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Differential Roles of MAPKs in Thermal Injury Induced Endothelial Cell Dysfunction

Author: WuZuo
Tutor: HuangQiaoBing;ZhaoMing
School: Southern Medical University,
Course: Pathology and Pathophysiology
Keywords: Human skin micro- vein endothelial cells Burns plasma Mitogen-activated protein kinase MAPKs F-actin ZO-1 ICAM-1
CLC: R644
Type: Master's thesis
Year: 2009
Downloads: 45
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Research purposes: increased vascular permeability and leukocyte adhesion is important in many pathological processes such as trauma, burns, shock and inflammatory changes, especially in burn shock has an important impact on the development and outcome of the disease. Because of its mechanism has not been fully elucidated, has yet to find the effective measures of prevention and treatment of burns vascular permeability disorders therefore extremely important to explore the signal transduction mechanisms of vascular permeability. Our laboratory proof of work in the past, blocking G protein receptor kinase, Rho kinase, can block the burn serum vascular endothelial cell stress fiber formation and improve the burns caused by increased vascular permeability. We also found that PKC, ERK and p38 are involved in the burns caused by endothelial cell actin reorganization, especially p38α, p38δ played a relatively more important role. Burns caused by endothelial cell function changes, increased vascular permeability and leukocyte adhesion to play the role in order to further study the p38 upstream and its downstream signaling pathways, this study using molecular biology and histological means, at the molecular level, cellular level observe normal and burns caused by stimulation of plasma endothelial cytoskeletal proteins Claudin changes in vascular permeability relationships; investigate the expression and regulation of ICAM-1 observed the role of p38 signaling pathway regulation of endothelial cell adhesion molecules, endothelial cell cytoskeletal proteins; mechanism and look forward to provide a basis to elucidate the mechanism of burns endothelial cell dysfunction, and to provide new therapeutic targets for the prevention and treatment of disorders of the vascular permeability. Method: 1, normal preparation burns the plasma: my room, to 200g-250gSD rats were given burns greater than the surface area of ??1/3, followed by the carotid blood, centrifugal retained supernatant spare; rat plasma Preparation of 37 ° C warm water bath 30s I rats with burns. 2, according to the experimental groups, respectively, with a specific inhibitor of MAPKs and adenovirus vectors Pretreatment of endothelial cells, and then give the burn plasma stimulation observed inhibit MAPKs, and its downstream pathway after burn plasma induced endothelial cell response; 3 , labeled with a fluorescent probe burns endothelial cell F-actin, ICAM-1 and ZO-1, and then using the laser scanning confocal microscope to observe their changes and reconstruction; 4, the observed activation of MAPKs protein level and burns caused by Western blot endothelial cell dysfunction. Results: 1 burn serum effectively activation the MAPKs pathway of endothelial cells. The burn serum 5min, JNK, p38, ERK phosphorylation level began to increase. p38 phosphorylation levels peaked at 10 min and the highest level of ERK phosphorylation in 15min 20min peak level of phosphorylation of JNK. 2, F-actin in normal endothelial cells surrounding the cells surrounding tightly packed and slick. The the burn plasma stimulate 1h endothelial cells, the outer periphery of the F-actin cytoskeleton dense band edges become rough irregular cells the Central with scattered stress fiber formation. Give p38 inhibitor SB203580 or ERK inhibitor PD98059 pretreatment 30 min, can significantly reduce the formation of stress fibers of endothelial cells; However, blocking JNK pathway, no significant change. P38 downstream kinase MAPKAPK2 (MK2), and PRAK all involved burn plasma caused the endothelial cytoskeletal protein reorganization. MK2 sustained activation using adenoviral vectors can obviously make muscle actin remodeling, the formation of stress fibers; continuous activation the PRAK also can play a similar role, but it is not obvious. Similarly, the use of viral vectors inhibits activation of MK2, can significantly reduce burn plasma caused by the stress fibers; blocking of PRAK role better blocking MK2 obvious. By inference, burn, MK2 and PRAK are involved in p38-mediated endothelial cell actin reconstruction, but the role of the MK2 may be more important. P38-mediated endothelial cell actin cytoskeleton remodeling, MK2 and PRAK HSP27 phosphorylation. MK2 can burn serum stimulation of endothelial cells, can significantly increase HSP27 phosphorylation levels; sustained activation of MK2 adenoviral vectors can be significantly increased HSP27 phosphorylation level, persistent activation of the role the PRAK of HSP27 phosphorylation levels not very obvious; blocking inhibit HSP27 phosphorylation levels of burn plasma caused by the increase, the blocking the PRAK also have some of the similar role. Moreover, the morphology of the above results with endothelial cell actin cytoskeleton corresponding. 5 the endothelial cytoskeletal protein, blocking PKC and Rho kinase caused varying degrees of ease burn derangement. The use of a specific inhibitor of PKC and Rho, can reduce the formation of stress fibers burn endothelial cells, inhibition of Rho kinase activity can also reduce the burn plasma caused the level of p38 phosphorylation. Infer PKC and Rho kinase involved in burn plasma caused by the activation of endothelial cells. P38 involved in burns caused by endothelial cell tight junctions destruction. Normal endothelial cells of ZO-1 distribution in the cells nearly endometrial Department, tightly packed, smooth edges. Burns stimulate 1h, ZO-1 distribution was ring punctate cells blurring, ZO-1 protein is dispersed in the cytoplasm. 30 min after treatment with SB203580, can inhibit the burn plasma induced endothelial cell tight junction ZO-1 distribution change, clear cell outline, ZO-1 still uniformly distributed in the cell periphery; inhibition of the ERK pathway there is no protective effect. 7, normal endothelial cell ICAM-1 expression rarely; while in the burn plasma stimulate 24h, ICAM-1 expression was significantly increased, and give a specific inhibitor of JNK pretreatment significantly inhibited after burn plasma caused by the expression of ICAM-1 . Tip JNK is involved in the regulation of burns caused by endothelial cell ICAM-1 expression may play a certain role in leukocyte adhesion and migration. Conclusion: We found that in burn plasma stimulated endothelial cells, MAPKs are activated; 2, PKC and Rho kinase involved in the regulation of the burn human skin micro-vein endothelial cell stress fiber formation; 3, p38, and ERK burns involved in the regulation of endothelial reconstruction of the actin cytoskeleton and stress fiber formation, p38 is also involved in the process of burns endothelial cell tight junctions destruction; 4, p38, through its downstream kinase MK2 HSP27 phosphorylation to change the arrangement of the actin cytoskeleton; another a downstream kinase PRAK may also play a certain synergy. 5 of JNK pathway is involved in regulation of burns adhesion protein molecules ICAM-1 expression.

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CLC: > Medicine, health > Surgery > Traumatology > Burns and scalds ( burns )
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