| BACKGROUNDAtherosclerosis is a kind of chronic inflammatory disease, which is characterized by intimal lipid deposition, smooth muscle cells and collagen proliferation, foam cell formation, it can be viewed as common pathological basis for cardiovascular and cerebrovascular diseases. Vascular endothelial cell monolayer covers the vessel wall, which is located between the blood flow and vascular smooth muscle, and plays an essential role in the homeostasis of the blood vessel. Atherosclerosis has a close relationship with endothelial cells, this injury to endothelial cells may contribute to the pathogenesis of atherosclerosis. The first evidence that endothelial cell apoptosis may contribute to the initiation of atherogenesis was given by the finding that all classical risk factors, which are known to promote endothelial dysfunction and atherosclerotic lesion formation, induce endothelial cell apoptosis. Furthermore, the induction of endothelial cell apoptosis may also contribute to plaque erosion and enhance thrombus formation, the major players for the development of acute coronary syndromes.Endothelial cells are as an important cells in atherosclerotic lesion formation, further elucidation of relationship between endothelial cells apoptosis and atherosclerotic pathological process, moreover, the article will highlight its specific molecular mechanisms, which will contribute to a better understanding of atherosclerosis and the development of novel treatment modalities. As a therapeutic target with apoptosis, intervention in apoptotic signaling pathways, it will provide a theoretical basis and new ideas for the development of new drugs and gene therapy.Hyperlipidemia is one of the common traditional risk factors affecting atherosclerosis, its main causative agent is oxidized low-density lipoprotein (ox-LDL). Lectin-like oxidized low-density lipoprotein receptor-1(LOX-1) was originally described via molecular cloning in1997by Sawamura et al as the major receptor for ox-LDL in endothelial cells of large arteries. LOX-1is a membrane protein that belongs structurally to the C-type selectin family and is expressed in vivo in vascular endothelium and in vascular-rich organs. Atherosclerosis pathological factors can upregulate LOX-1expression, LOX-1is responsible for the binding, internalization and degradation of ox-LDL in endothelial cells, activates the nuclear transcription factor nuclear factor-kappaB (NF-kB) from the cytoplasm into the nucleus, and combines with the target gene of promoter and enhancer, induces related genes expression, which leads to activation, injury and apoptosis of endothelial cells, lipid aggregation in smooth muscle cells and macrophages, activate platelets to promote thrombus formation and increase plaque instability, so that induces the occurrence and development of atherosclerosis[3]. LOX-1expression has been detected in atherosclerotic lesions of animals and humans, luminal endothelial cells covering early atherosclerotic lesions are more frequently positive for LOX-I expression than those in advanced atherosclerotic lesions. Macrophages, smooth muscle cells and micro vessels in the intima of advanced atherosclerotic plaques are positive for LOX-1, suggesting that LOX-1might play a role in the early stages. The persistent accumulation of the LOX-1protein in the atherosclerotic lesion implies longterm effects, which may be involved in atherosclerotic complications, such as plaque rupture and thrombosis formation, however, downregulation its expression can prevent plaque formation and development. Taken together, LOX-1may be a novel, exciting molecular target for endothelial cell dysfunction and atherosclerotic lesion, offer a new approach for the diagnosis and treatment of related diseases.Pioglitazone is a high-affinity ligand activator of artificial synthetic peroxisome proliferator-activated receptor gamma (PPARy), belongs to thiazolidinediones insulin sensitizers. It is widely used in type2diabetic patients owing to metabolic and insulin resistance improved. More and more scholars at home and abroad pay close attention to anti-atherosclerotic role of pioglitazone, it may not only improve glucose and lipid metabolism, but also protect endothelial function from injury, reduce inflammatory markers, inhibit smooth muscle cell proliferation, anti-thrombosis and stable plaque, which improve atherosclerotic pathological processes. But the role of LOX-1-NF-κB signaling pathways in the pioglitazone affecting on anti-atherosclerotic pathological processes and vascular protection is unclear. Thus, in this study, we will establish hyperlipidemia atherosclerosis model rat with high-fat diet, and LOX-1for the intervention target, to investigate effects of pioglitazone on LOX-1-NF-κB signaling pathways of aorta in hyperlipidemic rat, as well as endothelial cell apoptotic protein Bcl-2, Bax expression. Then we will further explore the mechanism of pioglitazone to protect blood vessels, to provide a theoretical basis for its anti-atherosclerotic effect, to offer new ideas for improve the formation mechanisms of atherosclerotsis and the development of new therapies.OBJECTIVETo investigate effects of pioglitazone on LOX-1-NF-κB signaling pathways of aorta in hyperlipidemic rat, as well as endothelial cell apoptotic protein Bcl-2, Bax expression, and also to study the potential role of LOX-1-NF-κB signaling pathway, so as to provide a theoretical basis for pioglitazone of anti-atherosclerotic and vascular protective effects.METHODS27male Sprague Dawley rats were randomly divided into control group (n=9) and high-fat diet group (n=18), respectively raised for12weeks with normal diet and high-fat diet.Then high-fat diet group was randomly divided into model group and pioglitazone treated group. Control group and model group were gavaged with distilled water, pioglitazone treated group was gavaged with pioglitazone10mg/(kg’d), once a day for4weeks. After16weeks, serum lipid and glucose levels of all rats were measured, using hematoxylin eosin staining to observe aortic early atherosclerotic pathological changes, structure of aortic endothelial cells was observed by electron microscopy. LOX-1, NF-κB p65and apoptosis protein of Bax, Bcl-2on the aorta were analyzed by immunohistochemical method. Apoptotic cells in aortic intimal area was stained by terminal deoxynucleotidyl acyltransferase-mediated dUTP nick end labeling, and then to calculate apoptosis index. Statistical analysis: datas were analyzed by statistical software of SPSS13.0, values were presented as means±SD. Compared with between two groups, detection homogeneity and normality of the variance, if variance was equalized datas were analyzed with independent sampler t test, otherwise Satterthwaite t test with correction. Compared with before and after medicine intervention, paired-sample test was used. Comparison among groups were performed by single factor analysis of varianc, if variance was equalized, datas were analyzed with LSD, otherwise Dunnett T3.P≤0.05was statistically significant.RESULTS1. Levels of plasma lipid and blood glucose in each group after12and16weeksCompared with the control group, TG, TC, LDL-C were significantly higher in high-fat diet group after12weeks (P<0.01). Intervention with drug for4weeks, TC, TG were statistically significant in each group after16weeks (P<0.01). Compared with the control group, TC, TG, were significantly increased in model group (P<0.01). Compared with model group, TC, TG were significantly lower in pioglitazone treated group (P<0.01). Moreover, the serem levels of TG, TC, HDL-C and LDL-C in pioglitazone treated group were markedly lower than before (P<0.01), however the model group was no significant difference (P>0.05). Blood glucose was no significant difference between each group after12and16weeks (P>0.05).2. Changes of aortic histological morphology in each group2.1Aortal pathological morphology by HE staining in each groupSample from control group showed that structure of vessel wall was clear, complete and smooth; endothelial cells in intimal layer were integrity with no loss; smooth muscle cells in medial layer were running clearly with no proliferation. While the model group was observed that vessel wall was thicken, most of endothelial cells were missing, smooth muscle cell in medial layer were proliferated and arranged disorderly. Pioglitazone treated group suggested that intimal layer was smooth, endothelial cells were occasionally fell off, proliferation with smooth muscle cell in the medial layer was not obvious, these cells were well-distributed thickness, arranged regularly, and run the form with no obvious abnormalities.2.2Structure of aortic endothelial cells observed by electron microscopy in each groupThe control group was showed that boundaries of aortic endothelial cells were clear, structure of cell membrane surface was distinct, the nucleus structure was normal, mitochondria in the cytoplasm was common, and internal elastic membrane was well-distributed thickness, continuous. While the model group was observed that structure of endothelial cells was obvious abnormality, cell membrane ruptured, chromatin in the nucleus was loose, nuclear membrane is not complete, organelles dissolved or disappeared, loosed degeneration; collagen fibers in subendothelial layer were increased, fibroblast cells were proliferated; internal elastic membrane was inequable thick with interruption and focal dissolved. Pioglitazone treated group suggested that aortic endothelial cells were basic integrity, some cells defected, mitochondrial was occasional observed swelled.3. Expression of LOX-1and NF-κB p65on aorta in each groupAortic wall was observed by light microscope under400times, LOX-1was expressed mainly in the cytoplasm, NF-κB p65in the nucleus, and positive cells were stained with brown-yellow or brown. Compared with the control group, model group showed that LOX-1, NF-κB p65protein-positive cells were significantly increased in aortic endothelial and neointimal area, the average optical density was significantly higher (P<0.01). And compared with model group, positive cells with LOX-1, NF-κB p65protein were decreased, the average optical density was significantly lower in the pioglitazone treated group (P<0.01).4. Expression of Bcl-2and Bax on aorta in each groupAortic wall was observed by light microscope under400times, Bax and Bcl-2protein were expressed in the cytoplasm, and positive cells were stained brown-yellow or brown. Compared with the control group, Average optical density of Bax was significantly increased (P<0.01), average optical density of Bcl-2and the ratio of Bcl-2/Bax were significantly lower in model group (P<0.01). Compared with model group, pioglitazone treated group showed that the average optical density of Bax was significantly lower, average optical density of Bcl-2and the ratio of Bcl-2/Bax were significantly increased (P<0.01).5. Apoptosis of endothelial cells on aorta in each groupTUNEL detected aortic endothelial cells, positive cells were brown. Compared with the control group, apoptotic cells were increased in the aortic intimal area of model group, while the pioglitazone treated group showed that apoptotic cells were reduced. These cells were observed by light microscope to count cell and calculate the apoptotic index. Compared with the control group, apoptotic index was significantly increased in model group (P<0.01). While apoptotic index of aortic intima was significantly lower in pioglitazone treated group than the model group (P<0.01).CONCLUSIONS1. Rats raised with High-fat diet can be successfully induced hyperlipidemia atherosclerotic animal models. The model group showed that expression of LOX-1, NF-κB p65, Bax protein on aorta were increased, and Bcl-2protein, the ratio of Bcl-2/Bax were reduced. Furthermore, apoptotic cells were increased in aortic intimal area, which promoted the early atherosclerotic pathological changes.2. Pioglitazone can reduce lipid levels in hyperlipidemia rats, and improve atherosclerotic pathological changes, which is independent of hypoglycemic effect.3. Pioglitazone can decrease expression of LOX-1, NF-κB p56protein on aorta in hyperlipidemia rats, weaken vascular injury changes induced by LOX-1-NF-κB signaling pathway.4. Pioglitazone has an effect on expression of apoptosis protein Bax, Bcl-2on aorta in hyperlipidemic rats, down-regulating Bax protein, up-regulating Bcl-2and the ratio of Bcl-2/Bax, reducing apoptosis of aortic intimal area, so as to play a role in protection of vascular endothelium. |
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