| Objective:Diabetic nephropathy(DN), one of the most common and serious chronic complications of diabetes. The specific pathogenesis mechanism of diabetic nephropathy is not entirely elucidated, but many studies have shown that the formation of persistent high glucose in diabetes induced excessive reactive oxygen species, the body biological macromolecules can be attacked directly, producing peroxide degeneration, crosslinking, or rupture, causing damage to cell structure and function, thus to promote the occurrence and development of diabetic nephropathy. When the body cope with ROS damage, antioxidant response elements(ARE) of some protective gene upstream regulatory region will promot a series of protective protein expression, to alleviate cellular damage. Studies have demonstrated that ARE can be actived by Nrf2 a critical transcription factor regulating antioxidant response in cells. Nrf2/ARE signaling pathway is primary to maintain the balance of oxidizing environment- reduced state in vivo. As a result, the activation of Nrf2 and downstream antioxidant protein expression may provide new targets for prevention and control of diabetic nephropathy. In this study, we established the diabetic rats model and given hydrogen rich saline to explore whether it can improve the state of oxidative stress in diabetic rats through activation of Nrf2/ ARE. We detected gmalondialdehyde(MDA) content, glutathione hormone(GSH)level and superoxide dismutase(SOD)activity in kidney tissues to indicate oxidative stress level of diabetic rats and used immunohistochemical and western blot to measure Nrf2?HO-1 expression in kidney tissues. In general, this experiment provides a new theoretical basis for the prevention and treatment of diabetic nephropathy.Methods : 36 healthy male SD rats of SPF level, with weight of 180~220g were selected. After rats were feed adaptively 1 week, all rats fast 12 hours, were randomly divided into two groups, 12 normal group(NC group), the others were made into diabetes mellitus model, given intraperitoneal injection STZ(65mg/kg), and NC group the equally amount of citric acid buffer. The model of diabetes was considered to be successful when the blood glucose was?16.7mmol/L after 72 hours of the injection. Then the 24 diabetic rats were randomly divided into two groups, DM group and HRS group. HRS group rats were lavaged with 12ml/kg HRS, and other groups were given the same dose of saline every day. During the experiment the rats drunk and ate freely, and were not given insulin and other hypoglycemic agents. Six rats of each group were randomly chosen after the onset of diabetes at the eighth and twelfth weeks. Heart blood and 24 h urines were collected to measure serum creatinine, blood urea nitrogen and urine protein. The kidney was sliced into pieces, and we observed kidney pathological changes under the light microscopy after hematoxylin and eosin(HE) and periodic acid shciff(PAS) stain. We observed the expression of Nrf2, HO-1with immunehistochemistry and western blot and measured the content of indicators of oxidative stresss like MDA, GSH and SOD. All the experimental data were expressed with mean ± standard deviatio( ?x ±S), one-way analysis of variance was used in date analysis using SPSS20.0. If the data did not meet the normality or homogeneity, we used non-parametric tests. It was considered statistically significant when P-value was less than 0.05.Results: 1 General situation Rats spirit in NC group was good, body weight grew stably, response was rapid, hair was glossy, urine was no obvious change. DM group and HRS group showed diabetes symptoms, such as polydipsia, polyphagia, polyuria, and other symptoms after moding. Along with the progression of diabetic, rats emerged listlessness, slow response, hair without burnish and weight loss. This phenomenon was most obvious in DM group.2 Biochemical indicators At 8 and 12 weekends, compared with NC group, 24 h urine protein, blood glucose,serum creatinine and blood urea nitrogen in DM and HRS group were significantly higher(P<0.05); HRS group was significantly lower than DM group(P<0.05). 3 Oxidative stress indicators Compared with NC group, the MDA content of DM group increased in the kidney of rats, with intervention of hydrogen rich saline, the MDA level was reduced(P<0.05). Compared with NC, the SOD activity and GSH level of DM group decreased significantly, after given hydrogen rich saline, the SOD activity and GSH level was increased(P<0.05). 4 Renal morphologic changes Light microscope displayed that the renal tissue of rats in NC group was structure clear and no obvious pathological changes;Throughout DM group, glomerular volume was increased, glomerular mesangial matrix increased, mesangial cells proliferated, mesangial area broadened, basement membrane was thickened, some renal tubular expanded and some renal tubular epithelial cells were in vacuoles degeneration. Kidney pathological structure was most apparent and improved after rats given hydrogen rich saline. 5 The expression of Nrf2 and HO-1 The results of immunohistochemical staining showed that Nrf2, HO-1 mainly expressed in intrinsic tubular epithelial cells, the expression intensity: HRSgroup > DMgroup > NCgroup(P < 0.05); The variation tendency of western blot result corresponded with immunohistochemical result.Conclusions: 1 GSH level and SOD activity of rat renal tissue decreased, MDA content and Nrf2,HO-1 expression are distinctly increased when diabetes, which show that oxidative stress are obvious in diabetic rats kidney tissue and the Nrf2/ARE pathway was activated. 2 Hydrogen rich saline can reduce the levels of blood glucose, serum creatinine, blood urea nitrogen and urinary protein in diabetic rats, bring down MDA content, improve GSH level and SOD activity of renal tissue, and increase Nrf2, HO-1 expression. It is supposed that the protective effect hydrogen rich saline on the renal may be related to activation of Nrf2/ARE and reduced oxidative stress injury. |
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