| BackgroundCardiovascular disease (CVD) is a series of diseases which cause long duration and entirely poor prognosis. Effective measures should be conducted to reduce the prevalence rate of the disease based on various risk factors that lead to vascular lesion. Therefore, researchers have conducted more etiological studies of cardiovascular disease, in which many new pathogenic factors were found, and based on these findings new measures were designed for preventing cardiovascular disease.Mycotoxins are toxic metabolites produced by fungi, which mainly exist in the grains and relevant products. According to statement of FAO, about 1/4-1/3 of grains in the world is contaminated by mycotoxins. CIT is one of the most important mycotoxins which are relative to cardiovascular damage. Previous experimental researches demonstrat that CIT inhibits proliferation of vascular endothelial cells, affects physiological functions of endothelial cells, and causes the release of inflammatory cytokines, which are the initial pathologies in atherosclerosis process. We propose the following hypothesis that CIT leads to vascular endothelial damage, and contributes to the pathogenesis of atherosclerosis. We conduct this study to clarify the effects of CIT on vascular endothelial cells and atherosclerosis. This study observed the effects of CIT on vascular endothelial cells adhesion functions based on the cell culture experiment in vitro and animal experiment in vivo and analysed the role of CIT in the pathogenesis of atherosclerosis.The combinations of drugs with biological macromolecules (e.g. protein) induce structure changes, and affect the occurrence and development of pharmacology and toxicology effects induced by these drugs, even influence the absorption and metabolism of drugs in human bodies. This theory helps us to explore drug interactions with biological macromolecules through a variety of methods and to clarify the mechanism of biological effects. Human Serum Albumin (HSA),Immunoglobulin (IgG) andLysozyme (LOM) are often used to analyzed the effects of drugs on human bodies. Fluorescence spectroscopy(FS),ultraviolet spectroscopy(UVS), infrared spectroscopy(IR) and circular dichroism(CD) are also used in this study.Ecological study is applied to explore the contact between disease and exposure at the group level, based on the comparison among different regions, different time or different populations. Ecological study is simple in the research process and the data analysis, and can gain research conclusion quickly. Although there are many defects of methodology, such as disability to evaluate exposure factors at individual level, ecological fallacy, The advantage of ecological study can help us to explore many risk factors at population level. Through an ecology study, this study compared the difference of incidence rates of coronary heart disease (CHD) in two areas, and detected the potential relationship of CIT pollution levels in the food with coronary atery.Objective1. To assess investigate the effect of CIT on adhesion of human umbilical vein endothelial cells (HUVECs) induced by tumor necrosis factor-alpha (TNF-a).2. To analyze the effect of CIT on the atherosclerotic lesions development in apolipoprotein E knockout (apoE-/-) mice.3. To analyze the binding of CIT to human serum albumin (HSA), immunoglobulin (IgG) and Lysozyme (LOM). The binding constants and binding-site of the combinations were determined.4. To investigate the relationship between CIT and coronary heart disease in an ecological study.MethodsAdhesion of HUVECs to monocytes was analyzed by co-culture experiments using U937 cells. The expression of Intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, E-selectin and monocyte chemoattractant protein (MCP)-1 was determined by Western blotting, Enzyme-Linked Immunosorbent Assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). The activation of nuclear factor-kappa B (NF-κB) was assessed by Western blotting and immunofluorescence staining.The mice were fed with high fat diets and treated with CIT for 15 weeks, atherosclerotic lesions in the aorta of mice were determined with oil red O staining. Vascular endothelial growth factor (VEGF) and endothelin (ET)-1 in serum were detected with ELISA assay. The levels of ICAM-1, VCAM-1, VEGF and ET-1 in plaque areas of artery walls were assayed by immunohistochemical analysis. Expressions of ICAM-1 and VCAM-1 mRNA were determined by Real Time PCR.Fluorescence spectroscopy, ultraviolet-visible (UV-Vis) absorption, circular dichroism (CD) methods and molecular models were used to analyze the binding sites of the combinations and types of binding forces. Molecular docking study was carried out to get further understanding about the binding modes of the three compoundsThe mortality data of coronary heart disease in Yian and Huachuan were collected. The samples of rice, flour and millet were obtained in the areas, and levels of CIT in the samples were determined by HLPC method. We assess the relationship between CIT and coronary heart disease with ecological study.Results1. The effect of HUVECs adhesion to monocytes was higher in CIT treated groups than controls. The expression levels of ICAM-1, VCAM-1, E-selectin and MCP-1 are higher in in CIT groups than controls. The nuclear translocation of NF-κB in HUVECs was significantly activated in CIT group.2. The lesions in aortic sinus were aggravated in CIT groups compared to vehicle-treated group. Compared to vehicle-treated group, the areas of aortic lesion were increased in CIT treatment. CIT increased the expressions of VEGF, ET, ICAM-1 and VCAM-1 in plaques of aortic roots. The levels of VEGF and ET-1 in serum were higher in CIT groups than control. Expressions of ICAM-1 and VCAM-1 RNA were upregulated.3. The binding of CIT to HSA, IgG and LOM were detected. CIT can bind with HSA and quench the fluorescence of HSA, the quenching mechanism of CIT and IgG is static quenching, and CIT can strengthen fluorescence of LOM. The distance "r" between binding sites of CIT with HSA, IgG and LOM and tryptophan residues are 3.25,3.32 and 3.61nm, which indicating that energy transfer happens. The binding forces between CIT and HSA are hydrophobic force and hydrogen bonds, the binding forces between CIT and LOM or IgG are hydrophobic force and electrostatic interaction, and hydrogen bonds also exist.4. The mortality rates of coronary heart disease in Yian were higher than Huachuan in the years from 2008 to 2011. The CIT levels of grains in Yian were higher than Huachuan.Conclusions1. CIT markedly increased TNF-a-induced HUVECs adhesion to monocytes and expression levels of ICAM-1, VCAM-1, E-selectin and MCP-1. TNF-α-induced nuclear translocation of NF-κB in HUVECs was significantly elevated by CIT.2. CIT caused dysfunction of vascular endothelial cells, enhanced inflammatory response and increased the development of atherosclerotic lesions in apoE-/- mice.3. The interacton with CIT altered the secondary structures of HSA, IgG and LOM, the binding parameters of the combination were determined.4. CIT contamination may be one of atherosclerotic causes, for which mortality rate of coronary heart disease in Yian is higer than Huachuan. |
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