Protective Effects of Et-1mrna Antisense Oligodeoxynucleotide on Diabetic Nephropathy in Diabetic Rats
|School||Ningxia Medical University|
|Keywords||Endothelin -1mRNA antisense oligonucleotide Diabetic nephropathy Endothelin Transforming growth factor - beta1|
Objective: antisense oligonucleotides (antisense oligodeoxynucleotide, AS-ODN) technology rat model of diabetes mellitus (DM) endothelin -1 (endothelin, ET-1) gene expression inhibition test, endothelin Antisense The protective effect and mechanism of nucleotide (ET-1 As-ODN) in early diabetic nephropathy (diabetic-nephropathy, DN) kidney. Subjects and methods: 1.; Selected 89 healthy SD rats were randomly divided into three groups: normal control group (NC) (saline 0.5ml/rat/wk of intravenous injection), diabetic group (DM) (saline 0.5ml/rat/wk, intravenous injection), endothelin antisense oligonucleotide treatment group (ET-1AS-ODN 6 OD / kg / wk, intravenous injection), model group and treatment group 65 rats with streptozotocin (STZ) diabetic model preparation method of 60mg/kg intraperitoneally. Rats after modeling success, then the above three groups each group is divided into 2, 4, and 8 weeks, three subgroups, dynamic observation and measurement of the general state of the rats, body weight, urine volume, fasting blood glucose (FBG); 2. chemical colorimetric analysis at 2, 4 and 8 weeks dynamically detect rat blood urea nitrogen, creatinine renal function; 3 were measured by radioimmunoassay in urine albumin (UAER), beta 2-micro-globulin (beta2 -MG) and endothelin ET-1 content to observe the different points in time ET-1 AS-ODN treatment before and after the changes in renal function; 4. HE staining under light and electron microscopic examination of kidney tissue morphology changes; 5. transforming growth factor (TGF-beta1) mRNA gene expression in the kidney tissue using real-time PCR technology to make quantitative analysis of the observed ET-1AS-ODN whether through regulation of endothelin-1 levels to reduce the transforming growth factor (TGF- content beta1) gene expression. Results: After the modeling of experimental groups, blood glucose, body weight and changes in urine glucose levels in diabetic rats basically stable in the 19 to 40 mmol / L, urine output increased significantly (P lt; 0.01), but the urine 8 weeks significantly reduced (P lt; 0.05) compared to four weeks ago; weight of the normal group on the 2, 4 and 8 weeks of observation points in ascending order, 8 weeks, compared with two weeks an increase of 47.7% (P lt; 0.01); model group decreased gradually, at different observation points and 8 weeks compared with modeling two weeks significantly decreased by 31.2% (P lt; 0.01). After ET-1 AS-ODN treatment, the treatment group weight gradually increased after 4 weeks, 8 weeks, body weight compared to two weeks, an increase of 17.2% (P lt; 0.01), increased urine output compared with the model group (P lt; 0.01) , blood glucose no significant change compared with the model group (P gt; 0.05) (2) The renal function (1) BUN, Scr, the CCR changes in model group were made two weeks after the model was successful not yet apparent renal abnormalities, increased BUN, Scr, CCR, respectively, than the normal group in the 4 weeks 43.8% (P lt; 0.01), 11.4% (P lt; 0.01), 93% (P lt; 0.01), 8 weeks, increased BUN, SCr, CCR, respectively, than the normal group 54% (P lt; 0.01), 22.4 % (P lt; 0.01) and 84.1% (P lt; 0.01). 8 weeks after ET-1 AS-ODN treatment, BUN, Scr relative model group were reduced by a 17.7% (P lt; 0.01), 13.7% (P lt; 0.01) CCR relative to the model group increased 42.4% (P lt; 0.01) (P lt; 0.01). ② The urinary albumin and beta2-MG excretion rate comparison 24-hour urinary albumin excretion rate (UAER), model group the early small amount, at 2, 4 and 8 weeks UAER than the normal group in turn increased (P lt; 0.01); urinary beta2-MG content changes, the model group than the normal group at 2, 4 and 8 weeks persistent increased (P lt; 0.01) (see Table 1-3). After ET-1 AS-ODN treatment, 2 weeks, 4 weeks of treatment of urinary UAER level close to the level of the normal group, 8 weeks, compared with model group decreased by 76.8% (P lt; 0.01); urinary beta2-MG content 8 weeks beta2-MG content compared with the model group decreased 65% (P lt; 0.01). The above indicators suggest a protective effect of ET-1 AS-ODN drugs DN renal function. 3 rat glomerular pathological changes in electron microscopy results show that the model rats after 2 weeks, glomerular pathological changes is not obvious, after 4 weeks of the glomerular basement membrane mild uneven thickening, mesangial cells and mild endothelial cell proliferation, partial fusion of foot processes, glomerular pathological changes after 8 weeks significantly increased basement membrane dysplasia, partial rupture, foot process fusion or even disappear. ET-1 AS-ODN after 8 weeks of treatment, the treatment group showed thickening of the glomerular GBM by model group 535.16 with disabilities 301.23nm down to the treatment group 244.72 ± 58.18nm, the relative model group decreased by 54.3% (P lt; 0.01) mesangial area compared with the model group was significantly narrowed (see Figure 2). Prompt ET-1AS-ODN treatment of renal pathological changes were markedly reduced. ET-1 and TGF-beta1 expression changes in the content of radioimmunoassay and real-time PCR technology: 8 weeks, ET-1 levels in the renal cortex of the model group than in normal group increased by 58.1%, TGF-beta1 gene The expression level than the normal group increased 3.20-3.66 times. After ET-1 AS-ODN treatment, renal tissue endothelin levels compared with the model group was significantly reduced by 60.1% (P lt; 0.05), target gene TGF-beta1 expression levels compared with the model group decreased by 2.11-2.55 times. TIP ET-1AS-ODN indirect reduction in renal tissue expression of TGF-beta1 gene content. Conclusion: ET-1AS-ODN can effectively improve kidney function in diabetic rats, reduce urinary protein excretion, slowing the progress of glomerulosclerosis and glomerular fibrosis, kidney protective effect. 2. ET-1 AS-ODN kidney of diabetic rats the molecular mechanisms of the protective effect may be indirect regulation of TGF-beta1 gene expression by inhibiting the expression of ET-1 content, and to play a role, which is following the ET-1 by another target of action of the drug in the body after blocker.