| Background and ObjectiveDeep vein thrombosis is a common vascular thrombosis disease. It is easy to cause pulmonary embolism in the acute phase. Owing to its potential deadly, more and more scholars pay attention to it at home and abroad. The incidence rates of deep vein thrombosis was0.05%～0.10%, which show a rising trend at the stage of pregnancy and puerperium in recent years, the high incidence of deep vein thrombosis is closely related to physiological acquired thrombophilic factors and hypercoagulability of pregnancy patients. Now currently accepted Virchow, vein injury, slow blood flow and blood hypercoagulability are three major factors to the formation of DVT. In recent years, Studies show that inflammation have a close relationship with deep vein thrombus formation and development. Inflammation mediated vessel wall damage and blood flowed stagnant promote venous thrombosis development. Pathology and serology provide a strong basis on the relationship between inflammation and venous thrombosis. Therefore, To explore protein related to inflammation have great significance for inhibit the formation of thrombosis and judgement of prognosis. Nuclear factor-kappa B discovered by Sen and Baltimore, which is named for specific binding with B cell immunoglobulin Klight chain gene enhancer kB sequence (GGGACTTTCC). NF-κB played an important role in inflammation and detected on cell was a specific DNA sequence protein in recent years. It have an important regulator of gene transcription. In unstimulated cells, NF-κB resides in the cytoplasm as an inactive NF-κB-IκB complex. NF-κB is released by the degradation of IκB, then free to enter cell nucleus and activate transcription; when it was stimulated by ultraviolet radiation, cytokines, oxygen radical and so on. Currently, NF-κB protein functions was found in many disease, such as activated lymphocyte, sepsis, acute inflammatory response syndrome (AIRS), liver ischemia-reperfusion injury, tumor angiogenesis aspects.More and more studies have shown that NF-κB activated may be a central role in a cytokine network of inflammatory response. Stimulation of neutrophils and endothelial cells is mediated by NF-κB signaling pathway, then produce a series of adhesion molecules, cytokines, enzymes and so on, consequently leads to cell and tissue injury. NF-κB controls inflammation and immune-related genes, these genes are involved in toxic shock, acute phase response, radiation damage, asthma, rheumatoid arthritis, atherosclerosis, cancer and other diseases. It is likely to suppress NF-κB activation as a therapeutic target for that diseases. Pyrrolidine dithiocarbamate (PDTC) as a specific inhibitor of NF-κB, can suppress activation of NF-κB which reduce it enter cell nucleus. On these base, To explore the expression and characteristics of NF-κB protein in vascular endothelial cells of DVT. Improving the inflammatory response and affecting the formation of thrombosis whether to suppress NF-κB activation and reduce cytokines produced. NF-κB may be as a new drug targets for venous thrombosis by provided the theory basis.Therefore, Study on DVT models were induced by inferior vena cava ligation. To investigate the expression of NF-κB protein in vascular endothelial cells of deep venous thrombosis by qualitative observation, the relationship between NF-κB and deep venous thrombosis in rats at different times. Correlation analysis between NF-κB and thrombus weight to length ratio. To explore the effects of NF-κB and the thrombus formation that PDTC intervened in venous thrombosis.Meterials and Methods1.54Sprague-Dawley (SD) rats were purchased from animal center in southern medical university. All rats were feed for a week, then you take the2female rats and1male rat into a cage. Pregnancy for10or15days, of which48rats were randomly divided into the model group and the PDTC treated group. According to6h,12h,24h,72h time points divided into four groups, each point only6, the other6rats were sham-operated group.2. The DVT models were induced by inferior vena cava ligation. The model group (treatment with normal saline) and PDTC treated group (treatment with PDTC) at1hour before surgery. The rats were injected every24h,3times were injected at72h.3. The rats were sacrificed at6h,12h,24h and72h after operation. The thrombus weight to length ratio of DVT were measured and compared between the two groups. The vein tissue put to10%marin fluid, Hematoxylin-eosin and Immunohistochemical methods were used to detect.4. We observed the thrombosis tissue about pathological morphology change and thrombosis inflammatory cells by HE dyeing. Immunohistochemical methods were used to detect NF-κB expression. Application of imageproplus image analysis system test NF-κB protein-mean optical density (IOD) value, then analyze the characteristic of NF-κB at different time points of tissues. Correlation analysis of NF-κB expression and thrombus weight to length ratio. 5. Statistical analysis: SPSS13.0statistical analysis software application for processing. The data shows by average add and subtract standard deviation. Comparison of NF-κB protein IOD value and thrombus weight to length ratio were used to factorial design ANOVA test. Between the two groups use the independent-sample t-test. Correlation analysis used bivariate correlation analysis, Pearson correlation was selected. P value of <0.05was considered statistically significant.ResultsI. The deep venous thrombosis model were established and detected1. The deep venous thrombosis model were successfully established by inferior vena cava and its branch of iliolumbar ligation in pregnancy rats.2. At different time points, inferior vena cava can be seen venous thrombosis formation. At6、12、24hour after surgery form red thrombus and mixed thrombus. The thrombosis was soft and flexibility, it was easy to separate from venous wall and without obvious adhesion. At72hour it formed layer thrombosis, the thrombosis was hard and had closely adhere with venous wall. With time prolonging, the thrombus weight to length ratio of DVT was rose.3. At6、12、24hour can be seen red thrombus or mixed thrombus by HE staining, It can be seen layer thrombosis at72hour. The venous wall around inflammatory cells significantly increased at24hour and72hour.Ⅱ. The quantitative of NF-κB p65protein in endothelial cells of DVT1. The expression of NF-κB protein in normal vascular endothelial cells is low. The expression of NF-κB protein increased at6hour, peaked at24hour, decreased at72hour after DVT.2. NF-κB p65IOD value (0.72±0.23) in endothelial cells of DVT is higher than that (0.12±0.04) in normal vascular endothelial cells (t=-6.379, P=0.000) Ⅲ. The thrombus weight and length of DVT after PDTC intervenedThe thrombus weight to length ratio in model group is higher than in PDTC treated group (F=5.755, P=0.021). At different time points, the thrombus weight to length ratio were significantly different after PDTC intervened (F=4.394, P=0.009). The thrombus weight to length ratio was significantly lower than that in model group at72hour (P=0.047), But it were not significantly different comparison between the others time points.Ⅳ. The expression of NF-κB p65protein in endothelial cells of DVT after PDTC intervened1. NF-κB p65IOD value in PDTC treated group is lower than that in model group (F=17.276, P=0.000). At different time points, NF-κB p65IOD value were significantly different after PDTC intervened (F=15.685, P=0.000)2. The expression of NF-κB p65protein in PDTC treated group is lower than that in model group. NF-κBp65IOD value after PDTC intervention at12hour (P=0.046), at24hour (P=0.044), at72hour (P=0.016) were significantly decreased in model group.Ⅴ. Correlation analysis between NF-κB and thrombus weight to length ratioNF-κB expression in vascular endothelial cells of normal, DVT model group and PDTC treated group was significantly different (F=28.704, P=0.000). The level of NF-κB was significantly positively correlation with the thrombus weight to length ratio (r=0.392, P=0.006)Conclusions1. The deep venous thrombosis model were successfully established by inferior vena cava ligation in pregnancy rats. At6h,12h,24h,72h time points can be seen venous thrombosis and around vessel wall inflammation. It means inflammation take part in development of DVT. 2. It have low level expression of NF-κB protein in normal vascular endothelial cells and high level expression in vascular endothelial cells with DVT. NF-κB was significantly activated in vascular endothelial cells after DVT. The expression of NF-κB p65protein decreased and inflammatory reaction suppressed after PDTC intervened.3. The expression of NF-κB protein was positively correlation with the thrombus weight to length ratio. The activation of NF-κB influenced formation and development of DVT.4. NF-κB inhibitors of PDTC can reduce the thrombus weight to length ratio and alleviate thrombus formation, But which has changed over time. The beneficial effect mainly lies in inhibit of NF-κB signaling pathway in vascular endothelial cells, which suppressed inflammatory reaction and reduced vessel wall damage. |
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