普罗布考联合山莨菪碱对糖尿病大鼠对比剂肾损伤的影响

Effect of probucol combined with anisodamine on contrast-induced nephropathy in diabetic rats

目的探讨普罗布考联合山莨菪碱对糖尿病大鼠对比剂肾病(CIN)的影响。方法选取130只大鼠制备糖尿病大鼠模型,将造模成功大鼠随机分为模型组、普罗布考组、山莨菪碱组、联合组各21只。另选10只正常大鼠为正常对照组。糖尿病造模9周后,普罗布考组及联合组给予普罗布考500 mg/kg灌胃,连续6 d;第8天时,于制备CIN模型前,山莨菪碱组及联合组经腹腔注射山莨菪碱100μg/100 g,15 min后,模型组、普罗布考组、山莨菪碱组、联合组经尾静脉注射复方泛影葡胺注射液0.8 m L/100 g制备CIN模型;1 h后,山莨菪碱组、联合组经腹腔注射山莨菪碱100μg/100 g共7次。注射对比剂前及24 h后,采用CBB法测定尿蛋白。内眦静脉取血,测定血清BUN、Scr水平。称重,开腹后采集左右肾脏并称重,计算肾指数。HE染色法观察肾脏组织病理变化。取肾脏组织制成肾组织匀浆,测定氧化应激指标包括一氧化氮(NO)、一氧化氮合成酶(NOS)、超氧化物歧化酶(SOD)、丙二醛(MDA)及总抗氧化能力(T-AOC)。荧光定量PCR法检测肾组织Bcl-2相关X蛋白质(Bax)和B细胞淋巴瘤/白血病-2基因(Bcl-2)mRNA表达。结果 (1)模型组BUN、Scr、UAE均高于正常对照组(P均<0.05)。普罗布考组及联合组BUN、Scr、UAE均低于模型组,联合组BUN、Scr、UAE均低于山莨菪碱组(P均<0.05)。(2)模型组肾指数高于正常对照组,普罗布考组及联合组肾指数低于模型组(P均<0.05)。(3)与模型组比较,普罗布考组、山莨菪碱组肾脏NOS水平升高,MDA水平降低(P均<0.05)。联合组肾脏SOD、T-AOC均高于普罗布考组和山莨菪碱组(P均<0.05)。(4)与模型组比较,普罗布考组、联合组肾脏Bax表达水平降低,Bcl-2及Bcl-2/Bax升高(P均<0.05);山莨菪碱组肾脏Bcl-2/Bax升高(P<0.05),联合组肾脏Bcl-2/Bax高于山莨菪碱组(P<0.05)。结论普罗布考、山莨菪碱联用可减轻糖尿病CIN大鼠的肾脏损伤,其机制可能与抑制氧化应激反应、上调Bcl-2/Bax比值有关,且效果优于单药应用。

Objective To explore the synergistic effect of probucol combined with anisodamine on contrast-induced nephropathy（ CIN） in diabetic rats. Methods Totally 130 rats were selected to make the models of diabetic rats. The diabetic rat models were randomly divided into the model group,probucol group,anisodamine group,and the combination group（ probucol combined with anisodamine）,with 21 rats in each group. In addition,10 normal rats were selected as the control group. After 9-week feeding,the diabetic rats in the probucol group and the combination group received intragastric administration of probucol（ 500 mg/kg） for 6 d. On the eighth day,rats in the anisodamine group and combination group were injected with anisodamine 100 μg/100 g intraperitoneally before the preparation of CIN model. After 15 min,all diabetic rats were injected with urografin 0. 8 m L/100 g intravenously to prepare the CIN models. One hour later,rats in the anisodamine group and combination group were given anisodamine 100 μg/100 g intraperitoneally for 7 times. The coomassie brilliant blue（ CBB） was used to determine the urinary protein before and after injection with contrast medium 24 h.Blood samples were taken from the inner canthus vein for measuring the serum BUN and Scr levels. After weighed,the left and right kidneys of all rats were also weighed for calculating the renal index. Pathological changes of kidneys were observed by HE staining. The renal tissues were homogenized for testing nitric oxide（ NO）,nitric oxide synthase（ NOS）,superoxide dismutase（ SOD）,malondialdehyde（ MDA） and total antioxidant capacity（ T-AOC）. The expression levels of B cell lymphoma/leukemia-2 gene（ Bcl-2） and Bcl-2-related X protein（ Bax） mRNA were detected by fluorescent quantitative PCR. Results（1）The levels of BUN,Scr,and UAE in the model group were higher than those in the control group.The levels of BUN,Scr and UAE in the probucol group and combination group were lower than those in the model group,and the levels of BUN,Scr,and UAE in the combination group were lower than those of the anisodamine group（ all P〈0. 05）.（2）The renal index in the model group was higher than that of the control group,and the renal index in the probucol group and the combination group was lower than that in the model group（ all P〈0. 05）.（3） Compared with the model group,the NOS level increased and MDA decreased in the probucol group and anisodamine group（ both P〈0. 05）. The levels of SOD and T-AOC in the combination group were higher than those in the probucol group and anisodamine group（ both P〈0. 05）.（4）Compared with the model group,the Bax expression decreased,the levels of Bcl-2 and the ratio of Bcl-2/Bax increased in the probucol group and combination group（ all P〈0. 05）,and the ratio of Bcl-2/Bax in the anisodamine group increased（ P〈0. 05）. The ratio of Bcl-2/Bax in the combination group was higher than that in the anisodamine group（ P〈0. 05）. Conclusion The probucol combined with anisodamine can alleviate the renal injury of diabetic CIN rats. The mechanism may be related to the inhibition of oxidative stress and up-regulation of Bcl-2/Bax ratio,and the effect is superior to single drug application.