| Objective: Diabetic nephropathy was one main complication of the diabetes and the important cause of end-stage renal failure. Its clinical characteristics were increased albuminuria and renal dysfunction. Pathological features were thickening of basement membranes, expansion of mesangial matrix and interstitial fibrosis. Oxidative stress, inflammatory response and apoptosis of renal inherent cells played important role in the occurrence and development of renal injury. Hyperglycemia environment could induce the body to produce excessive reactive oxygen species, activate diabetic nephropathy related signaling pathways, and cause imbalance of cytokines and growth factors. Nrf2 was a critical transcription factor regulating antioxidant response. Nrf2 played a role of antioxidant stress by activating gene sequences of antioxidant response elements and promoting the expression of downstream antioxidants and Ⅱ phase detoxification enzymes. Currently, researches about Nrf2/ARE pathway were mainly in anti-aging, anti-apoptosis, anti-tumor and so on. There were few studies about the role of oxidative stress in diabetic nephropathy. Astaxanthin, as one kind of carotenoids, had perfect antioxidant activity. And this made it possible to prevent and treat various diseases, such as such as cancers, diabetes, diabetic nephropathy and cardiovascular diseases. Research on whether astaxanthin could reduce the oxidative stress of diabetic nephropathy through activating Nrf2/ARE signaling pathway was relatively rare at home and abroad. In this experiment by modeling the experimental diabetic rats induced by streptozotocin and given astaxanthin, we observe the changes of Nrf2, NQO1 and MDA in rats kidney tissue through molecular biology detection method. We explore whether astaxanthin can improve the state of oxidative stress of rat sand delay the development of kidney damage, so as to provide theoretical foundation to prevent and treat diabetic nephropathy.Methods: 36 healthy male SD rats with weight of 180~220g were selected. We choose randomly 24 rats made into diabetes mellitus model, the others were as normal control group(NC group, n=12). After 1 week adaptive feeding, before intraperitoneal injection of streptozotocin(STZ),all rats required fasting 12 hours, then the 24 SD rats were given STZ 65mg/kg. The model of diabetes was considered to be successful when the blood glucose was≥16.7mmol/L after 72 hours of the injection. The NC group were given the equally amount of citric acid- sodium citrate buffer intraperitoneal injection. Then the 24 diebetic rats were randomly divided into two groups, DM group and ATX group. ATX group rats were lavaged with 25 mg/kg ATX once a day, and other groups were given the same dose of vegetable oil every day. During the experiment the rats drunk and ate freely, and were not given any agent, for 12 weeks. Six rats of each group were randomly chosen after intervention at the eighth and twelfth weeks. 24 h urines were collected in metabolic cage for the measurement of urine protein. After weighting, the rats were given intraperitoneal injection of 10% chloral hydrate anesthesia, we got the heart blood and separated serum to detect serum creatinine, blood urea nitrogen and blood glucose. After remove the envelope, we weighed the kidneys. 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 measured the content of lipid peroxidation marker malondialdehyde(MDA) and the activity of T-AOC and observed the expression of Nrf2, NQO1 with RT-PCR and immune- histochemistry. All the data were expressed with mean ± standard deviation( x ±S), one-way analysis of variance was used in date analysis using SPSS19.0.If the data did not meet the normality and homogeneity, we used non-parametric tests. It was considered statistically significant when P-value was less than 0.05.Results:Rats hair in NC group was glossy and body weight grew stably.DM group and ATX group emerged polydipsia, polyphagia, polyuria,listlessness after molding. And along with the progression of diabetic,diarrhea, weight loss, hair without burnish, lags in response and othersymptoms appeared gradually. But ATX group was better than DM group.Compared with NC group, 24 h urine protein quantification,creatinine, bloodurea nitrogen and blood glucose in DM and ATX group were significantlyhigher(P<0.05). The index except for blood glucose in ATX group wereower than DM group(P<0.05). Blood glucose in ATX group was mildlydecreased, but there was no significant(P>0.05). From Light microscope inkidney tissue of each group, HE and PAS staining showed that with thecontinuous presence of hyperglycemia, NC group rat kidney had nosignificant lesion. We observed glomerular hypertrophy, basementmembrane thickening and mesangial matrix hyperplasia in DM group.Comparing with DM group, ATX group had milder renal lesion. MDAcontent in DM group increased significantly than NC group in 8th and 12thweeks, and ATX group declined when comparing with DM group(P<0.05).T-AOC level in DM group and ATX group were lower than NC group, butATX group was obviously higher than DM group. At 8th and 12 th weeks, theimmunohistochemical results showed that the expression of Nrf2 and NQO1in DM group were significantly higher than those in NC group of the sametime, but lower than that in ATX group(P<0.05). The variation tendency ofPCR result corresponded with immunohistochemical result.Conclusion: The oxidative stress response enhances and antioxidant products increase in rats renal when diabetes. Astaxanthin can activate Nrf2, and up regulate the expression of NQO1, in order to reduce hyperglycemia induced oxidative stress reaction and play a role of kidney protection in STZ induced diabetic nephropathy. |
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