| Cardiac and cerebrovascular disease which caused by atherosclerosis is detrimented to people's health and life. The morbidity and mortality is front row of various kinds of diseases. The precisely multiplicity etiopathogenisis is not clear up to now. There are many doctrine about this etiopathogenisis in hundreds years, including lipid infiltration, thrombogenesis and damage reactology. For the past few years, the hypothesis of reactology is generally acknowledged, namely, atherosclerosis lesion is beginning the endodermis damage. Vascular endothelial cell is to serve as semi-permeability barrier between blood and tissue fluid. The exchange materials in exterior and interior of blood must be preference through vascular endothelial cell. The endothelial cell integrality of its morphous and function is very crucial for the normal physiological function. Accordingly, it has important significance for medical science to lucubrate the molecular mechanism of endothelial and to look for the medicine to protect the formation of atherosclerosis.Melatonin is a hormone secretaried by pineal gland. In recently years, the investigations have domestranted that melatonin have many physiological functions, such as degrade permeability of blood-braid barrier, regulate lipid metabolism and atherosclerosis formation. There are considerable basic and clinical research work to do although people believed that melatonin is benefical for the development of atherosclerosis. This study is based on duplicate hypolipoidemia and diabetes mellitus model and study the relationship between MLCK expression and mitogen-activated protein kinases signal transduction pathway. On this fundament, duplicate AS and to investigate the effect of MLT on the relationship between MLCK and MAPK signal transduction pathway on endothelial in AS rabbit artery. Then to elucidate the molecular mechanism of MLT on MLCK via MAPK signal transduction pathway.AIM: To study the relationship between MLCK expression and MAPK signal transduction pathway of hyperlipoidemia rabbits and diabetes mellitus rats. On this fundament, to investigated the effect of MLT on the relationship between MLCK and MAPK signal transduction on endothelial in AS rabbit artery. And then to elucidate the molecular mechanism of MLT on MLCK via MAPK signal transduction pathway.METHODS: (1) hyperlipoidemia rabbits model was induced by hypsi-cholesterol and diabetes mellitus rats model was induced by streptozotocin. The phosphorylation of extracellular signal-regulated kinase,p38 and c-jun-terminal kinase was assayed by Western blot. MLCK expression was detected by Western blot and immunohistochemical method. (2) AS rabbits model was induced by hypsi-cholesterol. The AS rabbits were treated with MLT(10mg/kg/d d42-d84). Blood lipids were analyzed by correspondence kits. The phosphorylation of extracellular signal-regulated kinase, p38 and c-jun-terminal kinase was assayed by Western blot. MLCK expression was detected by Western blot and immunohistochemical method. MLCK activity was measured by -32P-ATP incorporation into myosin light chain. The permeability of endothelial was detected by immunofluorescence (surface biotinylation techinique). (3)HUVEC were cultured in vitro. The expression, transcription, activity of MLCK and phosphorylation of MAPK were detected in HUVEC induced by oxLDL and treated with MLT, PMA(a selective activator of extracellular signal-regulated kinase), PD98059(a selective inhibitor of extracellular signal-regulated kinase), SB203580(a selective inhibitor of p38), SP600125(a selective inhibitor of c-jun-terminal kinase), respectively. The proliferation of HUVEC induced by oxLDL was assayed by MTT, MLCK expression induced by MLT was measured by Western blot, the morphous of HUVEC were observed by light microscope. The phosphorylation of MAPK was assayed by Western blot. The expression, transcription, activity of MLCK was detected by Western blot and immunihistochemical method, 32p-ATP incorporation, revere transcription-polymerase chain reaction, respectively.RESULTS:(1) The relationship between hyperlipoidemia rabbits artery and diabetes mellitus rats kidney and MAPK.The model of hypolipoidemia rabbits and diabetes mellitus rats were established successfully. MLCK expression was increased obviously in hyperlipoidemia rabbits artery which was associated with MAPK signal transduction pathway. MLCK expression was also increased in diabetes mellitus rats kidney which was not associated with MAPK signal transduction pathway.(2) The effect of MLT on MLCK in AS rabbits artery The AS model was established successfully. The levels of lipids and permeability of artery were decreased by MLT treated. The phosphorylation of MAPK increased during the formation of AS. It is maybe the mechanism of phosphorylation to elevate MLCK activity and MLC phosphorylation which contribute to the endothelial permeability. MLT decreased the phosphorylation of MAPK which regulate the expression and activity of MLCK and endothelial permeability. The results indicated that MAPK signal transduction pathway participated the formation of AS. (3) The effect of MLT and PMA on MLCK in HUVECThe expression and activity of MLCK was promoted by PMA and oxLDL, MLT shows a negative feedback of MLCK expression. The expression and activity of MLCK induced by PMA and oxLDL could decreased in HUVEC coexisted with PMA and MLT via the phosphorylation of ERK.(4) The effect of MLT and PD98059 on MLCK in HUVECThe expression and activity of MLCK were decreased by MLT and PD98059, respectively. The expression and activity of MLCK induced by oxLDL have synergistic effects in HUVEC coexisted with PD98059 and MLT which was associated with the phosphorylation of ERK.(4) The effect of MLT and SB203580 on MLCK in HUVECThe expression and activity of MLCK were decreased by MLT and SB203580, respectively. The expression and activity of MLCK induced by oxLDL have synergistic effects in HUVEC coexisted with SB203580 and MLT which was associated with the phosphorylation of p38.(5) The effect of MLT and SP600125 on MLCK in HUVECThe expression and activity of MLCK were decreased by MLT and SB203580, respectively. The activity of MLCK decreased and the expression was not changed induced by oxLDL in HUVEC coexisted with SB203580 and MLT. It is not through JNK/MAPK signal transduction pathway to regulate MLCK expression.CONCLUSIONS:(1) The expression and activity of MLCK in hyperlipoidemia and AS rabbits artery were increased obviously via MAPK signal transduction pathway.(2) MLT regulated the expression and activity of MLCK and endothelial permeability by the phosphorylation of MAPK signal transduction pathway, which could be inhibit the development of AS. (3) The mechanism of MLT to regulate MLCK expression and activity maybe via p38 and ERK/MAPK signal transduction pathway and via JNK/MAPK signal transduction pathway to regulate MLCK activity.
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