Effects of Pioglitazone on NOS-NO and AdipoR of Aorta in Hyperlipidemia Rats
|School||Southern Medical University,|
|Course||Cardiovascular within science|
|Keywords||Hyperlipidemia Pioglitazone Nitric oxide Nitric oxide synthase Adiponectin Adiponectin receptor|
Hyperlipidemia (Hyperlipemia, HL) is an important risk factor for atherosclerosis formation, atherosclerosis early increase in the oxidation of low-density lipoprotein and cholesterol on artery intima functional damage, which affects endothelial function. Endothelial plays an important role in maintaining vascular integrity, originating the role of endothelial injury in the course of atherosclerosis, atherosclerosis, acute coronary syndrome, hypertension and other cardiovascular disease development and pathology has close contact. Progressive development of the atherosclerotic process causes serious cardiovascular and cerebrovascular incidents seriously affect the survival and quality of life for patients with poor prognosis. Arterial injury due to blood lipids in early intervention, and repair of blood vessels, blocking atherosclerosis progression, plays an important role in the control of cardiovascular acute events. Atherosclerosis early changes in endothelial damage, nitric oxide synthase (NOS) expression and cofactor system change, leading to increased destruction of nitric oxide (NO), decrease in the synthesis of NO, endothelium-dependent vasodilation regulation imbalance can be resulting in atherosclerosis formation. Reasonable increase NO levels can improve endothelial function, thus preventing atherosclerosis progression. Adiponectin (ADP) is a negative correlation with body fat content was adipocytokines, has confirmed that it has a wide range of biological activity, such as anti-inflammatory, increase insulin sensitivity, hypoadiponectinemia becoming cardiovascular an independent risk factor for the disease. Adiponectin receptor (AdipoR) 1/2 regulating insulin sensitivity in regulation of adiponectin, AdipoR1 / 2 expression in the skeletal muscle and liver, which may in the regulation of ADP anti-inflammatory effects, improve endothelial cell dysfunction. Peroxisome proliferator-activated receptor γ (PPARγ) as transcription factors belonging to the nuclear receptor superfamily. PPARγ agonist pioglitazone not only regulate lipid metabolism, but also by inhibiting vascular inflammation, increase the activity of the fibrinolytic system, improve endothelial function, reduce the level of C-reactive protein. Improved aortic vascular function mechanism is unclear. Therefore, the author uses the high-fat diet hyperlipidemia rat aorta injury model to explore the role and mechanism of pioglitazone intervention on the of hyperlipidemia rat aortic injury process, provide theoretical support for pioglitazone anti-atherosclerosis. Objective: To investigate pioglitazone hyperlipidemia rat plasma adiponectin hormone (ADP), aortic nitric oxide (NO), inhibition of nitric oxide synthase (iNOS), constitutive nitric oxide synthase (of cNOS) of total nitric oxide synthase (tNOS) the changes and lipid hormone receptor (AdipoR) expression change and topiramate grid column one of the intervention impact explore pioglitazone possible role of mechanisms, theoretical basis for pioglitazone rosiglitazone anti-atherosclerosis. Methods: the first 12 weeks, 27 male Sprague-Dawley rats by the random number table, were randomly divided into two groups, were randomly divided into normal control group of 9, 18 fat diet group, the modeling experiments. After 12 weeks, the survival made module 17 rats were randomly divided into the model group by random number table, adjacent pioglitazone group. Control group fed standard rat chow, the model based on high fat diet (2% cholesterol, 0.5% cholate, methyl thiouracil fed 0.2% and 72.3% of diet) fed for 12 weeks, 12 weekend from the orbital venous blood testing blood lipid levels to determine whether the model successfully reproduced, and detection of blood sugar (blood glucose, BG) level. Subsequent fat diet group rats were randomly divided into the model group and the adjacent cell column ketone group, pioglitazone ketone groups: pioglitazone 10mg · kg-1 · d-1 continuous gavage control group and model group: double distilled water orally, 1.5 ml/100g weight, 1 times / day; gavage 4 w. After 4 weeks, 24 hours of fasting, and at 8:00 the next day, with 20% ketamine dose of 1 ml/100g anesthesia, cut open the chest and abdomen cavity, exposed the heart, the left ventricle puncture blood mll static at room temperature set 3000 rpm / separation for 5 minutes, the serum was separated at -20 ℃ refrigerator to save the tested serum markers; ice intraday separation of the aorta, and drawn in the junction of the aortic arch and thoracic aorta, with 10% sexual formaldehyde fixed for HE pathological detection, the remaining aorta tissue stored in liquid nitrogen the DUT other indicators. Pioglitazone-one intervention 4 weeks to detect groups lipids level triacyl glycerol (TG) TPO-PAP method to detect, total cholesterol (TC) CHOD-PAP method to detect low-density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) is detected by selective precipitation, and detecting BG. Eosin staining, immunohistochemical staining vascular endothelial cells observed by light microscopy, electron microscopy endothelial cell microstructure. Enzyme-linked immunosorbent assay serum ADP, nitrate reductase measured serum and aortic NO, arginine the catalytic assay aorta tNOS, iNOS and of cNOS (equal tNOS the level with the difference of iNOS). Western blot hybridization measured aortic adiponectin receptor 1 and 2 protein level, real-time quantitative PCR method to measure aortic adiponectin receptor 1 and 2 gene expression. Light and electron microscopy aortic morphological change. Aortic NOS-NO, adiponectin and its receptors decline may intervene hyperlipidemia vascular injury, pioglitazone has anti-atherosclerosis effect, this effect may be related to the increase serum ADP and the aorta the vascular AdipoR expression. Statistical methods: statistical software SPSS13.0 analysis, quantitative data are presented as mean ± standard deviation (x ± s) said; two sets of data homogeneity of variance, independent samples t-test, if unequal variances with Welch correction t test; among groups with completely randomized design information if homogeneity of variance, LSD test for pairwise comparisons, analysis of variance, heterogeneity of variance with Dunnett's T3 test. P ≤ 0.05 was considered statistically significant. Results: 1. Serum lipids, blood glucose, pioglitazone rosiglitazone intervention model after 12 weeks of high-fat diet group TG, TC, LDL-C compared with the control group was significantly higher (P lt; 0.01), HDL-C, BG fat diet group than in the control group showed no statistical significance (P = 0.091, P = 0.884). By pioglitazone ketone intervention in four weeks, the model group than the normal control group TG, the TC was significantly higher (P lt; 0.01), pioglitazone ketone group compared with the model group TG, the TC significantly with decreased (P lt; 0.01); TG, the TC pioglitazone ketone group compared with the control group difference was not statistically significant (P = 0.562, P = 0.931). Control group, model group, pioglitazone ketone group of HDL-C, LDL-C, BG differences without statistical significance (P = 0.884, P = 0.177 P = 0.860), but pioglitazone ketone group HDL-C have tended to increase. Rat aorta morphological changes 2.1 the aortic pathology HE staining morphology aortic endothelial change control group did not change significantly, endothelial maintain continuity, endometrial integrity, no smooth muscle thickening; model group lipidoses, part intimal thickening, smooth muscle disorders, hyperplasia, endothelial fracture, absent; pioglitazone group was no significant change in the intima and smooth muscle. The 2.2 light microscopy showed morphological changes in factor VIII-related CD31 antibody to vascular endothelial cells observed by light microscopy, the control group rat aortic endothelial and smooth, smooth muscle tissue evenly distributed. The shedding model group rat aortic endothelial tissue most obvious proliferation of smooth muscle, disorganized, cell hypertrophy, perpendicular to the endometrium. The pioglitazone group aortic endothelial largely intact, occasional off, smooth muscle cell proliferation is not obvious, regular arrangement normal morphology. 2.3 electron microscopy of vascular endothelial cell ultrastructural changes in the control group rat vascular endothelial cells normal intracytoplasmic mitochondrial normal, normal structure of the nucleus, the internal elastic membrane continuous, uniform thickness, smooth muscle cells is normal. Lesions heavier model rats endothelial cells, membrane rupture, cytoplasmic depigmentation spills or loose degeneration, dissolution or disappearance of organelles in the cytoplasm, nucleus, loose chromatin, reduce the electron density incomplete karyotheca; subendothelial fibers The mother cell hyperplasia Variable collagen fibers. Interrupted internal elastic membrane of varying thickness, multifocal dissolved in the film smooth muscle cells lipid deposition, a foam-like change. Pioglitazone group rat vascular epithelium neat, almost normal endothelial cells, occasionally endothelial cell mitochondria edema, smooth muscle cells of the intima occasional mitochondrial edema and foamy change. 3. Aortic NO and NOS vitality of changing the model group aortic NO level lower than the control group (P lt; 0.01); while pioglitazone ketone group and the control group between differences without statistical significance (P = 0.490): pioglitazone ketone group and model group between differences have statistical significance (P lt; 0.01): the model group than the control group cNOS decreased (P lt; 0.01), the difference between pioglitazone ketone group and the control group no significant difference (P = 0.444) pioglitazone ketone group than in model group increased , the difference was statistically significant (P lt; 0.05). Groups between the iNOS, tNOS vitality changed completely randomized analysis of variance derived differences not statistically significant (P = 0.389, P = 0.189). Plasma ADP levels decreased adiponectin levels compared with the control group (P lt; 0.01), plasma ADP change the model group the pioglitazone group plasma ADP levels were significantly increased (P lt; 0.05). Pioglitazone group compared with the control group the difference was not statistically significant (P lt; 0.518). 5. Aortic AdipoRmRNA and AdipoR protein expression in model group AdipoR1mRNA level low control group (P lt; 0.01), pioglitazone ketone group and model group differences have statistical significance, higher than the model group (P lt; 0.01) and the control group with topiramate grid column one no significant statistical significance (P = 0.062) in the group. The model group AdipoR2mRNA level lower than that of the control group (P lt; 0.01), statistically significant difference between the pioglitazone group compared with control group no significant differences (P lt; 0.191), the glitazone group and the model group than the model group (P lt; 0.01). Low control group (P lt; the model group AdipoRl protein level of 0.01), the difference between the pioglitazone group and model group were statistically significant, higher than the model group (P lt; 0.05). No statistically significant differences between the pioglitazone group and control group (P = 0.182). Model group AdipoR2 protein level is lower than the control group (P lt; 0.01), pioglitazone ketone group and the control group comparison was no statistical difference (P = 0.068), pioglitazone ketone group and model group there are statistically significant, higher than the model group (P lt; 0.01). Conclusion: 1. Caused by high-fat diet in hyperlipidemia rat TG, TC, LDL-C was significantly higher, and successfully copied a rat model of hyperlipidemia; the pathological vascular endothelial fracture, absent intimal thickening smooth muscle disorders, hyperplasia. Model Aortic NO observed of cNOS levels significantly decreased plasma ADP concentration, quantitative real-time PCR and Western Blotting results reduce the aorta AdipoRmRNA its protein synthesis; 3. Pioglitazone significantly improve fat hyperlipidemia rat aortic injury, may lower blood lipid levels (TG, TC), plasma ADP, aortic NO, NOS levels and aortic AdipoRmRNA of expression and protein synthesis; 4. pioglitazone protect vascular tissue, inhibition of endothelial Injury, which may be related to plasma ADP levels, promote aortic cNOS-NO generation and to increase aortic AdipoR expression.