Effects Of Pioglitazone On NOS-NO And AdipoR Of Aorta In Hyperlipidemia Rats

BACKGROUNDHyperlipemia and the subsequent formation of atherosclerosis is one of the most risk factors for ischemic heart disease. Considerable evidence exists that hyperlipemia causes endothelial dysfunction a pre-requisititc of atherosclerosis which relate with cholesterol and ox-LDL. Endothelial cells (ECs) exert critical functions in the maintenance of vascular homeostasis, which impairment will result in atherosclerosis, acute coronary artery syndrome and hypertension. The incidence of atherosclerosis increases quickly due to aging and cardiovascular dangerous factors, which progressively develop and lead to decreased heart function even severe cerebral vessels and cardiovascular incidents, dysprognosis, low quality of life. Early improve impairment of vascular endothelial which can inhibit the process of atherosclerosis. Endothelial dysfunction is an early pivotal event of atherosclerosis. Alterations of nitric oxide synthase (NOS) expression or one of the cofactors involved in NOS activation, and increased destruction of nitric oxide (NO). NO has been thought to be the most important mechanism of endothelial dysfunction in hyperlipemia.when the quantity of NO decrease will lead to diastolic dysfunction which resulted in atherosclerosis. Adiponectin, know as adipocyte complement-related protein which is negatively with body fat. Adiponectin plays important roles in the regulation of insulin sensitivity, anti-inflammatory and metabolism. A decreased plasma level of adiponectin is one of the most risk factors for ischemic heart disease. Peroxisome proliferator activated receptors (PPARs) are transcription factors belonging to the nuclear receptor superfamily. Pioglitazone as PPARγagonists which not only can decrease the blood lipid but also inhabit the inflammatory of vascular, elevate the activity of fibronolysis, improve endothelial cell function and decrease the level of C reactive protein. Whether or not PPAR agonists may preserve vascular endothelial function and mechanism is not clear, and few research in AdipoR of aorta in hyperlipidemia rats. Thus, we established hyperlipidemia rat with high fat diet to research mechanism of Pioglitazone in the model and provided theory support for clinic curing.OBJECTIVEIn order to provide academic foundation on anti-atherosclerosis effect by pioglitazone, we have been investigated the effects of pioglitazone on NOS-NO and AdipoR of aorta in hyperlipidemia rats.METHODS27 healthy male SD rats were randomly divided, by random digits table, into 2 groups:control group, high-fat diet group. Control group fed with typical forage. The high-fat diet group fed with high fat diet (2% cholesterol, cholate 0.5% thyreostat 0.2%) for 12 weeks. Then to draw blood from vena orbitalis posterior, the level of serum lipid and blood glucose were measured. An ideal experimental rat model of hyperlipidemia was made when the level of lipid was higher in high fat diet group. The high-fat diet group was randomly divided, by random digits table, into 2 groups: model group, pioglitazone group. Pioglitazone group:lavage with Pioglitazone (10mg/kg, qd)for four weeks, control group and model group were lavaged with tales doses of distilled water for four weeks(1.5 ml/100g,qd). Rats were continuously lavaged for 4weeks in every group and then were dissected, we anesthetized injection by 20% Ketamine (1ml/100g), routinely sterilized, unfolding splanchnocoel.We got blood 5ml from left ventricle, then to put with room temperature for 1 hour, centrifuge for 5 minutes with 3000 rpm. We separated the aorta from juncture of arch of aorta and thoracic aorta in disk with ice.The formalin fixation tissue was made for HE stain.4 weeks after the intervention of pioglitazone, we observed alterations of level of blood lipid. triglyceride (TG) was measured by CHOD-PAP method, total cholesterol (TC) was detected by CHOD-PAP. Low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) was measured by selectivity precipitation method. we also observed alterations of level of blood glucose. The aorta was taken for pathologic analysis with HE stain, immunohistochemical method and electron microscope. ADP was measured by euzymelinked immunosorbent assay. NO was determined by nitric acid reductase method, and iNOS and tNOS were determined by catalytic method (cNOS activity was obtained by subtracting iNOS activity from the total NOS activity). The expression of AdipoR was measured by Western Bolt and fluorescent quantitation reverse transcription polymerise chain reaction. We observed the change of aorta with light microscope and electron microscope. Dcreased NOS-NO, adiponectin and its receptors may be involved in vascular impairment in rats with high-fat diet induced hyperlipidemia. Pioglitazone has anti-atherosclerotic effects, the effect may be related to increased NO, cNOS, serum adiponectin and vascular expression of adiponectin receptor.Statistical method:The data was analyzed by SPSS 13.0 statistic software.Values are presented as means±SD.If variance to be equalized when two sets data to compared with each other, data were analyzed with independent sampler t test. If variance to be not equalized, data were analyzed with Welt t test. Comparisons among groups were performed by one way ANOVA analysis. if the variance to be equalized, two sets data were analyzed with LSD test. if the variance to be not equalized, two sets data were analyzed with Dunnett's T3 test.A P<0.05 was considered significant.1. Alterations of blood lipid and blood glucoseBefore the rats in model group underwent intervention with Pioglitazone for 12 weeks, After 12 weeks of feeding, The levels of TG, TG,and LDL-C in high fat diet group significantly increased (P<0.001, vs control group). The level of HDL-C and blood glucose (BG) in high fat diet group and control group had no statistical difference (P=0.091, P=0.884). After 4 weeks'treatment with Pioglitazone, the levels of TG,TC in Pioglitazone group were significantly different from model group (P<0.01), and were roughly identical to control group (P=0.562, P=0.931).The levels of HDL-C, LDL-C and BG in Pioglitazone, model group and model group had no statistical difference(P=0.884,P=0.177, P=0.860), The level of HDL-C had upgrade tendency.2. Alterations of aorta morphology.2.1 aorta HE stainingSample from control group showed intact endothelial layer and no smooth muscle proliferation. Endothelial cell sloughing, and smooth muscle proliferation and irregular cell form were observed in model group. Pioglitazone group showed attenuated morphologic changes of endothelial and smooth muscle cell.2.2 Alterations of aorta morphology with light microscopeThe vascular endothelial of aorta was stained by CD31. It was showed that the control group with intact endothelial layer, smooth muscle well-distributed and no proliferation in. Lots of endothelial layer ablated, smooth muscle proliferation, irregular cell form and hypertrophy were observed in model group. The smooth muscle proliferation in pioglitazone group was not obvious. Pioglitazone also showed fundamentally intact endothelial layer.2.3 Alterations of vascular endothelial cell morphology with electron microscopeIntact vascular endothelial cell, intracytoplasm chondriosome and nucelus structure smooth muscle well-distributed and no proliferation in control group. The results which were observed by electron microscope showed that hypochromatosis, plasmatorrhexis, organelle diminished or disappeared, vacuolar degeneration in vascular endothelial cells, even adipose capsule in model group. Few lipid droplet vacuoles in cytoplasm of the endothelial cells, and various cellorgans and elastic membrane were seen in model group. Pioglitazone showed fundamentally intact endothelial layer. A sprinkle of endothelial cell showed with dropsical chondriosome and a sprinkle smooth muscle showed with foam apomorphosis.3. Alterations of NO and NOS of the aorta The levels of NO,cNOS of aorta of model group was decreased(P<0.001) compared with that of control group.The level of NO in Pioglitazone group was increased significant compared with model group (P<0.01) and had no visible difference with control group (P=0.490). The level of cNOS in Pioglitazone group was increased significant compared with model group (P<0.05) and had no visible difference with control group (P=0.444). The levels of iNOS and tNOS in Pioglitazone, model group and control group had no statistical difference(P=0.389,P=0.189).4. Alterations of plasma of ADPPlasm level of ADP in model group was decreased(P<0.01) compared with that in control group. Plasm level of ADP in Pioglitazone group was increased compared with model group(P<0.05)Tthere was no visible disparity between Pioglitazone group and control group(P=0.518).5. AdipoR mRNA expressions and AdipoR protein of aorta.AdipoR1 and AdipoR2mRNA expressions of model group were significantly decreased (P<0.01), compared with control group. AdipoR1mRNA in Pioglitazone group was increased compared with model group (P<0.01), and had no difference to control group (P=0.062). AdipoR2mRNA in Pioglitazone group was increased compared with model group (P<0.01), there was no visible disparity between control group and Pioglitazone group (P=0.191).AdipoR1 protein expressions of model group were significantly decreased (P<0.01), compared with control group. AdipoR1 protein in Pioglitazone group were increased compared with model group (P<0.05) and had no obvious difference to control group (P=0.182). AdipoR2 protein expressions of model group were significantly decreased (P<0.01), compared with control group. AdipoR2 protein in Pioglitazone group were increased compared with model group (P<0.01), and had no obvious difference to control group (P=0.068)CONCLUSIONS 1. The results show that TG, TC and LDL-C with hyperlipemia were significantly increased. Endothelial cell sloughing, absence and smooth muscle proliferation and irregular cell form were observed in model group. We copy model rats with high fat diet successfully.2. The results show that levels of NO and cNOS of aorta and plasm ADP were decreased obviously in model group, whenever AdipoRmRNA expressions and AdipoR protein receptors of aorta detected by real time fluorescence quantitation polymerase chain reaction and Western Blotting.3. Pioglitazone could improve vascular impairment in hyperlipemia rats which might be related to increase plasm level of ADP, the levels of NO and cNOS of aorta, AdipoRmRNA expressions and AdipoR protein receptors.4. Pioglitazone can inhibit process of vascular impairment in hyperlipemia rats, which may increase AdipoR expression, then strengthen the ADP increasement result in the quantity of NO and cNOS of aorta increased.