were detected by FCM.5.The expression of Bcl-2of BMNC were detected by FCM.6.The expression of P53of BMNC were detected by immunocytochemistry and Western Blot(WB).7.The expression of Caspase-3of BMNC were detected by WB.Results:1.Evaluation of acute radiation injury in mice model:After the model had been successfully established,the mice in NS group showed the symptoms that they had more water,eat less, had diarrhea occasional, had bloody stool and at the same time their furs were somewhat chaotic and easy to fall off. Meanwhile their activity significantly delayed and they became sleepiness, moved closer to each other. However,the mice treated with APS were lighter.2.Effects of the APS on the activity of SOD and content of MDA of BMNC in radiation injuryed mice:After1,3and7days of irradiation,compared with the normal group, the activity of SOD was significantly decreased(P<0.05),the content of MDA significantly elevated in all NS group(P<0.05).Compared2mg/kg APS group with NS group in the same time, the activity of SOD increased and the content of MDA decreased in the before group(P>0.05), while in8mg/kg APS group the activity of SOD significantly increased and the content of MDA significantly decreased (P<0.05), but they were still not close to the normal level.3.Effects of APS on cell cycle of BMNC in radiation injured mice: After irradiation G0/G1phases were blocked in NS groups compared with the normal group and the differences were significant (P<0.05). Compared with2mg/kg APS group and NS group (1,3d), the G0/G1phase decreased, but it was no significant differences (P>0.05), while compared with2mg/kg APS group (7d),8mg/kg APS group and NS group at the same time, the G0/G1phase significantly decreased(P<0.05), but still higher than normal group.4.Effects of APS on apoptosis rate of BMNC in radiation injured mice:After irradiation apoptosis rate was significantly increased in NS groups compared with the normal group (P<0.05). Compared with2mg/kg APS group and NS group (1,3d), the apoptosis rate decreased, while compared with2mg/kg APS group (7d),8mg/kg APS group and NS group at the same time, the apoptosis rate significantly decreased(P<0.05), but still higher than normal.5.Effects of APS on the expression of Bcl-2of BMNC in radiation injured mice:After irradiation the expression of Bcl-2were decreased in NS groups compared with the normal group and the differences were statistically significant (P<0.05). Compared with2mg/kg APS group and NS group (1,3d), the expression of Bcl-2increased, but it was no significant differences (P>0.05), while with in2mg/kg APS group (7d),8mg/kg APS group and NS group at the same time, the expression rate of Bcl-2significantly increased (P<0.05), but still lower than normal.6.Effects of APS on the expression of P53of BMNC in radiation injured mice:The results of immunocytochemical showed:There was no expression of P53in BMNC of the normal mice, while expression of P53increased significantly in the NS group,2mg/kg APS and8mg/kg APS group. However, expression were no significant differences among the three groups at same time. The results of WB showed:After irradiation with1,3and7days, the expression of P53were significantly higher in NS group than normal group (P<0.05). Compared with2mg/kg APS group and NS group at the same time, the expression of P53protein decreased, but the difference was not significant (P>0.05). Compared with8mg/kg APS group and NS group at the same time, the expression of P53protein decreased significantly (P<0.05). 7.Effects of APS on the expression of Caspase-3of BMNC in radiation injured mice:Caspase-3was not activated in the normal group so there were only the expression of the full-length fragments (35KD), but no expression of activated fragment (12+17KD). On the other hand, after irradiation in NS group the expression of Caspase-3with full-length fragments were significantly less than that of normal groups, and activated fragment significantly increased (P<0.05). Compared with2mg/kg APS group and NS group at the same time, expression of full length fragment increased, activated fragment was reduced lightly, so the differences were not significant (P>0.05). However compared with8mg/kg APS group and NS group at the same time, expression of full length fragment increased, activated fragment decreased significantly (P<0.05).Conclusion: APS can improve the activity of SOD and reduce the content of MDA of BMNC in radiation injured mice. It can also reduce the cell cycle block, apoptosis rate, the expression of P53protein and activation fragment of Caspase-3, while increase the expression rate of Bcl-2, finally protect radiational injured mice.8mg/kg APS group is more obvious than2mg/kg APS group in resist radiation. The mechanism may be:APS can increase the activity of SOD and reduce the content of MDA to alleviate the content of free radical in BMNC after radiation, thereby reduce damage of DNA. At the same time, APS also can reduce the expression of P53and the cell cycle block, up-regulate expression of Bcl-2, reduce activation of Caspase-3and ultimately attenuate apoptosis. |
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