Expression Of NF-κB In Insulin-resistance And Diabetic Rats And The Influence Of Its Level Treated With Gliquidone

Establishment of insulin resistance model in the rats induced by high-fatty diet [Abstract]Objective:To establish an insulin resistance animal model. Methods:Thirty healthy male Wistar rats at 2-month-old were randomly divided into control group(group A,n=15)and insulin resistance group(group B,n=15)and 30 healthy male 12-month-old Wistar rats were also randomly divided into control group(group C,n=15)and insulin resistance group(group D,n=15).The rats in insulin resistance group were fed with high-fatty diet that contained unsaturated fatty acid for 8 weeks.The rats in control group were fed with normal diet.Body weight,blood glucose,blood lipid,blood insulin and other parameters were detected.The insulin resistance index(HOMA-IR)in each group was calculated and the change of GIR was measured by euglycemic clamp both to test the validity of the model.Results:Fasting glucose was increased in IR group,but without statistical significance compared to the control group.Fasting insulin was much higher in insulin resistance group compared to the control group with the same age(p＜0.01).High fatty diet resulted in a pronounced reduction in insulin-stimulated glucose disposal rate [GIR:(26.02±5.66)vs(18.96±4.76)mg/kg/min in 4-month-old IR group rats and control group rats(Group A and B);(19.16±2.58)vs(14.43±3.18)mg/kg/min in 14-month-old IR group rats and control group(Group C and D),p＜0.01]. HOMA-IR was 3.09±0.8,7.72±0.65,8.34±0.72 and 12.89±1.08 respectively in group A,B,C and D.Conclusion:Insulin resistance model was successfully reproduced by using the high fatty diet in rats.Rats in old ages also have the characteristics of insulin resistance.High fatty diet and old ages are both risk factors for insulin resistance.Expression of NF-κB and Downstream Inflammatory Factors in the kidney and myocardium of insulin resistance rat[Abstract]Objective:1.To investigate the variation and significance of NF-κB, cyclooxygenase-2(COX-2)and inducible nitric oxide synthase(iNOS) expression in the renal lesions of insulin-resistant rat.2.To investigate the variation and significance of NF-κB,monocyte chemoattractant protein-1 (MCP-1),intercellular adhesion molecule-1(ICAM-1)expression in the myocardial lesions of insulin-resistant rat.Methods:Thirty healthy male Wistar rats were bred since 2-month old,they were randomly divided into normal control group and insulin-resistant group(15 rats in each group).Insulin resistace rat model was constructed by feeding with high fat and sucrose diet.The fasting blood glucose,serum insulin,and serum lipid of the rats in each group were tested two months later.Hyperinsulinemic-Euglycemic Clamp test was used to verify the efficiency of the model.The kidney and heart of the rats were obtained after the successful construction of the model.The structure variations of the kidney and myocardium were observed(HE),and the protein expressions of NF-κB,iNOS and COX-2 in the kidney were observed by immunohistochemistry staining.The protein expressions of NF-κB,MCP-1 and ICAM-1 in the cardiomyocyte were observed.The mRNA expression of NF-κB, iNOS and COX-2 in the kidney,as well as the mRNA expression of NF-κB, ICAM-1 and MCP-1 in the cardiac tissues were detected by RT-PCR.DNA binding activity of NF-κB in the rat kidney from every group was tested by EMSA.Results:1.Euglycemic clamp test was applied to the insulin resistace model,and compared with the control group,the GIR level in the IR group decreased significantly,which indicated that the model was constructed successfully.2.There were no pathological changes in the glomeruli and renal tubular interstitial of the control group;the early lesions of the renal tissue such as glomerular enlargement and mesangial region broadening can be seen in the insulin-resistant model group.The myocardial pathological changes of the insulin-resistant group were not obvious(HE) compared with that of control group.3.Immunohistochemistry stain:only a few glomerular cells expressed NF-κB can be seen in the control group,while the NF-κB was expressed at high level on the glomerular cells in the insulin resistance group.The positive expressions of COX-2 and iNOS on the glomerular cells of the insulin resistance group also increased significantly,and the difference of the mean absorbance between this group and the control group was significant(p＜0.05).Compared with the control group,the expressions of the NF-κB,ICAM-1 and MCP-1 in the cardiac tissue of the insulin resistance group increased significantly,and the difference of the mean absorbance was significant(p＜0.05).4.RT-PCR results: the mRNA level of NF-κB was increased significantly in the insulin resistance group,and correspondingly,the expression level of the downstream inflammatory factors were also increased significantly in the insulin resistance group.5.EMSA results:Compared with the control group,the binding activity of NF-κB in the renal tissue of the insulin resistance group increased significantly.Conclusions:there were NF-κB activation in the kidney and cardiac tissue in the insulin resistance rat,which initiated the downstream target gene(such as MCP-1,ICAM-1,COX-2 and iNOS)expression increase,and then caused the kidney and cardiac tissue lesion.The activation of NF-κB may be one of the initiation factors that causing the kidney lesion of the insulin resistance rat,and may play an important role in the origin and development of the diabetic nephropathy and myocardial lesions.Expression of NF-κB in the Renal Tissue of Diabetic Rat Treated with Gliquidone[Abstract]Objective:To investigate the effect of gliquidone interference treatment on the glomerular pathological change in the diabetic nephropathy,as well as on the expressions of NF-κB,COX-2,iNOS.Methods:Twenty four 8-month old male Wistar rats were randomly divided into control group,diabetic group,gliquidone interference group,8 rats in each group respectively.The rats in the control group were fed with common diet.Diabetes was induced by feeding with high fat and sucrose diet+intraperitoneal injection of streptozotocin. The feed of the diabetic rat was added with gliquidone in the gliquidone interference group.The rats were executed after 2-months feeding,and the specimens of kidney of the rats in each group were obtained so as to observe the pathological changes.The expressions of NF-κB P65,iNOS and COX-2 in the kidney were analyzed by immunohistochemistry and Western blot.The mRNA expressions of NF-κB,COX-2 and iNOS in the kidney were detected by RT-PCR.The NF-κB binding activity in the kidney was detected by EMSA. Results:1.Random blood sugar was tested twice to the rats in the diabetic group a week after intraperitoneal injection of streptozotocin,and both of them were≥16.7mmol/l,which indicated that the diabetic model was constructed successfully.2.According to the pathological section,and compared with the control group,there were varying degrees of kidney lesions in diabetic group and gliquidone interference group.3.The results of immunohistochemistry analysis indicated that compared with the normal control group,the expressions of NF-κB,COX-2 and iNOS in the kidney increased significantly in the diabetic group and gliquidone interference group.Compared with diabetic group,the expression was milder in gliquidone treatment group.4.The results of RT-PCR indicated that compared with the control group,the mRNA expression level of NF-κB and downsteam inflammatory factors increased in the renal tissues in the diabetic group and gliquidone interference group.But compared with diabetic group,the mRNA expression was milder in gliquidone treatment group.5. EMSA results:Compared with the control group,the binding activity of NF-κB in the renal tissue of the diabetic group and gliquidone treatment group increased significantly.But when gliquidone treatment group was compared with diabetic group,the binding activity of NF-κB decreased significantly. 6.Western blot results:Compared with the control group,the expression of NF-κB in the renal tissue of the diabetic group and gliquidone treatment group increased significantly.The protein level in gliquidone treatment group decreased significantly compared with diabetic group.Conclusions:NF-κB and downstream target gene were activated in the diabetic rats.Treatment with gliquidone can attenuate the expression of NF-κB and downstream target gene.