Study On Relationship Between Serum Visfatin And Epcs Levels Of Obesity Rats Induced By High-fat Feeding

Endothelial progenitor cells (EPCs) have the ability to differentiate into mature endothelial cells.There is too much attention since EPCs were finded .EPCs participate in angiogenesis and vascular homeostasis by incorporating into the endothelium of damaged vessels .They are very important to maintain vascular endothelium function. However, there is impairement of EPCs in many diseases and endogenous vascular regenerative potential decreases. In recent years,epidemiological studies show that there is an obvious increase of obesity frequency worldwide. Many factors play major roles in the development of obesity and environmental factor is one of them. When calory is more than we needed, it will lead to excessive storage of adipose tissue. Obesity is related with many diseases such as type 2 diabetes mellitus, especially abdominal obesity. It is reported that the number of circulating EPCs is lower in obese compared with normal weight adults.The mechanisms responsible for the obesity-related reduction in circulating EPCs number are not clear. Recently,more and more studies indicate that adipokines,such as adiponectin and resistin ,are related with vascular diseases. Many adipokines secreted by adipocyte such as adiponectin, tumor necrosis factor-α(TNF-α)and leptin all have effect on EPCs. Visfatin, as a novel adipokine, is produced and secreted by adipose tissue of human and rodent, which has numerous biological functions .Moreover, visfain plays a critical role in vascular endothelium inflammation and obesity-related metabolic disorders.In the present study, we observe bone marrow EPCs level and serum visfatin level of the obesity rats induced by high fat diets . Investigate the relationship between EPCs level and serum visfatin , glucose and lipid metabolism ,hyperinsulinemia and obesity. We explore the relation between EPCs level and serum visfatin , providing a new study direction to improve EPCs level. The paper contains two parts below.Part one: EPCs level and its influence factors of obesity rats induced by high-fat feedingObjective: To establish obesity rat model induced by high-fat feeding; To isolate bone marrow mononuclear cells ,and then determinate endothelial progenitor cell numbers and cellular characterization;To observe bone marrow EPCs level in obesity rats which were induced by high fat diet and the relationship between serum visfatin level , blood glucose, insulin, lipid, body weight and visceral fat; To investigate the correlation between EPCs level and serum visfatin level ,hyperinsulinemia, obesity, glucose and lipid metabolism.Methods: Total 22 clean male Wistar rats weighed 200～250 grams were randomly divided into two groups: normal control group (NC) and high-fat- fed group (HF). The rats in NC group were fed with standard rat chow containing 10.3% fat, 24.2% protein, and 65.5% carbohydrate as percentage of total calories,and the total calories was 348 kcal per 100g rat chow. The rats in HF group were fed with high fat diet containing 60% fat, 20% protein and 20% carbohydrate as percentage of total calories, and the total calories was 501 kcal of per 100g rat chow. The rats were fed at the same time every day. Body weight was observed every week. 16 weeks later, determine body weight and Lee's index of all rats in the two groups to confirm whether the obesity rat model was duplicated successfully. After fasted for 10 hours, the rats were taken blood sample from carotid vein; the serum was separated for the detection of lipids, insulin and visfatin. The concentration of visfatin and CRP was determined by ELISA. In the end, the rats were sacrificed by depletion, the adipose pad around kidneys and epididymides was weighed exactly as the content of visceral fat. The renal or epididymal adipose tissues were removed respectively and flash-frozen in liquid nitrogen, then stored at -70°C for further detections. The expression of visfatin in visceral fat was measured by Western blot technology. Bone marrow mononuclear cells were harvested from bone marrow by douching marrow cavity of rat femur and tibia and then ficoll density gradient centrifugation was used to isolate them. BMMNS were cultured in EGM-2mv and the cells morphology were observed by inverted microscopy .9days later,confocal laser scanning microscopy was used to analyze EPCs.10 days later,immunocytochemistry was performed to analyze the expression of CD34,VEGFR-2. Count the stain cells.Results: 1. Compared with NC group, body weight , Lee's index , visceral fat , visfatin ,CRP,fasting blood glucose, fasting serum insulin, total cholesterol and triglyceride were higher in the HF group at the end of 16week(P<0.01).2. The EPCs level of HF group rats was lower than that of NC group and there were significant differences when compared with the NC group (P<0.01). 3. Compared with NC group, the mass of visceral fat was higher, and expression of visfatin was significantly increased in HC group. There were significant differences when compared with NC group (P<0.05). 4.The EPCs level was negatively correlated with visfatin,CRP,visceral fat, fasting blood glucose , HOMA-IR, total cholesterol, triglyceride and weight (r=-0.886,r=-0.849,r=-0.631,-0.753,r=-0.775,r=-0.744,r =- 0.821,r=-0.656, P<0.01; respectively). 5. In a multiple linear regression analysis, serum visfatin level and serumCRP level enters regression equation (P<0.05).Conclusions: 1 Body weight ,Lee's index and visceral fat were increased significantly in high-fat-fed group at the end of 16 week, which suggested the success of obesity rat model and the existence of the abdominal obesity rats in HF group. 2 Expression of visfatin was significantly increased in obesity rats . 3 The bone marrow EPCs level was lower in the obesity rats, which has a negative correlation with visfatin , CRP,visceral fat, fasting blood glucose , fasting serum insulin, total cholesterol, triglyceride and weight. We proposed visfatin may contribute to the development of the decrease of bone marrow EPCs level in obesity rats.Part two: Expression of NF-κB in EPCs incubated by visfatinObjective: To observe NF-κB expression in EPCs incubated by visfatin, and explore the role of the visfatin in the decrease of bone marrow EPCs level.Methods: The procedures of isolation and culture of EPCs derived from rat bone marrow were same to the first part. EPCs were incubated by visfatin in different concentration(0,50,100,150,200ng/ml).After 24 hours, the expression of NF-κB in EPCs nucelus was measured by Western blot technology.Results: Compared with 0ng/ml group, the expression of NF-κB in EPCs nucelus were significantly increased in other four groups. There were significant differences when compared with NC group (P<0.01).Conclusions: Visfatin can induce the expression of NF-κB in EPCs. Visfatin significantly increased NF-κB transcriptional activity .