白藜芦醇对脓毒症急性肾损伤大鼠肾小管上皮细胞线粒体功能的影响

Effect of resveratrol on mitochondrial function in renal tubular epithelial cells of rats with sepsis-induced acute kidney injury

目的评价白藜芦醇对脓毒症急性肾损伤大鼠肾小管上皮细胞线粒体功能的影响.方法健康SD大鼠96只,雌雄不拘,5~7周龄,体重180~220 g,采用随机数字表法分为4组(n=24):假手术组(Sham组)、脓毒症组(Sep组)、脓毒症+溶媒组(Sep+Ⅴ组)和脓毒症+白藜芦醇组(Sep+R组).采用盲肠结扎穿孔(CLP)法制备脓毒症模型.于CLP后6、12、18h时,Sep组、Sep+Ⅴ组和Sep+R组分别腹腔注射生理盐水0.5 ml、溶媒0.5 ml、白藜芦醇10 mg/kg.于CLP后24h时,测定血清Cr和BUN浓度;取肾组织,观察病理学结果,行肾小管损伤评分;分离纯化肾小管上皮细胞,采用JC-1荧光探针联合流式细胞仪分析技术检测线粒体跨膜电位,采用生物素-生物素酶发光技术测定细胞ATP含量,采用钙黄绿素-氯化钴技术联合流式细胞仪测定线粒体通透性转换孔开放的程度,采用过氧化物与亚铁离子的氧化还原反应直接测定细胞内过氧化脂质(LPO)含量,采用溶酶体摄取吖叮橙技术联合流式细胞仪测定溶酶体膜通透性.结果与Sham组比较,Sep组和Sep+Ⅴ组血清Cr和BUN浓度、肾小管损伤评分、肾小管上皮细胞线粒体通透性转换孔开放的程度、LPO含量和溶酶体膜通透性升高,线粒体跨膜电位和ATP含量降低(P<0.01).与Sep组和Sep+Ⅴ组比较,Sep+R组血清Cr和BUN浓度、肾小管损伤评分、肾小管上皮细胞线粒体通透性转换孔开放的程度、LPO含量和溶酶体膜通透性降低,线粒体跨膜电位和ATP含量升高(P<0.05).结论白藜芦醇可改善脓毒症大鼠肾小管上皮细胞线粒体功能,减轻急性肾损伤,机制可能与抑制氧化应激,降低溶酶体膜通透性有关.

Objective To evaluate the effect of resveratrol on mitochondrial function in renal tubular epithelial cells of rats with sepsis-induced acute kidney injury.Methods Ninety-six healthy Sprague-Dawley rats of both sexes,aged 5-7 weeks,weighing 180-220 g,were divided into 4 groups(n=24 each)using a random number table method:sham operation group(Sham group),sepsis group(group Sep),sepsis plus vehicle group(Sep+V group)and sepsis plus resveratrol group(Sep+R group).Sepsis was induced by cecal ligation and puncture(CLP).Normal saline 0.5 ml,vehicle 0.5 ml and resveratrol 10 mg/kg were intraperitoneally injected at 6,12 and 18 h after CLP in Sep,Sep+V and Sep+R groups,respectively.At 24 h after CLP,serum concentrations of creatinine(Cr)and blood urea nitrogen(BUN)were measured,and kidney tissues were obtained for examination of the pathological changes(using transmission electron microscopy),and the damage to the renal tubules was scored.The renal tubular epithelial cells(RTECs)were isolated from the kidney cortex at 24 h after CLP for determination of mitochondrial transmembrane potential(by flow cytometry with the fluorescent probe JC-1),intracellular ATP content,mitochondrial permeability transition pore(mPTP)opening,lipid peroxide(LPO)content,and lysosomal membrane permeability.Results Compared with group Sham,the serum concentrations of Cr and BUN,renal tubular damage score,mPTP opening,LPO content and lysosomal membrane permeability were significantly increased,and mitochondrial transmembrane potential and ATP content were decreased in Sep and Sep+V groups(P<0.01).Compared with Sep and Sep+V groups,the serum concentrations of Cr and BUN,renal tubular damage score,mPTP opening,LPO content and lysosomal membrane permeability were significantly decreased,and mitochondrial transmembrane potential and ATP content were increased in group Sep+R(P<0.05).Conclusion Resveratrol improves mitochondrial function in renal tubular epithelial cells of rats with sepsis and reduces acute kidney injury,and the mechanism may be related to inhibiting oxidative stress and decreasing the lysosomal membrane permeability.