| Backgroud:The pathogenesis of hypertension has not yet completely clear, it is mainly affect by genetic, environmental and lifestyle, and it is a kind of systemic disease that caused by multiple of interaction risk factors. In recent years, it is consider that hypertension is an important part of the metabolic syndrome (MS), and related to insulin resistance(IR), the theory of MS has established and expanded the research space of the pathogenesis of hypertension and to further improve its treatment strategy. The application of lifestyle intervention is the most often overlooked way but it can play an effective and efficient role in the prevention and treatment of heart and cerebrovascular diseases, the use of lifestyle interventions to rectify IR have offers a new way for the treatment of hypertension. Currently, there haven't got any relevant reports of using comprehensive measures combine with drug as a way to rectify the insulin resistance, and there haven't got any related research to use such comprehensive measures combine drug to observe the antihypertensive and target organ protection effects. To explore the use of drugs and lifestyle intervention to rectify IR on hypertension and hypertension-related target organ effects, we designed this study, our purpose is to further explore the internal mechanism of such comprehensive measures of antihypertensive and target organ protective effects.Methods:1. The main body of the experimental study is a total of 44 spontaneously hypertensive rats (SHR), aged 6 to 7 weeks, the body weight is 120-160 grams.The SHR were randomly divided into 4 groups:①SHR control group, this group was normal feeding, gave rat food and water.②Nifedipine drug control group(N), nifedipine was added in the drinking water, the concentration of 140mg/L.③Pioglitazone group(P1), pioglitazone was added in the drinking water, the concentration of 45mg/L.④Pioglitazone combine with lifestyle intervention group(P2). pioglitazone was added in the drinking water, the concentration of 45mg/L,while giving the rats lifestyle intervention everyday. We use the control of rats diet and add aerobic exercise (swimming) as lifestyle intervention, the study has lasted for 12 weeks.2. Diet control methods: Measured the SHR group for 24 h food intake once a week, and calculate the average daily food intake for each rat, every morning, the rats in P2 group were given 90% of the average daily intake of SHR group a week before.②Swimming methods and conditions: The rats in P2 Group were swam 6 ~ 15 min everyday, at the same time of the day and under the same quiet,conditions, treated the rats floating on the surface of the heads fixed and the emergence of sinking as the stop signal, the water temperature was 28℃~ 30℃, water depth was 50 cm, we ensure that rats can not touch the bottom and side walls of the pool to avoid exercise.3. Blood pressure measured was used by by tail indirect blood pressure measurement, we measured the blood pressure of the rats every 2 weeks, weight the rats every 4 weeks.4. At the end of the experiment, rats were fast for 8 hours, then collected the blood sample after anesthesia, the blood samples were centrifugation at 4000r/min speed for 5 minutes within 10 minutes after the blood samples were taken, and sent to the laboratory within 2 hours after the centrifugati. We detected total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol(LDL-c), high density lipoprotein cholesterol (HDL-c), fasting plasma glucose(FPG), fasting insulin levels (FINS), the concentration of eNOS and AngⅡ, calculated atherogenic index(AI)=(TC - HD)/HDL, insulin resistance index(HOMA-IR) =FPG×FINS/22.5, and insulin secretion index(HOMA-β)=FINS×20/(FPG-3.5).5. Take out of the left ventricular heart for 0.3cm×0.3cm, and the thoracic aorta for 1cm, fixed in 10% neutral formalin, conventional method to paraffin embedding and hematoxylin-eosin(HE) staining, using optical microscope to observe the morphological changes of the heart and aorta in every groups.6. Using immunohistochemistry(SP)and in situ hybridization method to detect the heart and aorta, focusing on the expression of eNOS and AT1 in protein and mRNA.7. The results of immunohistochemistry and in situ hybridization and the image analysis determined: we treated the cytoplasm showed brown particles, and the intensity of the color were harder than the background but non-specific staining was judged to be positive. We randomly selected 10 high magnification view (×200) in each slice, using LX70OLYMPUS Cooled CCD microscope to collected view photos, using quantitative analysis system (Image-Pro-Plus Analysis software) to analyze and image analysis system for image processing, analysis the selected field of view of the positive signal, calculated the average density of positive signals of myocardial and aortic tissue in each group.Results:1. During the experiment, the blood pressure of all the SHR began to increased, but different in amplitude, the rat in SHR group got the highest speed, the 1st to 6th weeks was a rapid increase, then enter the plateau; The blood pressure of N group was control better in the early experiments, but later got a rise trends; The blood pressure of the rats in P1 and P2 group was a slow increase in the 1st to 6th weeks, and then enter the plateau. at the end of the experiment, the blood pressure of the rats in P1 and P2 group have significant reduced, compared with SHR group, the difference was significant(P<0.05 and P<0.01), at the same time,the blood pressure of SHR in P2 group was better than in P1 and N group, the difference was significant (P<0.05).2. The weight of rats in each groups have grew over time, at the end of the experiment, compared to the SHR group, the weight of rats in N and P2 group have reduced, the difference was significant(P<0.05).3. Compared with SHR group, the TG levels in N and P1 group have reduced(P<0.05), rats in P2 group have lower TC levels (P<0.05), and higher HDL-c levels(P<0.05), compared with other three groups the atherogenic index decreased significantly by calculating, the difference was significant (P<0.05).4. Compared with SHR group, rats in P1 and P2 group have lower FPG (P<0.05), the FINS levels and the HOMA-IR were significantly reduced(P<0.05和P<0.01), the HOMA-βincreased significantly (P<0.05).5. Compared with SHR group, rats in P2 group have higher serum eNOS, the difference was significant (P<0.05), and the AngⅡof rats in P2 group were significantly reduced when compared with SHR group, the difference was significant (P<0.05).6. Using electron microscopy, all SHR have myocardial tissue edema and myocardial necrosis tiny inner in varying degrees, rats in SHR and N group were worse. Compared with SHR group, the myocardial tissue edema and myocardial necrosis tiny inner of rats in P2 group were significantly reduced; The degree of aortic wall were thickening in varying degrees, the elastic fibers and smooth muscle accrementition in varying degrees, and there are accumulation of foam cells under the subintimal. The degree of aortic wall of rats in P2 group have reduced, elastic fibers and smooth muscle arranged structured.7. The immunohistochemistry results showed that, there were expression of positive signals of eNOS in rats'heart and aorta endothelial in every SHR, but different in intensity, the SHR group and N group were weaker, showed as pale brown. Calculated by the gray scale scanning,compared with SHR group, the protein expression of eNOS in P1 and P2 group rats'heart and aorta endothelial were significantly increased, the difference was significant(P<0.01), the protein expression of eNOS in P2 group were higher than P1 group, the difference was significant(P<0.05). While there were expression of positive signals of AT1 in rats'heart and aorta endothelial in every SHR, the SHR group were higher, showed as brown. Calculated by the gray scale scanning, compared with SHR group, the mRNA expression of AT1 in P2 group rats were decreased significantly(P<0.01).8. The in situ hybridization results have the same trends with the immunohistochemistry and showed that, there were expression of positive signals of eNOS mRNA in rats'heart and aorta endothelial in every SHR. Calculated by the gray scale scanning, compared with SHR group, the protein expression of eNOS mRNA in P1 and P2 group rats'heart and aorta endothelial were significantly increased, the difference was significant(P<0.01), the mRNA expression of eNOS in P2 group were higher than P1 group, the difference was significant(P<0.05). While there were expression of positive signals of AT1 mRNA in rats'heart and aorta endothelial in every SHR. Calculated by the gray scale scanning, compared with SHR group, the mRNA expression of AT1 in P2 group rats were decreased significantly, the difference was significant (P<0.01), at the same time, compared with P1 group, the mRNA expression of AT1 in P2 group rats were decreased significantly(P<0.05).Conclusion:1. The methods of poglitazone combine with lifestyle intervention has a good blood pressure lowering effect in SHR.2. Pioglitazone combine with lifestyle intervention can improve glucose and lipid metabolism in SHR and have anti-atherosclerosis effect.3. Pioglitazone combine with lifestyle intervention can increase the protein and mRNA expression of eNOS in SHR'heart and aorta endothelial cell, while decreased the expression of AT1.4. Pioglitazone combine with lifestyle intervention has high protect effect in hypertension-related target organ (heart and aorta), the role of mechanism including rectify IR and increased the expression of eNOS and reduced AT1.5. Pioglitazone has blood pressure lowering effect and protect effect in hypertension-related target organ, however, pioglitazone combine with lifestyle intervention has better effect than only use pioglitazone.Innovation points:The first time to application diet control and swimming as a method of lifestyle intervention in animal experiments, and the first time to application insulin-sensitizing agent with the lifestyle intervention to rectify IR in research of hypertension,enhance the expression of eNOS and reduced the expression of AT1 are one of the mechanisms of antihypertensive and target organ protection.
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