Effect Of Resistin On Vascular Endothelium Secretion Dysfunction And Different Treatments On The Expression Of Resistin And Endothelial Function In Rats

Background and objectives:Atherosclerosis(AS),one of the cardiovascular diseases, is the most significant cause of mortality.Investigations in recent years have demonstrated that endothelial dysfunction is the initiating and early event in the development of atherosclerosis.Moreover,endothelial dysfunction exists in all periods of the development of AS and is a important marker for acute cardiovascular diseases.Therefore,it is crucial to detect the risk factor and mechanism of endothelial dysfunction earlier for the prevention of AS.Vascular endothelial cells play a central role in maintaining cardiovascular homeostasis in health.In addition to providing a physical barrier between the vessel wall and lumen, the endothelium secretes a number of mediators that regulate platelet aggregation,coagulation,fibrinolysis and vessel tone. The term endothelial dysfunction refers to an imbalance in the production of these mediators,which may promote vasospasm, thrombosis or vessel occlusion in experimental models and has been implicated in the pathogenesis of acute coronary syndromes and other cardiovascular disorders.The high risk factors for endothelial dysfunction include hyperlipidemia,hypertension, diabetes mellitus,inflammation,infection and smoking. Resistin was recently suggested to be involved in the development of endothelial dysfunction.Resistin is a recently described novel adipokine that has been suggested to play a role in the development of insulin resistance.Recent studies on the relationship between resistin and endothelial dysfunction have been predominantly in vitro.Some studies indicated resistin exerted direct effects to promote endothelial cell activation before the onset of overt AS.Incubation of porcine coronary rings with resistin impaired endothelium-dependent vasorelaxation,increased oxidative stress,and decreased eNOS expression.Endothelial cells(ECs)were activated by promoting ET-1 release,in part by inducing ET-1 promoter activity after incubated with human recombinant resistin.Furthermore, resistin upregulates adhesion molecules and monocyte chemotactic protein-1.However,the researches on the relationship between resistin and endothelial dysfunction were inadequate.Invivo, there are few investations and reports on whether resistin have regulating effects on endothelial function.And little is known about the mechanisms of how resistin signals in the endothelium. These problems are to be solved in the research of resistin. Whether the endothelium secretion factors changed with the variation of resistin? In this study,we established hyper-resistin model in rats by high-fat diet and resistin injection to observe the effect of resistin on the endothelial function invivo.At the same time,the present study was designed to investigate the protein and gene expression of phosphatidylinositol 3 kinase in the hyper-resistinmodel.The effects of rosiglitazone,swimming,diet control and resistin antibody on the resistin,endothelial function and PI3K were contrasted.Methods1.The establishment of extrinsic hyper-resistin modelThis study was conducted on 16 male 4 week old Sprague Dawley rats.The rats were divided into two groups randomly:resistin group(n=8 RS),normal saline group(n=8 NS).The rats in the resistin group received an infusion of rat recombinant resistin (Phoenix Pharmaceuticals Ltd,American)(10μg/rat)through the tail vein after 10 hours of fasting.A second infusion of rat recombinant resistin was given to the resistin group two hours after the first infusion.Sham operations were conducted on the NS group at the same time.Two hours after the second dose of resistin,the resistin group and the NS group were anesthetized and blood samples and thoracic arteries were collected for different detections.2.The establishment of intrinsic hyper-resistin modelThis study was conducted on 130 male 4 week old Sprague Dawley rats.The rats were divided into two groups randomly: high-fat diet group(n=122)and normal control group(NC n=8). All rats received high-fat chow except the rats in the normal control group,which received standard chow.After the high-fat diet group was fed for 8 weeks,blood samples of the two groups were collected from the right internal jugular vein for the measure of resistin.The rats with resistin levels 2 S.D.greater than the mean of resistin in the control group were defined as having diet-induced hyper-resistinemia model.There were 52 rats in the diet-induced hyper-resistinemia model.The 52 rats were divided randomly into hyper-resistinemia model group(HRE n=8) and other four groups(see method 3).The rats with resistin levels lower than X+1S.D.of resistin in the control group were defined as having diet-induced normal resistin model(24 rats). Eight rats were selected randomly from the diet-induced normal resistin model and defined as normal resistin goup(NRE n=8). Then the rats in the diet-induced hyper-resistinemia group(HRE), diet-induced normal resistin group(NRE)and normal control group(NC)were fed as before for 8 weeks.After 8 weeks,all rats in the three groups were anesthetized and blood samples and thoracic arteries were collected for different detections.3.The effects in different treatment methodsForty rats were selected randomly from diet-induced hyper-resistinemia model(see method 2)and randomized into diet-control group(DC n=8),swimming with diet-control group (SW n=8),rosiglitazone with diet-control group(RO n=8) antibody with diet-control group(AB n=8)and hyper-resistinemia group(HRE n=8).The rats in DC group were given standard chow without other treatments.The rats in SW group swam one hour per day,five days per week.The rats in RO group were received rosiglitazone 2mg/(Kg·d)The antibody group received two injections of resistin antibody(Biovendor Laboratory Medicine, Inc.Germany)through the tail vein for 3 days.The rats in SW, RO and AB group were given standard chow and the rats in HRE group were given high-fat diet as before.The treatments in RO,SW and DC group were lasted for 8 weeks.After the treatments,all rats in NC,DC,SW,RO,AB and HRE groups were anesthetized and blood samples and thoracic arteries were collected for different detections.4.Measurement of variables in serumSerum levels of resistin,PAI-1,and vWF were measured by using ELISA kits.Insulin(INS)and endothelin(ET)were measured by radioimmunoassay.Serum nitric oxide(NO)production was detected by measuring the final stable equimolar degradation products nitrite and nitrate by using a standard colormetric assay.Total cholesterol(TC)and triglycerides(TG)were determined by enzymatic method and low-density lipoprotein(LDL) and high-density lipoprotein(HDL)cholesterol were determined by immuno—turbidimetry assay respectively.Atherosclerotic index(AI)was calculated from TC and HDL:AI=(TC—HDL)/HDL.5.Determination of the mRNA expression of eNOS and ET by RT-PCR method:The relative expressing level was defined by optical density ratio of eNOS and ET to actin respectively(eNOS /actin,ET/actin).6.Immunohistochemistry was employed to examine the expression of phosphatidylinositol 3kinase(PI3K)p85αsubunit and protein kinase B(Akt)in thoracic artery endothelium.7.In situ hybridization was employed to examine the mRNA expression of PI3K p85αsubunit and Akt—1 in thoracic artery endothelium.8.Quantification was performed through a microscope with a computer-assisted image analysis system(image-pro plus 5.0).9.The data were indicated as mean±standard deviation((?)±s)and were analyzed by Student t test and one-way ANOVA using SPSS14.0 statistical software.The correlation between variables,which were considered to be normally distributed by normality plots,was analyzed using Person' s correlation and partial correlation.Value of P＜0.05 was considered significant.Results1.The effect of extrinsic resistin on endothelial function 1.1 After two injections of rat recombinant resistin,the levels of insulin and resistin in the resistin group were increased significantly,while the insulin sensitive indexs were lower in the resistin group than those in the NS group.In the resistin group,the levels of ET,PAI,and vWF were higher while the level of NO was lower than those in the NS group.There was no difference in body weight and lipids of the two groups.1.2 There was no difference in resistin mRNA expression of RE and NS group.The eNOS mRNA expression in RE group was lower and ET mRNA higher than those in NS group.1.3 The results in immunohistochemistry and In situ hybridization indicate that the expression levels of PI3K and Akt in the resistin group were much lower than that in the NS group,which had strong positive brown granules in the cytoplasm.2.The effect of intrinsic resistin on endothelium function2.1 The body weight,Lee index,TC,TG,LDL were much higher in high resistin group(HRE)and normal resistin group(NRE)than those in normal control group(NC),while the level of HDL in HRE and NRE group decreased.2.2 The level of resistin in HRE group was the highest among HRE,NRE and NS group.The level of insulin increased and the level of ISI decreased in HRE and NRE group compared with normal control group.There was markedly endothelial dysfunction in HRE and NRE group.Moreover,the rats in HRE group had worse endothelial dysfunction than NRE group.The level of NO decreased significantly and the levels of ET,PAI and vWF increased in HRE group.2.3 The resistin expression in HRE group was the highest among HRE,NRE and NC group.There was no difference between NRE and NC group in resistin expression.The eNOSmRNA expression in NRE group was higher than that in HRE group and lower than that in NC group.The ETmRNA expression in HRE group was the highest among HRE,NRE and NC group and the ETmRNA expression in NRE group was the second.2.4 The results in immunohistochemistry and In situ hybridization indicated that the expression levels of PI3K and Akt in the HRE group were much lower than that in the NRE group and NC group,which had strong positive brown granules in the cytoplasm.The IOD of PI3K and Akt in the HRE group were the lowest among HRE,NRE and NC group and those in NC group were the highest.3.The effects in different treatment methods3.1 After the treatments in RO and SW group lasted for 8 weeks, the body weight,Lee index were much lower in DC and SW group than those in HRE group.There was no difference in body weight, Lee index among RO,AB and HRE group.The lipids in SW group improved most and those in DC and RO group the second.There was no difference in lipids between HRE and NC group.3.2 The four treatments all decreased the level of resistin and insulin and increased ISI markedly.The levels of resistin and insulin in RO and SW decreased most among the four treatment groups and those in DC and AB group decreased secondly.3.3 The levels of ET,PAI,vWF in RO,SW and AB group were much lower and NO higher than those in HRE group.The levels of ET and PAI in De group decreased compared with the HRE group. There no difference in vWF and NO between DC and HRE group.3.4 The resistin mRNA expression in SW and RO group were decreased mostly.There was no difference in resistin mRNA expression between AB and HRE group.The four treatments all increased markedly the expression of eNOSmRNA and decreased the expression of ETmRNA.There was no difference between HRE and NC group.3.5 The results in immunohistochemistry and In situ hybridization indicated that the four treatments all increased markedly the expression of PI3Kp85αand Akt with the IOD increased significantly.RO and SW group improved the PI3Kp85αand Akt expression most among the four groups.4.CorrelationResistin levels and the expression of resistin mRNA were obviously correlated negatively with NO and eNOSmRNA expression and positively with ET,PAI,vWF and ETmRNA expression.Resistin levels and the expression of resistin mRNA were correlated negatively with the expression PI3Kp85αand Akt.Even after correction for weight,Lee index,insulin,ISI and lipids, resistin and the expression of resistin mRNA still correlated significantly with endothelial secretion markers,PI3K and Akt.Conclusions1.There were obvious endothelium dysfunction with PI3K,Akt mRNA and protein expression decreased after injection of resistin.2.High fat diet induced high resistin rats and normal resistin rats at the same time.The serum resistin and resistin mRNA expression in adipose tissue were higher markedly in high-resistinemia rats than those in normal resistin rats.There were obvious obesity,insulin resistance and abnormal lipid in both high resistin rats and normal resistin rats.3.The endothelial dysfunction was worse in high fat diet induced hyper-resistinemia rats than that in high fat diet induced normal resistin rats.The expression of PI3K and Akt in hyper-resistinemia rats were decreased significantly compared with the normal resistin rats.4.Resistin increased with the occurrence of endothelial dysfunction in obesity.The results indicate that resistin was an early marker for endothelium dysfunction.5.After correction for weight,Lee index,insulin,ISI and lipids,the level of resistin and resistin mRNA expression were correlated with endothelial secretion markers and the expression of PI3K and Akt.The result indicated that PI3K/Akt signaling pathway might contribute to the development of endothelial secretion dysfunction by resistin.6.Rosiglitazone,swimming,diet-control and resistin antibody can improve endothelial dysfunction separately with increased PI3K,Akt expression.Swimming with diet-control and rosiglitazone with diet-control were most effective treatment methods.Innovations and meanings:1.This study demonstrated in vivo the effect of resistin on endothelium function.The endothelium functions were different in the high fat diet rats with different resistin level. The result certified that resistin was the independent risk factor of endothelium dysfunction after correction for interferences.It was crucial to detect and treat resistin for the prevention of endothelium dysfunction.2.This study demonstrated in vivo the effect of resistin on PI3K/ Akt signaling pathway for the first time.There were different expressions of PI3K and Akt in individuals with different resistin.The result indicated the relationship among resistin,endothelium function and PI3K/Akt signaling pathway. The result indicated for the first time that PI3K/Akt signaling pathway might contribute to the development of endothelial secretion dysfunction by resistin.3.This study first contrasted and demonstrated the effects in different treatment methods on endothelium function.Swimming and diet control were two effective methods for child to decrease the expression of resistin and improve endothelium function.