The Study Of Lipopolysaccharide Stimulation Vein Endothelial Cells Participation Deep Vein Thrombosis

[Background and Objective]:Deep vein thrombosis is often diseases of the cardiovascular system, but also one of the common complications of orthopedic inpatients. In the three elements of thrombosis, the blood vessel wall to change is a key element of thrombosis. Including venous wall intima and adventitia, where direct contact with the inner membrane surrounding blood vein endothelial cell layer is formed from, it can stabilize vascular permeability, maintain normal endothelial barrier function, when the endothelial barrier function time of the injury, stored in the endothelial tissue factor underlying (Tissue factor, TF) abnormal release into the blood, start extrinsic coagulation pathway, and promote thrombosis. Vein endothelial cell permeability change and control mechanism has a very important practical significance Therefore clarify deep vein thrombosis.Studies have shown that the structure of the cytoskeleton and tight junctions (tight junction, TJ) is the main basis for the structure and function of the endothelial barrier. As the bacterial endotoxin LPS can cause severe sepsis body to promote fibrin abnormal increase of the biological role of LPS inflammatory occur in vivo microcirculation, thus resulting in diffuse intravascular coagulation (DIC), the nature of DIC is the tiny capillaries Homogeneity was eosinophilic fibrin plug, forming a "transparent thrombus." LPS more important role in biological functions reflect the impact on the stability and integrity of the cell membrane. Studies have found that LPS caused by intimal inflammation destroys the integrity of the endothelial cells in a mouse model thrombocytopenia bleeding occurs more reaction than normal mice, stability and integrity of intimal damage caused by inflammation Thrombocytopenia is the core cause of bleeding, LPS-mediated inflammation rely mainly on platelet ITAM pathway rather than classical GPCR pathway to regulate the stability of intima. DVT is also closely related to the stability and intimal disease, according to the previous study, we conclude that LPS induced intimal DVT may be involved in the process of change.Vein endothelial cell layer is a layer of a barrier layer functions neatly arranged, orderly connection between cells, effectively preventing the storage in the lower endothelial TF released into the blood clotting factor. Vein endothelial cells mainly through two cytoskeletal proteins-myosin and actin to maintain tension and cell morphology of each other, after the inflammatory reaction, vein inflammation leads to endothelial cell adhesion molecules secreted as E-selectin, ICAM-1 functional changes occur, endothelial cells, myosin and actin conformational changes in the endothelial cell surface and rearrangement occurs, "folds", a cell physical space, thereby causing damage to the endothelial barrier function. Vascular endothelial barrier function is maintained also relies on a structural basis that is important connections between cells, when small molecules through the blood vessels inside and outside the cell next to the way transport, mainly by way of regulating cell tight junctions, and the physical structure of tight junctions by several cross cytoplasmic membrane proteins and intracellular proteins, of which the most decisive is the biological role of intercellular junction protein-1. How LPS stimulation caused by vascular inflammation alter connexin protein expression between cells, thus affecting endothelial cell permeability?Taking into account the inflammatory response-deep vein thrombosis, there are some uncertain factors relationship, and lead to further explore LPS vein endothelial cell morphology and permeability changes in deep vein thrombosis role, this study focuses on LPS stimulation of cell adhesion adhesion molecule secretion, increase the adhesion of inflammatory cell morphology changes and endothelial permeability, promote the molecular mechanisms of thrombosis.[Method]:(1) Rat inferior vena cava thrombosis was observed two hours after the inferior vena cava vein stenosis -21 days of the inferior vena cava thrombosis, drawing weighing each time point comparison of the quality and length of thrombus and pathological examination, obtained in rats inferior vena cava thrombosis, development and time pattern subsided; collecting plasma samples to detect changes in the expression of cytokines IL-6, CRP, TLR, TNF-a; the vein wall qPCR detection of E-selectin and intercellular adhesion molecule-1 (ICAM-1) mRNA expression changes.(2) LPS intraperitoneal injection and inferior vena cava stenosis law creating toxic inflammation within the inferior vena cava thrombosis model, a separate group, 24h drawn weigh the inferior vena cava thrombosis wall+ quality, length, with 10% neutral formalin solution after modeling the whole+ vein thrombosis fixed segment (en bloc technique), dehydration embedded in paraffin, using morphometric measurement vein (Vein Wall Morphometrics) analysis techniques, depending on the cell morphology at high magnification, statistics neutrophils, monocytes, lymphoid cells, the total number of inflammatory cells; by Masson, vG special staining methods, semi-quantitative observe fibrin content in the vein wall and thrombus. SP immunohistochemical determination of E-selectin, P-selectin, ICAM-1 expression, and TF content determination.(3) Set different final concentrations of LPS (10ng/ml,50ng/ml, 100ng/ml, 200ng/ml,400ng/ml) and different LPS stimulation time (3h,6h,12h,24h,48h), qPCR method semiquantitative E-selectin under different stimulation conditions detected, ICAM-1mRNA transcriptional level. Selected LPS stimulation conditions: 200ng/ml,24h, set up the control group and LPS stimulation group were used to detect E-selectin by immunohistochemical staining, ICAM-1 expression; using Transwell, THP-1 cells were detected the adhesion and migration of HUVECs situation, understanding of LPS on endothelial cells and inflammatory cell adhesion, migration capabilities.(4) The HUVECs were seeded in 96-well cell culture plate,37℃,5% CO2 incubator incubated cultured 24h. Replacing without Green-fresh medium chain double antibody, adding Y-27632 (Sigma- Aldrich), its final concentration 10UM,37° C,5%CO2 incubator culture incubated 30min (This step procedure for signal pathway inhibitors pretreatment). Replacing without Green-fresh medium chain double antibody, adding LPS (Sigma-Aldrich), the final concentration of 200ng/mL, Another set up a group without LPS as blank control. Factors combine two treatments were incubated 24h. Changes immunofluorescence to detect the F-actin cytoskeleton; and blot to detect the tight junction protein Claudin-I expression.[Results]:(1) After modeling method to narrow the survival time of each rat was 100% coverage of, inferior vena cava thrombosis in rats stable form. Cytokine IL-6, CRP, TLR, TNF-a in the modeling have different degrees change. Vein wall qPCR detection E-selectin and intercellular adhesion (ICAM-1)-1 mRNA expression of molecules found in the modeling 6h, E-selectin and ICAM-1 expression was significantly changed their mRNA expression increased, change and maintain high levels until the 2Id.(2) In the 24 hours after LPS injection plasma concentration peak, Masson staining and vG severe liver and lung fibrosis and inflammation, LPS confirmed the inflammatory effect in rats. IVC thrombosis after LPS injection quality and thrombosis have undergone mass length ratio was significantly higher (P<0.05). Vein (thrombosis) Staining results confirmed thrombosis after LPS density increases, Masson and vG cause staining was found that LPS increased fibrin thrombi content. Intravenous injection of LPS wall morphometric found to increase the total number of inflammatory cells after which mononuclear cells increased most significantly (P <0.05). SP immunohistochemical assay was found after LPS stimulation, levels of expression of adhesion molecules E-selectin increased (P<0.05), P-selectin expression did not change significantly, TF content increased (P<0.05), the degree of increase in TF linearly related to fibrin.(3) LPS-stimulated HUVECs can make E-selectin and ICAM-1 mRNA levels increased, the difference was statistically significant, changes in the level of expression of the LPS stimulation time and stimulation showed a concentration-dependent manner. Immunofluorescence was found in the resting state HUVECs do not express E-selectin and ICAM-1, and in LPS-stimulated state, E-selectin and ICAM-1 were strongly expressed in the cytoplasm of HUVECs, indicating that LPS increases HUVECs cytoplasmic E-selectin, ICAM-1 protein levels. In the case of LPS stimulation, HUVECs and THP-1 adhesion rate lower than the vacancy situation LPS higher (53.2±4.8% vs 17.1±6.5%, P<0.05.); After LPS stimulation, the mobility of THP-1 cells was 50.2±6.3%, higher than the control group (17.8±4.6%).(4) Laser scanning confocal microscopy show, F-actin control group HUVECs are mainly concentrated in the surrounding membrane, evenly distributed; LPS stimulation group was significantly reduced membrane surrounding the F-actin, intensive stress fibers also appear in the cytoplasm; Y within -27 632 cells pretreated group compared with LPS stimulation stress fibers less than the control group, but there is still a certain degree of increase. Display LPS stimulation HUVECs cytoskeletal changes and rearrangements. With RhoA/ROCK signaling pathway inhibitor Y27632 pretreatment 30min, then add LPS stimulation HUVECs, HUVECs tight connection of high protein ZO-1 expression levels compared with LPS stimulation alone,, Description RhoA/ROCK signaling pathway involved in regulating permeability changes LPS-mediated cell.[Conclusion]:(1) Cytokine IL-6, CRP, TLR, TNF-a and adhesion molecules E-selectin, ICAM-1 is formed in the models of DVT process, LPS injected rats promotes the formation of the inferior vena cava thrombosis, inflammation and venous thrombosis presence association.(2) LPS stimulation concentration at certain times and stimulating role stimulation resulted in activation of HUVECs function, RhoA/ROCK signaling pathway is inhibited affect the distribution of F-actin expression, inhibition of RhoA/ROCK signaling pathway can be made on HUVECs LPS stimulation cytoskeletal rearrangement role reversal.(3) The molecular mechanisms of LPS change endothelial cell morphology and permeability:LPS by activating the RhoA/ROCK signaling pathway, downregulation of tight junction protein ZO-1 and cellular F-actin protein expression, increased cell permeability.