Rosiglitazone pretreatment alleviates renal oxidative stress in lipopolysaccharide-induced acute kidney injury
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摘要:
目的 探讨罗格列酮(rosiglitazone, RSG)抑制细菌脂多糖(lipopolysaccharide, LPS)所致急性肾损伤过程中氧化应激的机制。 方法 48只雄性CD-1小鼠随机分成对照组、RSG组、LPS 6 h组、LPS 24 h组、RSG+LPS 6 h组和RSG+LPS 24 h组共6组。LPS组小鼠经腹腔注射给予单剂量LPS(2 mg/kg); RSG+LPS组于LPS注射前经口灌胃给予RSG(10 mg/kg), 每天1次, 连续4 d。LPS注射后6 h和24 h取小鼠血清和肾脏组织。一侧肾脏用于苏木精-伊红染色法(hematoxylin-eosin staining, 简称HE染色法)并观察肾脏病理学损伤; 用全自动生化分析仪检测血清肌酐和尿素氮反映肾功能; 用生化方法检测肾脏还原型谷胱甘肽(glutathione, GSH)和丙二醛(malondialdehyde, MDA)水平; 用real time RT-PCR检测肾脏氧化应激相关基因(P47 phox, Sod-1, Sod-2, Sod-3)表达水平; 用蛋白质印迹(western blot, 简称WB)法检测肾脏NADPH氧化酶亚基NOX-2和NOX-4蛋白水平。 结果 RSG预处理明显减轻LPS所致肾小管破坏和肾功能损害, 抑制LPS所致肾脏炎性细胞浸润; RSG预处理抑制LPS所致肾脏MDA水平升高, LPS暴露下调了肾脏GSH水平, RSG预处理有改善LPS诱发GSH耗竭的趋势, 但无统计学意义; RSG阻断LPS上调肾脏P47 phox mRNA表达水平; RSG预处理抑制LPS诱导肾脏NOX-2和NOX-4蛋白表达水平升高。 结论 RSG预处理减轻LPS诱发肾脏氧化应激反应。 Abstract:Objective To investigate the effects of pretreatment with rosiglitazone(RSG) on oxidative stress in lipopolysaccharide(LPS)-induced acute kidney injury(AKI). Methods Forty-eight male ICR mice were divided into six groups: Control group, RSG group, LPS 6 h group, LPS 24 h group, RSG+LPS 6 h group, and RSG+LPS 24 h group. In the LPS and LPS+RSG groups, mice were intraperitoneal injected with LPS(2 mg/kg). In the RSG and RSG+LPS groups, mice were administered with RSG(10 mg/kg) by gavage four consecutive days before LPS injection. All mice were sacrificed at 6 h and 24 h after LPS injection. Some kidneys were collected for HE staining. Renal GSH and MDA were detected using biochemical method. Renal oxidant stress related genes were analyzed through real time RT-PCR. Renal NADPH oxidase-2 and NOX-4 were measured by Western blot. Serum was acquired for renal function. Results LPS-induced AKI, as determined renal pathological damage, renal function injury and inflammatory cell infiltration, were attenuated in RSG-pretreated mice. Although it did not affect renal GSH level, RSG pretreatment evidently inhibited the elevation of renal MDA content. Mechanistically, RSG pretreatment distinctly repressed LPS-induced upregulation of P47phox mRNA and nicotinamide adenine dinucleotide phosphate(NADPH) oxidase(NOX)-2 and NOX-4 in the kidney. Conclusion RSG pretreatment attenuates renal oxidative stress in LPS-induced acute kidney injury partially through inhibiting upregulation of NADPH oxidase. -
Key words:
- Rosiglitazone /
- Lipopolysaccharide /
- Acute kidney injury /
- Oxidative stress /
- NADPH oxidase
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图 1 小鼠肾脏病理和肾功能
注:图A显示小鼠肾脏HE染色; 图B表示小鼠肾脏病理学评分; 图C表示血清尿素氮水平; 图D表述血清肌酐水平。所有数据以(x±s)表示(N=8)。a表示P < 0.05, b表示P < 0.01。
Figure 1. Hematoxylin-eosin staining of renal histology and renal function in mice
图 2 小鼠肾脏GSH和MDA
注:图A表示小鼠肾脏GSH水平; 图B表示小鼠肾脏MDA水平。所有数据以(x±s)表示(N=8)。a表示P < 0.05, b表示P < 0.01。
Figure 2. Renal GSH and MDA in mice
图 3 小鼠肾脏组织氧化应激相关mRNAs
注:图A表示P47phox; 图B表示Sod-1;图C表示Sod-2;图D表示Sod-3。所有数据以(x±s)表示(N=8)。a表示P < 0.01。
Figure 3. Renal oxidative stress related mRNAs in mice
图 4 小鼠肾脏组织NOX-2和NOX-4蛋白表达
注:图A表示NOX-2和NOX-4蛋白条带; 图B表示NOX-2蛋白定量分析; 图C表示NOX-4蛋白定量分析。所有数据以(x±s)表示(N=8)。a表示P < 0.05, b表示P < 0.01。
Figure 4. Renal NOX-2 and NOX-4 protein in mice
表 1 引物序列
Table 1. Primers for real-time RT-PCR
基因名称 序列 长度(bp) 18S 正向: 5'- GTAACCCGTTGAACCCCATT-3' 151 反向: 5'- CCATCCAATCGGTAGTAGCG-3' P47phox 正向: 5'- CCAGGGCACTCTCACTGAATA -3' 100 反向: 5'- ATCAGGCCGCACTTTGAAGAA -3' Sod-1 正向: 5'-GCGATGAAAGCGGTGTGCGTG -3' 143 反向: 5'-TGGACGTGGAACCCATGCTGG -3' Sod-2 正向: 5'-TGGACGTGGAACCCATGCTGG -3' 162 反向: 5'-AGCGAACGGCCGTGTTCTGAG -3' Sod-3 正向: 5'- GAGAAGATAGGCGACACGCA -3' 155 反向: 5'- AGAACCAAGCCGGTGATCTG-3' 
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