| Objective: Diabetic nephropathy(DN) is one of the most common and most serious chronic microvascular complications of diabetes mellitus(DM), which has become one of the leading causes of end stage renal disease(ESR D). The pathogenesis of DN is complicated, many mechanisms contribute to the occurrence and development of it, including glycometabolism disorder, hemodynamic factors, endothelial cell dysfunction, oxidative stress, inflamm-atory, autophagy. The unifying mechanism of diabetic complications fully embodies the important function of hyperglycemia-induced oxidative stress. Recently, one approach that is attracting attention, the transcription factor NF-E2–related factor 2(Nrf2), together with its negative regulator, Kelch-like ECH-associated protein 1(Keap1), is considered one of the most important cellular defense mechanisms to combat oxidative stress with a particular role in the regulation of phase II detoxifying enzymes.In this study, we established streptozotocin-induced diabetic rat model, measured the level of malondiadehyde(MDA) and activity of total antioxidant capacity(T-AOC) in kidney tissue to estimate the oxidative status. The expression of Nrf2, heme oxygenase-1( HO-1) and vascular endothelial growth factor(VEGF) were detected with immunohistochemistry and realtime fluores-cence quantitative PCR(RTFQ-PCR). We observed the effects of sulforaphane on the expression of MDA, T-AOC, Nrf2, HO-1, VEGF to explore the relationship between Nrf2/ARE pathway and oxidative stress of DN, in order to provide experimental basis for prevention and treatment of DN.Methods: 36 male clean Sprague Dawley rats, weight 200±20g, were randomly divided into normal control group(NC group), diabetic model group(DM group) and drug intervention group(DM+SF group) with 12 rats in each group. Gave the rats of DM group and DM+SF group a single intraperitoneal injection of STZ 65 mg/kg(dissolved in 0.1 mol/L sodium citrate buffer, PH 4.2) and collected the tail tip blood to determine the blood glucose after 72 hours. The diabetic model was considered to be successful if the blood glucose were all≥16.7 mmol/L, urine glucose≥3+. The rats of NC group were given an equal amount of sodium citrate buffer. Feed the rats food and water normally without hypoglycemic drugs during the experiment. The DM+SF group was gavaged by SF 500 μg/kg(dissolved in vegetable oil) and the other two groups were given the same dose of vegetable oil once a day. At the end of 8 and 12 week,6 rats were randomly selected from each group to detect 24 hour urinary protein, blood glucose, serum creatinine and blood urea nitrogen. Renal cortical homogenate were made to determine MDA and T-AOC. We made histopathologic sections and stained the kidney tissue with HE and PAS to define the changes of renal pathology. Immunohistochemistry and realtime fluores-cence quantitative PCR methods were used to detect the expression of Nrf2, HO-1 and VEGF. All the experimental data were expressed as mean ± standard deviation by SPSS13.0 P-value less than 0.05 was considered statistically significant.Results:1 General conditionsFood-intake and urine of NC group had no significant change, the fur was lustrous, the reaction was agile and the weight increased obviously, the DM group and DM+SF group appeared polydipsia, polyphagia and urorrhagia gradually. The fur became dry and the weight decreased obviously compared with the NC group. The weight of DM and DM+SF group had no statistical significance at two time points(P>0.05).2 Biochemical indexesCompared with NC group,the serum creatinine, blood urea nitrogen, blood glucose, 24 hour urinary protein of DM and DM+SF group increased significantly(P<0.05). The blood glucose between DM group and DM+SF group had no difference(P>0.05), at two time points. Compared with DM group the serum creatinine, blood urea nitrogen, 24 hour urinary protein of DM+SF group were decreased(P<0.05), at two time points.3 Levels of MDA and T-AOCConcentration of MDA in renal cortical homogenate of DM and DM+SF group was higher than NC group(P<0.05), while the T-AOC activity was lower than NC group(P<0.05). Compared with DM group the MDA level of DM+SF group was lower, the T-AOC activity was higher(P<0.05).4 Renal histopathological changeGlomerular structure of NC group was normal, renal tubular epithelial cells were aligned and structure clear, the staining of basement membrane was homogeneous, the increase of mesangial cells and mesangial matrix had not found. In DM group, part of glomerulars became larger, the mesangial cells and mesangial matrix increased, mesangial region broadened, renal tubule distended in some area, part of renal tubular epithelial cells appeared vacuolar degeneration. The change was more notable at the end of 12 th week, and the DM+SF group were lighter than the DM group.5 The immunohistochemical resultsNrf2, HO-1 and VEGF expressed lightly in renal tissue of NC group. At two time points, the expression of Nrf2、HO-1 and VEGF in DM group was higher than NC group(P<0.05). Compared with DM group the expression of Nrf2 and HO-1were higher(P<0.05), while the expression of VEGF was lower in DM+SF group.6 RTFQ-PCRThe expression of Nrf2, HO-1 and VEGF in DM group was more than NC group(P<0.05). Compared with DM group, the expression of Nrf2 and HO-1 was increased(P<0.05), after SF intervention, the expression of VEGF was decreased in DM+SF group(P<0.05).Conclusion:1 There was oxidative stress in the kidney tissue of DM rats, the expression of antioxidant proteins regulated by Nrf2/ARE pathway were increased in oxidative stress status.2 SF could decrease the content of MDA, increase the activity of T-AOC in renal tissue, and could induce the expression of Nrf2 and HO-1 to improve the oxidative stress status of diabetic rats. It could also suppress the expression of VEGF to improve the endothelial dysfunction of diabetic rats.3 SF had protective effects of diabetic rats, the mechanisms might be through alleviating the renal damage by oxidative stress and the endothelial dysfunction. |
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