| BackgroundAtherosclerosis is one of the most common cardiovascular diseases. Over the past century, the cardiovascular diseases have become the first cause of death, making great changes in categories of the world diseases. Atherosclerosis belongs to arteriosclerosis, mainly involving large and medium arteries in the whole body. It has experienced lipid deposition, fibrosis and atheromatous plaque formation in artery intima, which makes the artery wall hardening and lumen stenosed. When the collateral circulation can't compensate for narrow arteries, blood flow of important organs such as heart, brain and kidney will be interrupted, causing ischemia or necrosis in these organs. The pathogenesis of AS is very complex. Researches during more than one hundred of years have suggested lipid infiltration, oxidative stress, inflammation theories. But recently some researchers have found that damage in endothelial cells is also one important mechanism. What's more, research has said that atherosclerosis may begin with vascular endothelial damage.The phosphorylation of myosin light chain(MLC) mediated by myosin light chain kinase(MLCK) plays an important role in changes of endothelial permeability. MLC whose serine and threonine were phosphorylated can activate ATPase in the head of myosin heavy chain, which can provide energy for interacting between myosin II and actin. But the interaction may cause intercellular space enlarged and endothelial permeability increased. Increased vascular endothelial permeability may accelerate the formation of AS by facilitating lipids permeate into the subendothelium.Mitogen-activated protein kinases(MAPK) which belong to serine/threonine protein kinases play an important role in signaling pathways. Extracellular regulated protein kinases(ERK) are main members of MAPK family(including JNK, ERK and p38). It has been reported that ERK can improve MLCK activity and MLC phosphorylation, causing connection between cells damaged and intercellular space enlarged. In addition, researches say that the role of MAPK can be restrained by progesterone.Tanaproget(TNPR) which is a new nonsteroidal progesterone receptor agonist can combine with progesterone receptor tightly in many species. Although the protective effects of sex hormones on cardiovascular diseases have been widely studied, as a new drug, study about TNPR was few. Therefore, the present study aimed to explore whether TNPR could regulate MLCK expression and endothelial permeability through MAPK pathway in AS rabbits. ObjectiveThis study was to investigate the effect of TNPR on MLCK expression and endothelial permeability in AS rabbits. In addition, it aimed to explore whether the effect of TNPR on MLCK expression was mediated by MAPK pathway in the endothelium of AS rabbits. Methods1 Set up the AS model in rabbits. 24 New Zealand white rabbits were randomly divided into 3 groups: normal group, AS model group and TNPR group. The normal group was given ordinary diet, while the AS model group was given high cholesterol diet plus intravenous injection of bovine serum albumin and the TNPR group was given TNPR(10 mg/kg/day) on the basis of high cholesterol diet plus intravenous injection of bovine serum albumin. After 12 weeks, tissues were collected for other tests.2 Aortic oil red O staining was to observe atheromatous plaques in the aorta.3 The endothelial permeability was analyzed by frozen section immunofluorescence(surface biotinylation technique).4 Immunohistochemical method and Western blot were used to detect MLCK protein level in aorta. In addition, MLCK mRNA level was measured by qRT-PCR. Besides, Western blot was also used to detect the phosphorylation levels of JNK, ERK and p38. Results1 After 12 weeks the AS model in New Zealand rabbit was set up successfully: oil red O staining showed that no plaques occurred in the aorta of normal group, and there were obvious plaques in the AS model group. Although there were plaques in TNPR group, the plaques were much smaller than those in AS group.2 The analysis of aortic endothelial permeability: there was no fluorescent dye penetrant in the aortic intima of normal group, but in AS model group fluorescent dye penetrant significantly increased. Compared with AS model group, the extent of fluorescent dye penetration reduced in TNPR group.3 The results of immunohistochemistry, Western blot and qRT-PCR showed that the expression of MLCK in AS model group was higher than that in normal group(P<0.05). But compared with AS model group, the expression of MLCK in TNPR group decreased significantly(P<0.05).4 The results of Western blot showed that the expressions of pERK, pJNK and pp38 in AS model group were higher than those in normal group(P<0.05). But compared with AS model group, the expressions of pERK, pJNK and pp38 were lower in TNPR group(P<0.05).ConclusionTNPR may down-regulate MLCK expression and improve endothelial permeability by inhibiting the phosphorylation level of MAPK pathway, which may prevent the development of AS. |
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