硫辛酸对糖尿病肾脏疾病大鼠肾脏组织氧化应激和SnoN蛋白表达影响的研究

Effect of Alpha lipoic acid on oxidative stress and expression of ski-related novel protein in diabetic kidney disease of rats

目的探讨硫辛酸(ALA)对DKD大鼠肾脏的氧化应激和SnoN蛋白表达的影响。方法 24只SD大鼠随机分为正常对照组(NC)、糖尿病组(T1DM)和ALA干预组(ALA),每组各8只,T1DM组和ALA组建立T1DM模型,模型建立成功2周后,ALA组给予ALA灌胃,NC组与T1DM组予同剂量生理盐水灌胃,于实验8周后处死大鼠,检测相应生化指标和氧化应激水平;Masson染色观察肾脏组织形态结构和阳性染色变化;RT-qPCR检测SnoN mRNA的表达;Western blot检测大鼠肾脏组织中SnoN、Smad泛素化调节因子2(Smurf2)蛋白表达水平;免疫共沉淀法检测SnoN泛素化降解程度。结果与NC组比较,T1DM组FPG[(5. 58±1. 02)vs(26. 78±1. 63)mmol/L]、Scr[(13. 75±1. 66)vs(34. 00±2. 78)μmol/L]、24 h尿蛋白(24 h UAlb)[(2. 99±0. 45)vs(20. 74±1. 07)mg/24 h]、丙二醛(MDA)含量[(2. 16±0. 19)vs(5. 82±0. 89)nmol/mg]、SnoN mRNA水平[(1. 00±0. 28)vs(3. 33±0. 29)]、Smurf2蛋白水平[(0. 42±0. 08)vs(1. 79±0. 27)]及SnoN泛素化降解升高(P<0. 05),总超氧化物歧化酶(T-SOD)活性[(88. 53±9. 76)vs(33. 41±7. 11)U/mg]、过氧化氢酶(CAT)活性[(34. 60±4. 51)vs(14. 36±2. 29)U/mg]、SnoN蛋白表达水平[(2. 64±0. 53)vs(0. 35±0. 11)]降低(P<0. 05),且T1DM组伴肾脏病变发生。与T1DM组比较,ALA组FPG[(26. 78±1. 63)vs(26. 63±2. 45)mmol/L,P>0. 05]差异无统计学意义,Scr[(34. 00±2. 78)vs(22. 57±1. 57)μmol/L]、24 h UAlb[(20. 74±1. 07)vs(15. 95±0. 56)mg/24 h]、MDA[(5. 82±0. 89)vs(3. 39±0. 38)nmol/mg]、Smurf2蛋白水平[(1. 79±0. 27)vs(1. 09±0. 17)]及SnoN泛素化降解降低(P<0. 05),T-SOD活性[(33. 41±7. 11)vs(50. 99±7. 52)U/mg]、CAT活性[(14. 36±2. 29)vs(27. 30±3. 17)U/mg]、SnoN蛋白水平[(0. 35±0. 11)vs(1. 04±0. 13)]升高(P<0. 05),SnoN mRNA水平[(3. 33±0. 29)vs(3. 13±0. 20)]差异无统计学意义(P>0. 05),且ALA组伴肾脏病变减轻。结论 ALA通过增强T1DM大鼠肾脏的抗氧化能力,减少Smurf2所介导SnoN的泛素化,恢复SnoN蛋白的表达,对DKD大鼠肾脏起保护作用。

Objective To verify effect of Alpha lipoic acid (ALA) on oxidative stress and expression of ski-related novel (SnoN) protein in diabetic nephropathy of rats. Methods 24 SD rats were randomly divided into normal control group (NC), diabetic group (T1DM) and ALA intervention group , with 8 rats in each group. Two weeks after successful T1DM modeling, the intervention group was given ALA gavage treatment. NC and T1DM group were given same dose of normal saline. Eight weeks after the experiment, the rats were sacrificed to detect the relevant biochemical indicators and oxidative stress levels. Masson staining was used to observe the pathological changes of the kidney. RT-qPCR and Western blotting was used to detect the expression of SnoN and Smurf2. Co - immunoprecipitation was used to detect the ubiquitin level of SnoN. Results Compared with NC group, the levels of fasting blood glucose (FPG)[(5. 58 ± 1. 02) vs (26. 78± 1. 63)mmol/L,P<0.05], Serum creatinine(Scr)[(13. 75±1. 66) m (34. 00 ±2. 78)pmol/L,P< 0.05], urine protein of 24 hours(24 h UAlb)[(2. 99±0.45) es (20. 74± 1. 07)mg/24 h,P<0. 05] and content of malondialdehyde(MDA)[(2. 16±0.19) vs (5. 82±0. 89)nmol/mg,P<0. 05] were increased in T1DM group, and the levels of total superoxided dismutase(T^SOD)[(88. 53 ± 9. 76) vs (33. 41 ± 7. ll)U/mg, P<0.05] and catalase (CAT)[(34. 60 ± 4. 51) vs (14. 36 ± 2. 29) U/mg, P<0.05] activities were decreased ,with the presentation of renal lesions. The expressions of SnoN [(2. 64±0. 53) vs (0. 35±0.11),P<0. 05] was decreased, SnoN mRNA[(l. 00±0. 28) m (3. 33±0. 29), P<0. 05], Smurf2[(0. 42±0. 08) vs (1. 79 ±0. 27), P<0. 05] and the ubiquitin level of SnoN were increased in T1DM group. Compared with T1DM group, FPG [(26. 63 ± 2.45) mmol/L, P>0. 05] had no significant difference, the levels of Scr [(22. 57± 1. 57)μmol/L, P<0. 05], 24 h UAlb [15. 95±0. 56)mg/24 h,P<0. 05] and content of MDA [(3. 39±0. 38)nmol/mgt,PV0.05] were decreased,T-SOD [(50. 99±7. 52)U/mg,P<0. 05] and CAT [(27. 30 ± 3.17)U/mg,P<0. 05] activities were increased in ALA group, with obvious alleviation in renal lesions. The expression of SnoN [(1. 04 ± 0.13), PV0.05] was increased, the level of SnoN mRNA [(3. 13 ±0. 20), P>0. 05] was not changed, Smurf2[(1. 09 ±0.17), P<0. 05] and the ubiquitin level of SnoN were decreased in ALA group. Conclusion ALA may promote the antioxidant ability of diabetic rats and increase the levels of SnoN protein through reducing the ubiquitin level of SnoN mediated by Smurf2, consequently play a role of protecting the kidney of diabetic nephropathy rats.