目的 探讨5-氨基水杨酸(5-ASA)对百草枯(PQ)中毒大鼠肾脏的保护作用及机制.方法将24只健康清洁级成年雄性SD大鼠按随机数字表法分为生理盐水(NS)对照组、5-ASA对照组、PQ模型组和5-ASA干预组,每组6只.采用一次性腹腔注射2% PQ溶液20 mg/kg复制PQ中毒大鼠模型;NS对照组和5-ASA对照组腹腔注射等量NS.5-ASA对照组和5-ASA干预组分别于注射NS或PQ溶液后2 h一次性灌胃1 mL的5-ASA(75 mg/kg);NS对照组和PQ模型组灌入1 mL双蒸水.观察大鼠行为学改变;于实验开始后24 h采集大鼠右心室血,检测肾损伤标志物及氧化应激指标.取左肾组织,经苏木素-伊红(HE)染色后,于光镜下观察肾组织病理学改变;采用蛋白质免疫印迹试验(Western Blot)检测核因子相关因子2(Nrf 2)、血红素氧合酶-1 (HO-1)蛋白表达.结果 PQ染毒30 min大鼠即出现明显的PQ中毒症状及体征;24 h光镜下观察显示,肾小球毛细血管网淤血,肾小管上皮细胞肿胀,出现空泡变性、坏死,部分核固缩;与NS对照组比较,大鼠尿素氮(BUN)、血肌酐(SCr)、超氧化物歧化酶(SOD)、丙二醛(MDA)、过氧化氢酶(CAT)和还原型谷胱甘肽(GSH)水平均出现明显异常,肾组织Nrf 2及HO-1蛋白表达明显升高.经5-ASA干预后,大鼠PQ中毒症状及肾组织病理学改变较PQ模型组明显改善;BUN、SCr和MDA较PQ模型组明显降低〔BUN(mmol/L):11.98±1.81比18.56±2.32,SCr(μmol/L):30.67±2.31比43.67±9.02,MDA(μmol/L):5.28±0.43比6.81±1.00〕,SOD活性及CAT、GSH含量较PQ模型组明显升高〔SOD(kU/L):125.49±7.63比106.76±7.94,CAT(ng/L):30.68±3.51比23.05±1.55,GSH(μmol/L):3.81±0.44比3.14±0.17〕,而肾组织Nrf 2和HO-1蛋白表达则均较PQ模型组进一步升高〔Nrf 2蛋白(灰度值):0.76±0.04比0.52±0.03,HO-1蛋白(灰度值):0.56±0.02比0.31±0.02,均P<0.05〕.单纯给予5-ASA干预对大鼠行为学、肾组织病理学、肾损伤标志物及氧化应激指标均无明显影响,但可诱导肾组织Nrf 2和HO-1蛋白表达明显升高,与NS对照组比较差异有统计学意义〔Nrf 2蛋白(灰度值):0.78±0.02比0.41±0.04,HO-1蛋白(灰度值):0.51±0.03比0.23±0.01,均P<0.01〕.结论 5-ASA可减轻PQ中毒导致的急性肾损伤(AKI),其机制可能与激活Nrf 2-抗氧化反应元件(ARE)信号通路有关.%Objective To investigate the protective effect of 5-aminosalicylic acid (5-ASA) on renal injury poisoned by paraquat (PQ) in rats and its mechanism. Methods Twenty-four healthy clean male Sprague-Dawley (SD) rats were randomly divided into four groups: normal saline (NS) control group, 5-ASA control group, PQ model group and 5-ASA treatment group, with 6 rats in each group. The rat model of PQ poisoning was reproduced by intraperitoneal injection of 2% PQ solution 20 mg/kg, and the same volume of NS was given in NS control group and 5-ASA control group. Two hours later, the rats in 5-ASA control group and 5-ASA treatment group were intragastrically administered with 1 mL 5-ASA (75 mg/kg) for one time after NS or PQ administration, and those in NS control group and PQ model group were administered with 1 mL double distilled water. Behavioral changes were observed in rats. Then the rats were sacrificed at 24 hours after starting of the experiment for cardiac blood harvest which could be used to detect the biomarkers of renal injury and oxidative stress parameters. The kidney tissue was collected, and the hematein-eosin (HE) staining was conducted for observation of pathological changes in renal tissue, and protein expressions of Nrf 2 and heme oxygenase-1 (HO-1) were determined by Western Blot. Results At 30 minutes after PQ poisoning, rats appeared obvious poisoning symptoms and signs. Twenty-four hours after PQ poisoned, hemocoel of glomerular capillary, swelling of renal tubular epithelial cell and serious micronecrosis appeared under the light microscope. Compared with NS control group, blood urea nitrogen (BUN), serum creatinine (SCr), superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT) and glutathione (GSH) levels were significantly abnormal in PQ model group, and Nrf 2 and HO-1 protein expressions in renal tissue were increased. After administration of 5-ASA, the morphological changes and pathological damage were mitigated as compared with those of PQ model group, the levels of BUN, SCr and MDA were decreased significantly [BUN (mmol/L): 11.98±1.81 vs. 18.56±2.32, SCr (μmol/L): 30.67±2.31 vs. 43.67±9.02, MDA (μmol/L):5.28±0.43 vs. 6.81±1.00], and the SOD activity, CAT and GSH contents were significantly increased [SOD (kU/L):125.49±7.63 vs. 106.76±7.94, CAT (ng/L): 30.68±3.51 vs. 23.05±1.55, GSH (μmol/L): 3.81±0.44 vs. 3.14±0.17], while the protein expressions of Nrf 2 and HO-1 were further increased [Nrf 2 protein (gray value): 0.76±0.04 vs. 0.52±0.03, HO-1 protein (gray value): 0.56±0.02 vs. 0.31±0.02, all P < 0.05]. Only 5-ASA intervention had no significant effect on behavior, pathology, renal injury markers and oxidative stress parameters, but it could induce the expressions of Nrf 2 and HO-1 protein in renal tissue, which were significantly higher than those of NS control group [Nrf 2 protein (gray value): 0.78±0.02 vs. 0.41±0.04, HO-1 protein (gray value): 0.51±0.03 vs. 0.23±0.01, both P < 0.01]. Conclusion 5-ASA attenuates the damage of acute renal injury (AKI) caused by PQ, which mechanism may be related with the activation of Nrf 2-antioxidant response element (ARE) signaling pathway.
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