| Objective: In this study, we established a type1diabetic rats modeland explored the effect of metallothinonein on oxidative stress state of diabeticrats and the development of kidney damage. We measured the level ofmalondiadehyde(MDA) and activity of total antioxidant capacity (T-AOC),which were oxidative stress indicators in the kidney. The expression of Nrf2and Trx in the kidney were detected with immunohistochemistry and reversetranscription PCR. In summary, this study provides experimental evidence forthe prevention and treatment of renal injury of diabetes.Diabetes could make the body in the oxidative stress state, increase thelevel of lipid peroxidation product and decrease the anti-oxidation ability ofcells.. Nrf2-ARE pathway is one of the most important antioxidant stresspathways. Nuclear factor EF-E2related factor (Nrf2) will bind withantioxidative response element ARE after stimulation of oxygen species(ROS), resulting in the transcription of protective protein genes, includingthioredoxin (Trx), quinone oxidoreductase (NQO1) and heme oxygenase1(Heine oxygen ase1, HO-1), to improve the body’s antioxidant ability andreduce oxidative stress injury.Methods:48male SD rats of clean level, with weight of180~220gwere selected. After1week adaptive feeding, the rats were randomly dividedinto normal control group (NC group, n=12), diabetic model group (DMgroup, n=12), diabetes (MT) high dose treatment group (MTH group, n=12),diabetes (MT) lower dose treatment groups (MTL group, n=12). The DMgroup, the MTH, MTL group rats should be fast intraperitoneal injected ofstreptozotocin (65mg/kg) to be diabetic rats model,and NC group the amountof citric acid buffer. After72h of intraperitoneal injection，sample caudalvein blood,measure the blood glucose with roche glucose meter. The diabetic model was considered to be successful when the blood glucose was≥16.7mmol/L. MTH group rats are lavage with0.5mg/kg MT,MTLgroup rats0.1mg/g k MT, and NC group, DM group the same amount ofsaline every day. At6and12weeks after the onset of diabetes,Five rats weresacrificed for each group respectively and measure their24h urinealbumin,serum creatinine(Scr) and blood urea nitrogen(BUN),the renalcortical tissues were removed and used for detecting rat kidney pathologicalchanges under the light microscopy,the expression of Nrf2,Trx withimmohistochemistry and rt-PCR and measuring the content of MDA and theactivity of T-AOC. All the experimental data were expressed as mean±standard deviation (x±S), one-way analysis of variance was used in dateanalysis using SPSS13.0, I t was considered statistically significant whenP-value was less than0.05.Results：1general situation DM, MT group showed polydipsia, polyphagia,polyuria and gradually emaciation after4~5days of injection of STZ. Thisphenomenon was most obvious in DM group and wasn’t observed in NCgroup.2Biochemical indicators2.124hours urinary protein By the end of6week, the24hoursurinary protein of DM group of rats was increased compared with NC,(p <0.05);24hours urinary protein of MTH group decreased (p <0.05), andMTL group has a downward trend compared with DM group.By the end of12week,24hours urinary protein of DM group rats were increased comparedwith NC,(p <0.05);each MT group showed decreased the levels of24hoursurinary protein Compared with DM group,(p <0.05).2.2the comparison of serum creatinine, blood urea nitrogen By the endof6week,,the serum creatinine, blood urea of DM group rats wassignificantly increased compared with NC group(p <0.01). Creatinine leveland blood urea of each MT group(p <0.05), compared with DM group. By theend of12week, compared with NC group, serum creatinine, blood urea levels of DM group rats increased (p <0.01); Compared with DM group, each MTgroup could significantly reduce the rat serum creatinine level and urea levels(p <0.01).3Oxidative stress indicators3.1the comparison of MDA by the end of6week, compared with NCgroup,the MDA level of DM group increased in the kidney of rats (p <0.05);Compared with DM group, the MDA level in each dosage group was varyingdegrees of decline. By the end of12week, compared with NC group, theMDA level of DM group increased significantly (p <0.05); Compared withDM group, each dose group could significantly decrease the MDA level in thekidney of rats (p <0.05).3.2The comparison of T-AOC6weekend, compared with NCgroup, T-AOC activity of DM group decreased significantly (p <0.05);compared with DM group, T-AOC activity Of each dosage group increasedsignificantly (p <0.05).12weekends, compared with NC group, T-AOCactivityof DM group decreased significantly (p <0.05); compared withDM group, T-AOC activities of renal tissues of MTL group increased(p <0.05), and of MTH group increased significantly (p <0.05).4chemical changes in the organization Light microscopy showed thatin every time the rat kidney tissue of NC group showed no obviouspathological changes.6weekend, compared with NC group, DM group andMT group did not showed no obvious difference.12weekends, compared withthe same period of NC group, DM group of glomerular volume increase,glomerular mesangial matrix was increased, mesangial cells increased. Renalinterstitial, renal tubule has no obvious pathological morphological changes.kidney pathological structure of MT groupis was improved at the same time.5the immunohistochemical results At6and12weekends, comparedwith NC group, Nrf2expression in rat kidney tissues of DM groupincreased,it was higher of MTL group than that of DM group,and it washigher of MTH than that of MTL group (p <0.05);6weekend,comparedwith NC group,Trx expression increased in DM group, but lower than that in MTL,MTH group at the same period,(p <0.05);12weekend, comparedwith NC group,Trx expression of DM group decreased, it is higher ofMTL group than that of DM group,and it is higher of MTH than that ofDM group (p <0.05).6the result of Reverse Transcription PCR (rt-PCR) Compared withNC group, Nrf2expression in rat kidney tissues of DM group and MT groupincreased, it increased more significantly by the end of12week (p <0.05);6weekend,Trx expression of DM group increased and lower than that ofMTL, MTH group at the same period(p <0.05);12weekend,compared withNC group,Trx expression of DM group decreased,Trx expression of MTLgroup is higher than that of DM group, it is higher of MTH group than that ofDM group (p <0.05).Conclusion:1MDA level of diabetes rat renal tissue increased, T-AOC activitydecreased, and Nrf2, Trx expression of diabetic kidney are significantlyincreased,because of obvious oxidative stress state and the Nrf2pathwaywas activated.2Metallothionein can reduce the occurrence of diabetic rats proteinuriaand renal pathological morphological changes, and decrease MDA contentof renal tissue, improve the activity of T-AOC. its mechanism may be relatedto increased Nrf2protein and its downstream antioxidants Trx expression, thusreducing oxidative stress, protecting the renal function. |
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