甘西鼠尾草总酚酸提取物抗嘌呤霉素氨基核苷诱导的足细胞氧化应激损伤

目的 研究甘西鼠尾草总酚酸提取物(SPE)在体内和体外对嘌呤霉素氨基核苷(PAN)肾病大鼠足细胞以及PAN诱导小鼠足细胞氧化应激损伤的作用。 方法 (1)建立PAN肾病大鼠动物模型, 给予SPE和他克莫司干预, 分别在第5、10、15、21天留取肾组织标本, WT1染色计数足细胞数目, 免疫荧光观察8-羟基脱氧鸟苷(8-OHdG)荧光强度。(2)体外采用PAN致足细胞损伤模型, PAN作用小鼠足细胞24 h, 分别加入含SPE、丹酚酸B(SalB)、迷迭香酸(RA)及他克莫司的培养基培养6、12、24、48 h, 观察足细胞骨架相关蛋白F-actin的表达, 流式细胞仪分析细胞内活性氧(ROS)荧光强度。 结果 (1)肾小球WT1细胞计数结果显示, PAN组第5天时足细胞数已开始下降, 第15天达(14.4±0.7)个/肾小球切面, 较正常组(37.2±1.5)个/肾小球切面减少(P<0.05), SPE组与阳性对照组(他克莫司组)各时间点足细胞数量高于PAN组;第15天时, 阳性对照组肾小球WT1细胞计数与SPE高剂量组较为接近(P>0.05)。第5天时PAN组大鼠肾组织8-OHdG荧光强度较正常组增强, 第10天上升至高峰, 而后开始减弱, 第15天时仍高于正常组;给予药物干预后, 大鼠肾组织的8-OHdG荧光强度降低, 其中阳性对照组与SPE高剂量组8-OHdG荧光强度较为接近。(2)体外研究发现, PAN作用24 h后, F-actin几乎完全解聚, 少数细胞尚有残存的被切断的丝状结构, 给予SPE、SalB、RA及他克莫司治疗后, PAN诱导的足细胞损伤明显减弱, 细胞内重新出现极性分布的微丝。与正常组相比, PAN作用小鼠足细胞24 h后ROS荧光强度增加(P<0.05)。给予药物干预后足细胞内ROS荧光强度降低, 24 h SPE低剂量组、SalB高剂量组和RA高剂量组与阳性对照组足细胞内ROS的荧光强度降低程度相近(P>0.05), 24 h SalB降低足细胞内ROS荧光强度效果优于RA, 且与药物剂量呈正相关。 结论 本研究从体内及体外证实, SPE对PAN所致足细胞氧化应激损伤具有保护作用。
Objective To determine the effect of Salvia przewalskii extract of total phenolic acids (SPE) on puromycin aminonucleoside (PAN)-induced oxidative stress in podocytes of rats in vivo and the effect of SPE on PAN-induced oxidative stress in podocytes of mice in vitro. Methods (1) Nephropathy rat model was established by PAN and was given intervention with SPE and tacrolimus.The renal tissue samples were obtained for WT1 staining to calculate the number of podocytes on the 5th, 10th, 15th and 21st day. The intensities of 8-hydroxy-2'-deoxyguanine (8-OHdG) were evaluated by immunofluorescence. (2)The podocytes of mice were exposed to PAN for 24 h in vitro, and then SPE, salvianolic acid B (SalB), rosmarinic acid (RA) or tacrolimus were added for 6, 12, 24, and 48 h culture. Then the cytoskeleton distribution of podocytes, indicated by F-actin, was observed by fluorescence microscopy, and the intracellular reactive oxygen species (ROS) production was measured by flow cytometry. Results (1)Decrease of podocytes per glomerular volume as measured by counting WT1-positive cells was started on day 5 in each group except normal control (NC) group, and on day 15 glomerular podocytes in PAN group was significantly less than that in the NC group ([14.4±0.7]/glomerular volume vs [37.2±1.5]/glomerular volume, P<0.05). The numbers of glomerular podocytes in SPE group and positive group (tacrolimus group) were more than that in PAN group at all time points. The glomerular podocyte count of high-dose SPE group was similar to that of positive group on day 15 ([21.7±1.0]/glomerular volume vs [23.6±1.2]/glomerular volume, P>0.05). After