摘要
目的探讨5-羟基-1-甲基海因(HMH)对百草枯(PQ)中毒所致肾损伤的防护作用及机制。方法采用随机数字表法将15只SPF级健康昆明小鼠分为生理盐水(NS)对照组、PQ中毒模型组和HMH干预组,每组5只。采用一次性灌胃30mg/kgPQ溶液制备PQ中毒模型;NS组灌胃等量NS;HMH组于制模后立即给予100mg/kg的HMH继续灌胃。于HMH灌胃后1d处死各组小鼠并留取心脏血和肾脏组织备检。采用苏木素-伊红(HE)染色,光镜下观察肾组织形态学改变;按照试剂盒说明书步骤检测肾组织丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性;采用蛋白质免疫印迹试验(WesternBlot)检测肾组织血红素氧合酶-1(HO-1)、白细胞介素-1β(IL-1β)蛋白表达;采用气相色谱质谱联用法(GC-TOF-MS)检测血清代谢产物,用主成分分析法(PCA)观察各组样本之间的整体分布状况,用多维分析法正交偏最小二乘法-判别分析(OPLS-DA)评价模型的准确性,以重要性投影(VIP)值>1来筛选有差异的代谢物。结果光镜观察显示:NS组肾小球结构清晰,肾间质及血管周围无充血、无炎性细胞浸润;PQ组部分肾小球萎缩坏死,肾小球内毛细血管充血,周围炎性细胞浸润,肾小管上皮细胞肿胀,管腔轻度狭窄,偶尔出现上皮细胞坏死脱落;HMH组肾损伤程度较PQ组明显减轻。与NS组比较,PQ组肾组织MDA含量显著增加(nmol/g:6.70±0.84比2.70±0.43,P<0.01),SOD活性显著降低(kU/L:33.30±4.66比50.20±3.23,P<0.05),肾组织HO-1、IL-1β蛋白表达水平显著增加(HO-1/β-actin:1.11±0.12比0.61±0.13,IL-1β/β-actin:0.93±0.13比0.32±0.06,均P<0.05)。与PQ组比较,HMH组MDA含量显著下降(nmol/g:5.10±0.93比6.70±0.84,P<0.05),SOD活性显著增加(kU/L:61.00±9.02比33.30±4.66,P<0.05),HO-1蛋白表达水平显著下降(HO-1/β-actin:0.77±0.07比1.11±0.12,P<0.05),而IL-1β蛋白表达水平差异无统计学意义(IL-1β/β-actin:0.87±0.13比0.93±0.13,P>0.05)。代谢产物检测结果显示:与NS组比较,PQ组肌酐、甘氨酸、琥珀酸、富马酸、柠檬酸水平显著增加(VIP值分别为1.50、1.58、1.64、1.74、1.95,均P<0.05),而软脂酸、α-生育酚和6-磷酸葡糖酸水平显著下降(VIP值分别为1.10、1.55、1.56,均P<0.05)。与PQ组比较,HMH组肌酐、柠檬酸水平显著下降(VIP值分别为1.50、1.86,均P<0.05),而反式4-羟基-脯氨酸、D-甘油酸、果糖-2,6-二磷酸、6-磷酸葡糖酸、氨基丙二酸水平显著增加(VIP值分别为1.36、1.55、1.63、1.68、1.76,均P<0.05)。结论HMH通过纠正三羧酸循环紊乱、脂质过氧化、能量代谢紊乱,从而防护PQ中毒致肾损伤,其机制与通过Nrf2通路调控HO-1蛋白表达有关。
Objective To investigate the effects of 5-hydroxy-1-methylhydantoin(HMH)on kidney injury induced by paraquat(PQ).Methods Fifteen SPF healthy Kunming mice were randomly divided into normal saline(NS)control group,PQ poisoning model group and HMH intervention group,with 5 mice in each group.PQ poisoning model was challenged by one-time gavage of 30 mg/kg PQ solution.The NS group received the same amount of NS by gavage.The HMH group was given 100 mg/kg of HMH immediately after the model was made and continued to be gavaged.Mice in each group were sacrificed 1 day after HMH gavage and heart blood and renal tissue were harvested for examination.The morphological changes of renal tissue were observed under light microscope by hematoxylin-eosin(HE)staining.The content of malondialdehyde(MDA)and the activity of superoxide dismutase(SOD)in renal tissue were detected according to the instructions of the kit.The expression of heme oxygenase-1(HO-1)and interleukin-1β(IL-1β)in renal tissues were detected by Western Blot.The serum metabolites were detected by gas chromatography time-of-flight mass spectrometry(GC-TOF-MS),the overall distribution of each sample was observed by principal component analysis(PCA),the accuracy of the model was evaluated by multidimensional analysis orthogonal partial least squares-discriminant analysis(OPLS-DA),and the difference metabolites were screened by variable importance in the projection(VIP)value>1.Results Light microscopic observation showed that:glomerular structure in NS group was clear,there was no hyperemia and inflammatory cell infiltration in renal interstitium and blood vessels.In PQ group,some glomeruli atrophy and necrosis,capillary congestion in glomeruli,infiltration of inflammatory cells around glomeruli,swelling of renal tubular epithelial cells,slight stenosis of lumen,and occasional necrosis and exfoliation of epithelial cells occurred.The degree of kidney injury in HMH group was significantly less than that in PQ group.Compared with the NS group,the content of MDA in the PQ group was significantly increased(nmol/g:6.70±0.84 vs.2.70±0.43,P<0.01)and the activity of SOD was significantly decreased(kU/L:33.30±4.66 vs.50.20±3.23,P<0.05),the protein expression of HO-1 and IL-1β were significantly increased(HO-1/β-actin:1.11±0.12 vs.0.61±0.13,IL-1β/β-actin:0.93±0.13 vs.0.32±0.06,both P<0.05).Compared with the PQ group,the content of MDA in the HMH group was significantly decreased(nmol/g:5.10±0.93 vs.6.70±0.84,P<0.05)and the activity of SOD was significantly increased(kU/L:61.00±9.02 vs.33.30±4.66,P<0.05),the protein expression of HO-1 was significantly decreased(HO-1/β-actin:0.77±0.07 vs.1.11±0.12,P<0.05),however,there was no significant difference in the protein expression of IL-1β(IL-1β/β-actin:0.87±0.13 vs.0.93±0.13,P>0.05).Metabolite detection results showed that:compared with NS group,the levels of creatinine,glycine,succinic acid,fumaric acid and citric acid were significantly increased in the PQ group(VIP value was 1.50,1.58,1.64,1.74 and 1.95 respectively,all P<0.05),while the levels of palmitic acid,α-tocopherol and 6-phosphogluconic acid were significantly decreased(VIP value was 1.10,1.55 and 1.56 respectively,all P<0.05).Compared with the PQ group,the levels of creatinine and citric acid were significantly decreased in the HMH group(VIP value was 1.50 and 1.86,both P<0.05),while trans-4-hydroxy-proline,D-glyceric acid,2,6-fructose phosphate,6-phosphate gluconic acid and aminomalonic acid were significantly increased(VIP value was 1.36,1.55,1.63,1.68 and 1.76 respectively,all P<0.05).Conclusions HMH protects kidney injury caused by PQ poisoning by correcting tricarboxylic acids cycle disturbance,lipid peroxidation and energy metabolism disturbance,and its mechanism is related to the regulation of HO-1 protein expression through Nrf2 pathway.
作者
高利娜
袁慧雅
曹志鹏
徐恩宇
刘俊亭
Gao Lina;Yuan Huiya;Cao Zhipeng;Xu Enyu;Liu Junting(School of Forensic Medicine,China Medical University,Shenyang 110014,Liaoning,China)
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2018年第12期1184-1189,共6页
Chinese Critical Care Medicine
基金
国家自然科学基金项目(81601644,81671864).
