| BackgroundRadiation is a natural phenomenon which is widely existed in the universe and human environment. According to the nature, radiation can be divided into two categories; one is electromagnetic radiation, the other is particle radiation. Electromagnetic radiation has energy but without rest mass; particle radiation not only has energy but also rest mass. According to the way it acts on substance, usually there are two types of radiation:ionizing radiation and non-ionizing radiation. Non-ionizing radiation can't ionize biological tissues. The direct effect of ionizing radiation is referred to rays transfer energy directly to biological molecule, causing ionization and excitation, changing molecular structure and losing of biological activity.Ionizing radiation effects on body. From the start of irradiation to visible injury was observed by cytology, the original and strengthening processes of radiation damages are proceeding in cells during this period. The original process includes three stages called physical, physical chemistry, and chemical stages. In this process, the absorption and transmission of radiant energy, the excitation and ionization of molecular, the generation of free radical, the breaking of chemical bonds and so on are all carried out in highly organized organisms. The absorption and transmission of energy make the well-ordered biological macromolecules in the cells stay in a state of excitation and ionization. And the special biological structure can also make the electron transmission and free radical chain reactions carry on. As a result, these lead to the initial biochemical damage. The destruction of subcellular causes the release of enzyme, and the direction and coordination of metabolic disorder prompts the further development of initial biochemical damage. As a consequence, a series of physiological and biochemical changes are induced and then turn into pathological changes.Ionizing radiation can cause the formation of free radical. The formation of free radical is an important factor for the biochemical process of chemical bonds' breakdown induced by ionizing radiation; as well as for the biological stage when cell tissue organ system lesions spread to whole organ system. NO is a new type biological information transmitter, discovered in recent years, with a unique physical and chemical, and biological properties. NO is highly unstable in vivo and it can be oxidized to the nitrogen and oxygen free radical of nitrate or nitrite, with simple structure, low content in body and low concentration. The NO produced by vivo is colorless, slightly soluble in water, high fat-soluble gas molecule with a half-life of 3-5s. It can go through the membrane freely in vivo. Using L2 arginine and reactive oxygen as the substrates, the organism generates NO under the action of nitric oxide synthase (NOS). The effect of NO in body is universal and bidirectional. It involves in a serious of biological processes under physiological and pathological condition, regulating circulation, nerve, immunity and a serious of physical activities, such as vasodilation, vascular permeability, platelet adhesion and aggregation, neural signal transduction, host defense reactions, but also participates in pathological process with tumor involved. Both the shortage and excess of NO can cause the abnormal of body system and diseases.Currently, three types of NOS are founded. Type 1 is neuronal NOS (nNOS), mainly in the nerve tissue; type2 is inducible NOS (iNOS), mainly in cell's cytoplasm of macrophage and neutrophil. Type3 is endothelial NOS (eNOS), mainly in the endothelial cell. nNOS and eNOS normally exist in cells known as structured NOS (cNOS). cNOS synthesize a little NO and it mainly involves in the maintenance of normal physiological function. iNOS can induce a lot of NO when stimulated by inflammation and it mainly involves in the pathophysiological process of the body.After subjected to certain stress factors, the synthesis of inducible NOS increases and further raise the NOS level in vivo. Ionizing radiation, as an external stimulating factor, when acts on body, it will raise the NOS level and further induce the synthesis of NO. After irradiation, how to determine the radiation dose of body has always been the focus of radiation medical research.Hematopoietic tissue is a radiation-sensitive tissue, and the severity of its change after irradiation is closely related to the radiation dose. Judging from its injury severity, the irradiation doses of patients can be roughly estimated. Some researches show that iNOS expression increases and leads to the increase of NO formations after macrophages, and smooth muscle cells are irradiated in vitro. The experiments in vivo also show that the significant increase of NO synthesis is found in parotid after large dose of irradiation. Maybe it is related to the xerostomia induced by radiation, and the NO of the liver of irradiated animals has changed. Some put forward that the blood NO concentration may also reflect the severity of irradiation, but in-depth studies have not done yet.Liver is the vital organ in NO synthesis in vivo, because all the liver parenchymal cells, Kupper cells, and sinusoidal endothelial cells are able to synthesize NO. iNOS doesn't express under normal conditions and it is induced to synthesize NO persistently under pathological conditions. NO can protect liver as well as doing harm to it. NO not only involves in many physiological processes but also pathological processes. Different amounts in different states make different effects. The research of NO expression in liver after radiation will have further prompting in liver physiological and pathological processes, and will find new ways to protect and treat liver diseases caused by radiation.Genomic DNA and the cell membrane are the target molecules of radiation. As a major target molecule of radiation, DNA structures can change easily when ionization occurs. They play a crucial role in cell mutations and cancer mechanisms, and closely relate to cells'aging and death. Ionizing radiation can delay all sorts of eukaryotic cell divisions, and blocking occurs in G1 and G2 phase, with accumulation and giant cell formation in S phase. Obviously blocking of G2 phrase occurs when radiation is in small dose (2Gy). Whether the blocking of G1 phrase after radiation occurs depends on P53 in cells. It is generally recognized that only the cells which express wild-type P53 gene can make blocking occur in G1 phase after radiation, and it doesn't occur when P53 gene is missing or mutates. When prolonged exposed NO causes DNA damage, wild-type P53 will be gathered here to protect cells from NO damage by inhibiting iNOS promoter's activity to decrease the expression. As a consequence, the researches on the relationship between iNOS and P53 have caught scholars'eyes increasingly. P53 mutation occurs in G:C-T:A transition and it is also the target functional spot of NO. NO and P53 constitute the negative feedback regulating pathways. It can induce DNA damage; lead to the gathering of wild-type protein; and inhibit the iNOS promoter's activity and its translation in order to decrease its expression. Excessive NO can change the structure of P53 protein, and can cause the activity loss of combination with DNA and biological function loss. Hence, P53 controls the transcription of DNA, while NO controls the activity of P53 after its transcription. The feedback regulation NO, iNOS, and P53 effect cells' proliferation and apoptosis.Research purposesThrough this research, we are expected to find the changes of NO concentration of peripheral blood and liver, as well as the rules of its changes after radiation damage, in order to acquire the effects of NO changes occurred in radiation's happening and developing. After recognized as radiation accident, it can provide scientific basses for the doses of radiation irradiate on organism s and can investigate the changes and purports of NO and iNOS in the processes of radioactive liver damage.Methods1. Breed and irradiate the healthy NIH miceHealthy NIH mice, male, weight (18-25) g, are irradiated by different doses of radiation source in Guangzhou irradiation center. The radiation source is 60Coy in Guangzhou radiation center and the dose rate is 0.3Gy/min. 2. Detect the changes of semi-lethal doses and lethal doses in peripheral of irradiated animals in NO levels.NO detection kits are used to detect the changes of peripheral blood in NO levels after a lethal dose and a semi-lethal dose of irradiation.3. Detect the changes of mice's serums and livers in NO levels after lethal doses of irradiation.NO detection kits are used to detect the changes of serums and liver tissues in NO levels after lethal doses of irradiation.4. Detect the expressions of iNOS and P53 in liver tissues by immunohistochemical SABC.Irradiate the mice with lethal doses and semi-lethal doses respectively; take the liver tissue in the 3rd,6th,9th,12th hour after irradiation respectively; and detect the protein expressions of iNOS and P53 by SABC staining methods with high magnification. Take 5 visions, count 300 cells per vision and calculate the total number of positive cells. Then SPSS statistical software is used to analyze the data, the correlation between iNOS and P53, and different variables in statistics.Results and discussionIn the doses of 6.5Gy and 9.0Gy, irradiated mice show a trend of increase in NO concentration of peripheral blood, and peaks in the 6th hour and then tens to the normal value. This shows that the NO concentration of mice's peripheral blood presents an excessive rise by certain stress modes after high-doses radiation. After a certain time, No concentration of peripheral blood returns to the normal range, which maybe caused by the decline of stress or depletion of body function.Under the irradiation of 6.5Gy and 9.0Gy doses, both the NO concentrations of peripheral blood have a rise in statistically significance 3 hours after irradiation, and peak in the 6th hour. But the variation is more obviously in 9.0Gy radiation dose than 6.5Gy, and the differences between each other are statistically significant. These show that the extents of body's responses to different doses of radiation are different. Under the radiation of lethal doses, the bodies' reactions are more intensive.So it is recognized that NO concentrations changing trends of irradiated animals' peripheral blood may reflect the extent of irradiation. Whether it can be a new biological dosimeter depends on the further research.After a certain time's irradiation with 9.0Gy doses, the NO concentrations of peripheral blood and liver cells are significantly higher before irradiation and can sustain for some time.24 hours after irradiation, both NO concentrations return to the original level before irradiation. This shows that ionizing radiation, as a stimulating factor, also can make vascular endothelial cells, macrophages, liver cells and so on produce stress response, leading to the significantly increment in NO concentration of peripheral blood and liver cells. Appropriate amount of NO can maintain the relaxation of sinusoidal; facilitate blood circulation and nutrient exchange.After irradiation, the expressions of hepatic iNOS and P53 increase as time goes by. It peaks in the 6th hour and remains in a high level for a while. So does NO expression. It indicates that iNOS, P53 and NO are closely related. Conducted from the count of positive cells, the number of positive cells of 9.0Gy is more than 6.5Gy group without statistical difference. It manifests that the expressions of iNOS and P53 is not related to the 6.5Gy and 9.0Gy doses in this study. However, whether it is related to other doses needs further studies to reveal. There are significant statistical differences at different points in the expressions of iNOS and P53 and it suggests that iNOS and P53 are closely related to the genesis and development of NO. The expressions between iNOS and P53 is positively closed (r=0.923, p<0.01). Moreover, the same positive correlation is existed in 6.5Gy and 9.0Gy dose group. When the body is exposed to ionizing radiation, the expressions of iNOS increases and NO is generated. The accumulation of P53 is triggered when cellular DNA damage is caused by NO. Through a combination with human iNOS gene transcription start site upstream region, P53 can suppress iNOS promoter activity, inhibit gene transcription, block the expression and decline NO level. The sustained NO expression can deform P53 protein directly; cause the loss of binding activity with DNA and biological function. But the experimental results show that the expressions of NO, iNOS and P53 decrease simultaneously after 6 hours. It indicates that there are other regulatory pathways, or the interaction between three regulatory mechanisms needs further study.
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